Journal of Chemical Ecology, VoL 22, No.

6, 1996

SEX PHEROMONE OF Stenoma cecropia MEYRICK

(LEPIDOPTERA: ELACHISTIDAE)

PIERRE ZAGATTI,I'* PHILIPPE LUCAS, I PHILIPPE GENTY, 2

SANDRA ARANGO, 3 CHRISTIAN MALOSSE, 1 and
FRI~DI~RIQUE TELLIER j

~INRA Unit~ de Phytopharmacie et M#diateurs Chimiques

78026 Versailles Cedex, France
'lndupalma SA
A.A. 6226 Bogot6, Colombia
'aPalmas de Tumaco Ltda.
A.A. 43906 Bogotd, Colombia

(Received May 12, 1995; accepted January 29. 1996)

Abstract--(Z,E)-9,1 l-tetradecadienal (Z9.E 11- 14 : Aid; I 1%), (Z,E)-

9,11,13-tetradecatdenal (Z9,EI 1, 13-14 : Aid; 67 %), (Z,E)-9,1 l-tetradeca-
dienyl acetate (Z9,E 11-14 : Ac. 5.5 %). and (Z,E)-9~ 11,13-tetradecatrienyI
acetate (Z9,E 11,13-14 : Ac: 16.5 %) were identified in the extracts of female
pheromone glands of Stenoma cecropia (Lepidoptera: Elachistidae). From
electroantennograms and single sensillum recordings, receptors to Z9,E 11,13-
14:Ald and Z9,EI 1-14:Ald were found on male antenna. Behavioral data
were obtained from olfactometric tests in the laboratory and field trapping
experiments in Colombia. It appeared that a blend of Z9,EI 1,13-14:Ald
(83 % ) and Z9,E 11-14 :Atd ( 17 %) was attractive to males. These aldehydes
are assumed to be components of the sex pheromone of S. cecropia, whereas
the acetates found in gland extracts might be precursors of the pheromone,

Key Words--Lepidoptera, Elachistidae, Stenoma cecropia, sex pheromone,

identification, electmantennography, single sensillum recording, field trapping
(Z,E )-9,1 l-tetradecadienal, (Z.E )-9 ,11.13-tetradecatrienal.

INTRODUCTION

T h e m o t h S t e n o r n a c e c r o p i a M e y r i c k b e l o n g s to a n e o t r o p i c a l s u b f a m i l y (Sten-
ominae) of the Gelechioidea. These moths were considered as Oecophoridae by

*To whom correspondence should be addressed.

1103

00t)8-0331/96/0600 1103509.50/0 (cj 1996 Plenum Publishing Corpt~ration

1104 P. ZAGATT!ET AL.

Hodges (1974). More recently, Minet revisited the taxonomic status of Sten-
ominae and included them in the family Elachistidae on the basis of the abdom-
inal articulation of the nymph (Minet, 1989). The adults of S, cecropia look
like colorful tortricids, but the larvae live in fixed bags and feed on several
forest trees, including the leaflets of Palmaceae. In oil palm plantations of central
Colombia, the populations of this insect can reach huge levels, and the threshold
for insecticidal treatment is 80 larvae per leaf (Genty, 1978). Severe defoliations
by this insect may decrease the yield of oil palm trees by as much as 15-20%.
We undertook the identification of the sex pheromone of S. cecropia to provide
a useful tool for monitoring the populations of the moth.
The chemical aspects relating to the synthesis of pheromone components
have been partly published (Ramiandrasoa and Tellier, 1990; Tetlier and Des-
coins, 1990). We present here the full identification study together with biolog-
ical data.

METHODS AND MATERIALS

Insects
Preliminary trials to send live insects to France were very unsatisfactory,
because 90% of the larvae or pupae died. It was thus decided in 1987 to extract
the pheromone in central Colombia by using a field laboratory in a 9000-ha
plantation (San Alberto, Cesar). The pupae were collected daily in their bags
and sexed in the laboratory. Half the pupae and emerging adults were then kept
under natural conditions (temperature varying between 28 and 40°C, 100%
relative humidity, and daylight from 06:30 to 18:30 hr). The other half of the
insects were kept in a room under similar conditions except that light was
provided from 04:30 to 16:30 hr.
The insects used for olfactometric tests, electroantennographic (EAG)
recordings, and single-sensillum recordings were collected as young pupae in
the field and brought back to France courtesy of traveling colleagues. They were
sexed and kept at 27°C with 75% relative humidity under a 12-12 hr photo-
period until emergence of the adults. The male moths used in these laboratory
studies were 1 or 2 days old.

Pheromone Extraction
As shown by observation of traps baited with virgin females, the period of
sexual activity of S. cecropia begins half an hour before dusk and lasts 2 hrs.
In San Alberto, the pheromone was extracted between 18:30 and 20:30 hr
(insects under natural photoperiod) or between 16:30 and 18:30 hr (insects under
shifted light regime).
SEX PHEROMONEOF Stenoma cecropia 1105

The last three abdominal segments of 1 or 2-day old virgin females were
clipped and soaked for 12 hr in 1 ml hexane (Carlo Erba Codex Depurato) at
- 5 ° C . The extract then was filtered through glass wool, reduced to ca. 100/zl
under a Nz flow and kept at - 3 0 ° C until used. Three extracts of, respectively,
684, 759, and 557 female abdominal tips were made using this method from
February 27 to March 18, 1987.

Pheromone Analysis
The sex pheromone extracts were analyzed by gas chromatography (GC)
and gas chromatography-mass spectrometry (GC-MS). GC analyses were per-
formed on a Hewlett-Packard 5890 chromatograph with flame ionization detector
(FID) and a Ross injector heated at 240°C, equipped either with a polar column
(30-m WCOT CPSil-88 CB, 0.22 mm ID; Chrompack, Middleburg, The Neth-
erlands) operated isothermally at 180°C, or with a nonpolar column (25-m
WCOT CPSil-5 CB, 0.22 mm ID; Chrompack) heated from 160 to 220°C at
5°C/min. GC-MS was performed on a Girdel 32 gas chromatograph linked to
a Nermag R 10-10C quadrupole mass spectrometer controlled by a SIDAR data
acquisition system. The chromatograph was fitted with a fused silica capillary
column (25-m WCOT CPSil-5 CB, 0.22 mm ID; Chrompack) and operated
isothermally at 180°C. Mass spectra were obtained by electron impact (El) at
70 eV scanning from 40 to 350 amu, and by chemical ionization (CI) at 92.5
eV with ammonia (NH 3) and isobutane (i-C4 H 10) as the reactant gas. Pheromone
compounds were identified by comparing their GC retention times and mass
spectra with those of chemicals synthesized in the laboratory (see below).

Synthesis of Chemicals
We report herein the synthesis of (Z,E)-9,11,13-tetradecatrien-l-al. For
some years, several laboratories have been interested in synthesis of 1,3,5-
undecatrienes (N/if et al., 1975; Ratovelomanana and Linstrumelle, 1987;
Andreini et al., 1987), which are reported to have odors highly appreciated in
perfumery. The need for regio- and stereochemicalty pure compounds for bio-
logical tests is well recognized. A number of stereoselective methods (Rato-
velomanana and Linstrumelle, 1987; Andreini et al., 1987) for obtaining these
nonfunctionalized trienes have been described. In our case the approach that has
given the best results was based upon the method of Rossi (Andreini et al.,
1987), which involved two sequential palladium-catalyzed cross-coupling reac-
tions. A first coupling between a molar excess of commercial E/Z 1,2-di-
bromoethylene and adequate alkynylzinc bromides led to (E)-l-bromo-l-en-3-
yne derivatives with a high stereoisomeric purity, since (E)-l,2-dibromoethylene
reacts preferentially when it is blended with the Z stereoisomer. These inter-
mediate bromoenynes were then coupled with vinylzinc bromide to afford the
1106 P. ZAGATTI ET AL.

corresponding 1,(E)-3-dien-5-ynes. The latter were allowed to undergo a Z

stereoselective reduction to give 1,(E)-3,(Z)-5-trienes. The actual steps for syn-
thesizing the desired trienal are shown in Figure 1. IH and '3C NMR spectra
were recorded on a Bruker AC 200 spectrometer [CDCI3; (5(ppm) from TMS,
J (Hz)]. Infrared spectra were measured on a Perkin-Elmer 397 spectrometer
[neat, 3' c m - ' ) ] . Stereoisomeric purity of all products was determined by gas
chromatographic analyses performed on a model 2900 Carlo Erba instrument
equipped with silica capillary polar column (25-m WCOT FFAP 0.32 ID, H 2
carrier gas flow 25 ml/min, 1.2 b).
(E)-1-Terbutoxy-12-bromo-9-dodecyn-11-ene. To a solution of 1-terbu-
toxy-9-decyne (Tellier and Descoins, 1990) [10.5 g (0.05 mol) in 25 ml Et20
and 25 ml THF] at 0°C were added 31 ml (0.05 mol) of 1.6 M n-BuLi in
hexane. After 30 min at 20°C, alkynyl lithium was obtained. To this reagent
was added at 0°C an anhydrous ZnBr 2 solution [12.4 g (0.055 mol)/50 ml THF].
The stirred mixture was wanned to 20°C for 30 min; then at 0°C were added
successively solutions of Pd(PPh3) 4 (3%) [1.7 g (1.5 mmol)/20 ml THF] and
46.5 g (0.25 mol) of E/Z (40/60) 1,2-dibromoethylene. The reaction mixture
was allowed to warm to room temperature for 16 hr and then hydrolyzed by
H2SO4 solution (1 N). After extraction with ether and evaporation of the solvent,
the crude mixture was passed through a small silica gel column in order to
remove the palladium catalyst (eluting with cyclohexane). The solvents were
evaporated and the residue distilled to afford 7.9 g of bromoynene (50% yield);
E purity _> 99%; bp, 120°C/0.1 Torr; m/z: 299, 301 (M-15); IR: 2250, 1200;
tH NMR: 1.18 (s, 9H), 1.33 (m, 12H), 2.22 (m, 2H), 3.32 (t, 2H), 6.13 (dt,
HI1), 6.56 (d, H 12) JH,~/H,~ = 14, JH~m,, = 2; '3C NMR: 19~5, 61.6, 72.3,
77.3, 93.2, 1t6.8, 118.1. Anat. calcd, for C,6H27BrO: C, 60.95; H, 8.63.
Found: C, 61.20; H, 8.77.
(E)-1-Acetoxy-12-bromo-9-dodecyn-11-ene. Ac20, 4.7 ml (0.05 mol), and
0.4 g (2.5 mmol) of FeC13 were successively added to a solution of 7.9 g (0.025

1) BuLL ZnBr2 = t'BuO(CH:DsC~C S B r Ac20, FeC}~

t-BuO(CH2)sC~CH
2) BrHC--CHBr, Pd°

//
///------Br CH;t__CHZnBr' pd o AcO(CH2)sC~C ///.....ff 1) KOH, MeOH
AcO(CH2)sC~C.._.~ ~" 2) Zn, KCN, PrO'H

H
OC
(H
s)z~ PDC,. OHC(CH2)7 __/
CH2CI2

FIG. 1. Synthesis of (Z,E)-9,11,13-tetradecatrien-l-al.

SEXPHEROMONEOF Stenorna cecropia 1107

mol) t-butyl ether in Et20 (50 hal). The stirred solution was left at room tem-
perature overnight. A saturated aqueous solution of Na2HPO4 (50 ml) was added
and the mixture was stirred for 1 hr. The solid precipitate was removed by
filtration. After extraction with ether and evaporation of the solvent, 6.9 g of
acetoxybromoynene were obtained (92 % crude yield); E purity (99 %); re~z: 221
(M-80); IR: 2250, 1740; ~H NMR: 1.2-1.7 (m, 12H), 2.03 (s, 3H), 2.25 (m,
2H), 4.04 (t, 2H), 6.16 (dt, Hit), 6.57 (d, H 12) JH,,H,-" = 14, JH"/H" = 2; ~3C
NMR: 19.4, 64.45, 77.4, 93.1, 116.9, 118.1, 170.6.
(E)-1-Acetoxv-11,13-tetradecadien-9-yne. A solution of ZnBr2 [24.8 g (0.11
mol)/100 ml THF] was added to a I M THF solution of vinyl magnesium
bromide (100 ml, 0.1 mol) cooled at 0°C. The reaction mixture was stirred at
20°C for 30 min, and after cooling to 0°C, 0.7 g (0.6 mmol) of Pd(PPh3)4 in
THF (20 ml) followed by 6 g (0.02 mol) of bromoynene were added. The stirred
mixture was allowed to warm to room temperature for 12 hr and then hydrolyzed
by H2SO4 solution (1 N). After extraction with ether and evaporation of the
solvent the crude mixture was passed through a small packed silica gel column
(eluting with cyclohexane). The solvent was evaporated and the residue distilled
to afford 3.7 g of acetoxydienyne (75% yield); E purity (96%); bp 115°C/0.1
Torr; m/z: 248 (M +, 3%), 91 (100%); IR: 2250, 1740, I620, 1245, 1000, 940,
905, 840, 745, 720, 700; ~H NMR: 1.33 (m, 12H), 2.02 (s, 3H), 2.30 (m,
2H), 4.04 (t, 2H), 5.09 (dd, HI4), 5.21 (dd, HI4'), 5.59 (d, H i l l 6.25 (dd,
HI2), 6.45 (ddd, H 13) JHI3/HI4" = 16.5, JH"/I-I'-"= 14.4, JM"-/H'~ = 9.9, JH'~m'~
= 9, JH,4/H,,. = 2; 13C NMR: 64.5, 79.6, 93.4, 112.7, 118.4, 136.4, 140.9,
17I. Anal. calcd, for C16H2402: C , 77.38; H, 9.74. Found: C, 77.33; H, 9.62.
(E)-11,13-Tetradecadien-9-yn-l-ol. This product was prepared by sapon-
ification of the corresponding acetate with 2 N KOH in MeOH; Crude yield
(98%); E purity (96%); m/z: 206 (M +, 4%), 91 (100%); IR: 3350, 2200, 1620,
1580, 1000, 940, 905,840, 740, 695; ~H NMR: 1.34 (m, 12H), 2.32 (m, 2H),
3.0 (m, 1H), 3.57 (t, 2H), 5.08 (dd, HI4), 5.20 (dd, HI4'), 5.58 (d, HI1), 6.24
(dd, HI2), 6.45 (ddd, H 13) JH,,/H,4. = 16.5, JH'~/H~-' = 14.4, Jn'-'m'~ = 10.8,
JH'~/H'4 = 9, Jn,,m,~. = 2. 13C NMR: 62.5, 79.6, 93.5, 112.7, 118.4, 136.3,
140.8.
(Z,E)-9,11,13-Tetradecatrien-1-oL A mixture of dienynol (2.9 g, 0.014
mol), 1-propanol-water (1/1) (600 ml), zinc powder (200 g), and potassium
cyanide (10.4 g, 0.16 tool) was stirred at room temperature for 24 hr under
argon in the dark. The reaction product was filtered through celite, and the
filtrate was extracted with Et20. After evaporation of the solvent, 2.6 g of trienol
were obtained (90% crude yield); ZE purity (98%); m/z: 208 (M ÷, 11%), IR:
3340, 1620, 1570, 1000, 935, 895, 740, 720, 695; ~H NMR: 1.33 (m, 12H),
1.99 (m, IH), 2.16 (m, 2H), 3.60 (t, 2H), 5.07 (dd, H14), 5.20 (dd, H14'), 5.46
(dt, Hg), 6.00 (ddt, Hm°), 6.19 (dd, Ht2), 6.41 (ddd, HI3), 6.51 (dd, H jl)
JH,3m,~ = 16.8, JH,,m,z = 14.6, JH~/H,~, = 11, JHIt,/tt,l = 10.6, JH"-/H'~ = 10.6,
1108 P. ZAGATTIET AL.

Jn,sm,, = 10, JHs/n9 = 7.7, JH,,/Hh4" = 2, JHS/H,O = 1.5; t3C NMR: 62.4, 116.6,
128.4, 128.6, 132.9, 133.2, 137.2. Anal. calcd, for C14H240: C, 80.71; H,
11.61. Found: C, 80.94; H, 11.45.
(Z,E)-1-Acetoxy-9,11,13-tetradecatriene. Ac~O, 1.4 ml (15 mmol), was
added to a stirred mixture of 1 g (5 mmol) of alcohol and 1.2 ml of pyridine.
Stirring was maintained for 3 hr at room temperature, and the mixture was
poured on crushed ice. After extraction with ether and evaporation of the solvent,
1.1 g of acetate were obtained (90% crude yield); ZE purity (98%); m/z: 250
(M ÷, 10%), 79 (100%); IR: 1745, 1235; ~H NMR: 1.32 (m, 12H), 2.02 (s,
3H), 2.16 (m, 2H), 4.04 (t, 2H). Data of the double-bond system are the same
as those of trienol. 13C NMR: 64.6, 116.85, 128.7, 128.8, 133.2, 133.3, 137.4,
170.8. Anal. calcd, for C16H2602: C, 76.75; H, 10.47. Found: C, 76.50; H,
10.36.
(Z,E)-9,11,13-Tetradecatrien-l-al. To a solution of alcohol, 1 g (5 mmol)
in CH2C12 (20 ml), was added 5.6 g (15 mmol) of PDC (pyridinium dichromate).
The reaction mixture was stirred at room temperature for 8 hr, mixed with Et20,
and filtered through a small column packed with Florisil to give, after evapo-
ration of solvents, the desired compound, 0.9 g (90% crude yield); ZE purity
(98%); re~z: 206 (M +, 6%), 70 (100%); IR: 2700, 1720; JH NMR" 1.31 (m,
10H), 2.15 (m, 2H), 2.40 (td, 2H), 9.72 (t, 1H) (J = 1.75). Data of the double-
bond system are identical to those of trienol. ~3C NMR: 43.8, 116.7, 128.5,
128.6, 133.0, 133.15, t37.2, 203.3. Anal. calcd, for CI4H220: C, 81.50; H,
10.75. Found: C, 81.71; H, 10.91.
Other Chemicals. The dienic compounds and (Z)-9,11-tetradecenyn-l-al
(Z9,Ynel 1-14: Ald) were prepared as described in Ramiandrasoa and Tellier
(1990). The other geometric isomers of trienic compounds, used in the identi-
fication work, were synthesized as described in Tellier et al. (1991) and Tellier
(1991).

Electrophysiology
Etectroantennograms were recorded on mate S. cecropia secured in a sty-
rofoam block. After a few antennal segments had been snipped off, the distal
end of one antenna was inserted into the tip of the recording electrode. The
neutral electrode was implanted in the neck of the insect. Both electrodes were
glass capillaries filled with Roeder's saline (NaCI: 9.0 g/liter; KCI: 0.2 g/liter;
glucose: 4.36 g/liter). Electrodes were connected to a WPI preamplifier through
chloridized silver wires. The filtered signal (DC to 300 Hz) was fed into an
oscilloscope and monitored directly on the screen.
To stimulate the antenna, air was puffed (0.5 sec; 1.5 liters/min) onto the
antenna through a glass cartridge containing a piece of filter paper impregnated
with 500 ng of synthetic compound. Ten compounds were tested, including
SEX PHEROMONEOF Stenoma cecropia 1109

those found in gland extracts of female S. cecropia, and a number of analogs.

All were synthesized in the laboratory, with an isomeric purity better than 98%.
The response profiles to synthetic compounds were recorded on 10 males (five
replicates only for the trienic aldehyde, acetate, and alcohol that had not been
synthesized at the beginning of the EAG screening).
Single sensillum recordings were carried out on excised antennae, using
the tip recording technique (Kaissling and Thorson, 1980; Lucas and Renou,
1989). The base of the antenna was inserted in a glass microelectrode filled with
hemolymph saline (Kaissling and Thorson, 1980). The tip of a few sensilla
trichodea were cut off with sharpened forceps, and the recording electrode, filled
with receptor lymph saline, was slipped over the cut end of a single hair. The
action potentials were filtered (150-5000 Hz), amplified ( × 1000), and recorded
on an audio tape recorder (Revox B77). The recordings were then played back
and digitized, with a 10-kHz conversion rate, by an A/D converter board (Dash
t6 of Metrabyte) integrated in a PC microcomputer. The responses were ana-
lyzed using a specific program written in Asyst (Macmillan Software Company)
language (Lucas and Renou, 1989).
The outlet of a Y-shaped glass tube was positioned 2 cm from the antenna,
and a flow of humidified pure air (1.5 liters/min) was continuously blown over
the antenna through one of the lateral branches. The stimulation cartridge con-
sisted of a Pasteur pipet containing a piece of filter paper (0.5 cm 2) on which
hexane extracts or hexane solutions of compounds were deposited. This cartridge
was introduced in the main branch of the Y tube. Stimulation was achieved by
blowing a puff of air (0.5 sec; 0.5 liters/min) through the cartridge. The same
compounds as those used for EAG were assayed on 14 sensilla trichodea belong-
ing to six different male moths.

Olfactometric Tests
The availability of tens of living pupae in the spring of 1989 enabled us to
develop a laboratory-based bioassay of pheromones. As we had insufficient time
to check the optimum conditions for wind-tunnel tests, we chose a dynamic,
linear olfactometer (Sower et al., 1973; Zagatti et al., 1987), consisting of a
glass tube (70 cm long, 3.5 cm diameter). Air was humidified by bubbling into
water and heated to 25°C, then flowed into the tube at 1.5 liters/min. The
olfactometer was situated in a room heated at 25°C and lighted by three incan-
descent red bulbs (40 W each).
The test started just at the beginning of the scotophase and lasted 1 hr.
Eight virgin males (1 or 2 days old) were placed at the downwind end of the
tube. Then the blend of synthetic compounds to be tested was deposited (1 /zl)
on a glass applicator at the upwind end of the tube. The males were scored
during 3 min according to whether they exhibited any of the following behavior
1110 P. ZAGATT!ET AL.

patterns: (1) wing fanning, (2) orientation toward the source by walking through
one half of the olfactometer length, and (3) contact with the source of attractant
with or without copulation attempts. These behavior patterns always occurred
in this order in a hierarchical sequence. Each male was tested only once, and
112 males were tested in 14 sets.
To test synthetic attractants, the compounds were diluted in hexane (Carlo
Erba Analyticals RPE) and the blends were prepared to obtain the doses (in 1
~1 hexane) shown in Table 1. These six blends were qualitatively the same as
those used during the 1989 field trapping experiment in San Alberto (Table 2).
In addition, the effect of natural pheromone was assayed by clipping off the
pheromone glands of two calling females and crushing them immediately to be
tested on the glass applicator.
The results were analyzed by a 7 × 2 chi-square test for global heteroge-
neity (P < 0.05), and then all blends or female glands were compared pairwise
using a 2 × 2 chi-square test. As this method multiplies the degrees of freedom,
the threshold of significance for the pairwise comparisons was fixed at P <
0,01, although the overall significance of the test was P < 0.05. The last step
of the behavioral sequence (contacts with source) was not analyzed, due to a
small number of responses,

Field Trapping
The first field-trapping experiments were conducted in August-October
1989 in San Alberto (estate of Indupalma S.A.). In the first part of this exper-
iment, the efficiency of pheromone trapping was studied by using traps baited
with virgin females. A trap containing two virgin females was hung 1.5 m above
the ground on a palm tree, This palm tree was examined every week to check
the number of living pupae on leaf number 17 and the number of eggs on 10
leaflets of leaf number 17, This is a standard method for monitoring populations
of S. cecropia (Genty et al., 1978). Three traps were used simultaneously and
the palm trees containing them subsequently sampled. The traps were made
from plastic cans (1 gallon vol) from which two windows (10 × 5 cm) were
cut. A small wire mesh cage was hung inside the trap in front of the windows,
and the bottom of the trap was filled with water and detergent. Two 1-day old
virgin females were held in the cages and replaced every two days. The trap
catches were recorded every day.
The second part of this experiment was conducted simultaneously to com-
pare the attractancy of synthetic chemicals. These chemicals were synthesized
in the laboratory with an isomeric purity greater than 98%. Six blends were
compared (Table 2). All tested blends were diluted in 1 ml hexane and loaded
on methanol extracted rubber septa (SNL, Paris, France). The trap design was
the same as in the virgin female trapping experiment, except that the pheromone
 X

t'rl
7~
o
o
z
o
TABLE 1. RESULTS OF OLFACTOMETRIC TESTS WITH MALE S. cecropia AND DIFFERENT SYNTHETIC BLENDS

Contacts
Compounds (ng) Wing Orientation with
fanning" to source" source ~'
Z9,EI t,13-14 : Aid Z9,EI 1-14: Aid Z9,EI 1,13-14: Ac Z9,EI 1-14: Ac Z9,Ynel 1-14: Aid N (%) (%) (%) ~"

1 50 10 30 10 32 62.5a 31.3ab 25
2 10 30 I0 50 16 31.3ab Ob 0
3 50 30 8 37.5ab Ob 0
4 10 10 8 Ob Ob 0
5 50 10 24 62.5a 33.3a 16.7
6 50 10 8 37.5ab 12.5ab 12.5
7 Two crushed glands of virgin ~males 16 62.5a 37.5a 18.8

"The results are expressed as percentages of males showing the behavior. In the same column, numbers followed by the same letter are not significantly different
(global heterogeneity tested as a 7 × 2 chi-square analysis at P = 0,05, then 2 x 2 single comparisons at P = 0.01).
~'Statistical analysis not available for this level of response.
 TABLE 2. TOTAL CATCHES OF ADULT S, cecropia IN TRAPS BAITED WITH DIFFERENT SYNTHETIC BLENDS FROM AUGUST 26 TO
SEVrEMBER 27, 1989 (SAN ALBERTO,CENTRALCOLOMBLA)

Compounds (~g)
Male Female
Blend Zg,EI 1,13-14: Ald Z9,EI 1-14 : Aid Z9,EII,13-14:Ac Z9,EI 1-14 : Ac Z9,Ynel 1-14: Atd catches" catches"

SC 1 250 50 150 50 28c 22a

SC2 50 150 50 250 19c 22a
SC3 250 150 29c 15a
SC4 50 50 16c 13a
SC5 250 50 88b 24a
SC6 250 50 20c 15a
2 virgin femalesb 458a 24a

"In the same column, numbers followed byr.Lhe same letter are not significantly different at P = 0.05 [one-way ANOVA on seven treatments and three
replicates after transformation of data to 4(x + 0.5) and Newman-Keuls test]. The differences between male catches and female catches in the same row
are not significant (P > 0.05), except for SC5 and for the virgin female traps (Wilcoxon's randomization test for matched pairs).
bSee Figure 3 for chronological data. "o
N

t-
 SEX PHEROMONEOF Stenoma cecropia 1113

TABLE 3. TOTAL CATCHES OF ADULT S. cecropia IN TRAPS BAITED WITH DIFFERENT SYNTHETIC
BLENDS FROM JUNE 6 TO JUNE 20, 1992 (TUMACO, COLOMBIA)

Compounds (gg)
Male Mean Female
Blend Z9,EI1,13-14 :Aid Z9,EI1-14 : Aid Z9-14: Aid catches" catch/day/trap catches

SC5 250 50 27b 0,6 0

SC7 250 0c 0 0
SC8 50 0c 0 0
SC9 250 50 10 9bc 0.2 0
SCI0 250 250 14bc 0.3 0
SCll 1000 200 l13a 2.7 0
SC12 250 5 0c 0 0

"Numbers followed by the same letter are not significantlydifferentat P = 0.05 (two-way ANOVAon three
blocks, after transformationof data to x/~ + 0.5) and Newman-Keulstest).

dispensers were hung inside the trap in front of the windows. The trap catches
were recorded daily for one month, with three replicates of each blend. The
pheromone dispensers were changed once, 18 days after the beginning of the
experiment.
A second trapping campaign was scheduled in June 1992 in Tumaco
(Narifio, south of Colombia) in a 3500-ha plantation (estate of Palmas de Tumaco
Ltda.). Synthetic chemicals, dispenser, and trap design were the same as in San
Alberto. Seven blends were compared (Table 3) with three replicates in a com-
pletely randomized block design. The trap catches were recorded daily for two
weeks.
The results were submitted to one-way (San Alberto) or two-way (Tumaco)
analysis of variance on data transformed by " , ~ + 0.5) to reach homoscedas-
ticity. The means were then compared by Newman-Keuls test with P < 0.05
(Dagnrlie, 1975). As some traps caught both males and females, the differences
between male and female catches were compared using Wilcoxon's random-
ization test for matched pairs (Siegel, 1956).

RESULTS

Pheromone Idenification
GC-MS analyses of the 557, 684, and 759 female gland extracts revealed
the same chromatographic pattern. In addition to saturated and unsaturated
hydrocarbons and fatty acids, GC-MS data (in electron impact mode) indicated
1114 P. ZAGATTIET AL.

two 14-carbon aldehydes, one of them diunsaturated [m/z 41, 55, 67 (100%),
82, 95, 105, 208 (M ÷ "; confirmed by CI-NH 3 with ions at m/z 209 and 226)]
and the other triunsaturated [m/z 41, 55, 67, 79 (100%), 80, 91, 107, 206
(M+-; ions at m/z 207 and 224 in CI-NH3) ]. The corresponding 14-carbon
acetates were also identified [one of them was diunsaturated with ions at m/z
43, 55, 67 (100%), 79, 82, 93, 110, 121, 192 (M-CH3COOH) and 252 (M +'"
ions at m/z 253 and 270 in CI-NH3) and the other triunsaturated with ions at
m/z 43, 55, 67, 79 (100%), 80, 91, 105, 119, and 250 ( M + ' ; ions at m/z 251
and 268 in CI-NH3)]. The diunsaturated aldehyde was identified by chemical
ionization analysis using isobutane to determine the position of the double bonds
(Einhom et al., 1985). The mass spectrum exhibited various molecular species:
M +" (m/z 208, 22%), (M + C4H9) ÷ (m/~ 265, 4%), MH + (m/z 209, 91%),
(MH-H20) + (m/z 191, 39%), and revealed the presence of abundant ions at m~
z 71 (33%), 169 (100%), and 151 (60%), which were clearly related to a 9,11
position of the diethylenic system. The stereochemistry was assigned to (Z,E)-
9,11 by comparison of the GC retention times with standards. Data on the trienic
system were completely lacking at the beginning of our work. The mass spec-
trum could be assigned either to a triene or to a triple bond, A9,Yne I l-enyne.
The triene hypothesis of a conjugated system with a terminal double bond was
suggested by comparing the mass spectrum of the natural product with spectra
of several chemicals sharing the same trienic motif, already prepared in our
laboratory as reaction intermediates during studies on moth pheromones. Finally,
all possible isomers of (A,A)-9,11,13 and A 9 . Y n e l l 14-carbon aldehydes and
acetates were synthesized, and comparison of GC retention times and mass
spectra proved that the natural products were (Z,E)-9,1 l, 13-tetradecatrienal and
(Z,E)-9,1 t, t3-tetradecatrienyl acetate. The extracts of pheromone glands of S.
cecropia contained: (Z,E)-9,11-tetradecadienal (Z9,E 11-14: Ald; 11%), (Z,E)-
9,11,13-tetradecatrienal (Z9,EII,13-14:AId; 67%), (Z,E)-9,11-tetradeca-
dienyl acetate (Z9,E11-14:Ac; 5.5%) and (Z,E)-9,11,13-tetradecatrienyl ace-
tate (Z9,E11,13-14:Ac, 16.5%).

Etectrophysiology
The EAG screening demonstrated that the two aldehydes found in the
female gland extracts were active on the antenna of male S. cecropia (Figure
2). The most active compound was the triene aldehyde, Z 9 , E l t , 1 3 - 1 4 : A I d ,
which elicited an EAG response of 1.9 +_ 0.7 mV (mean _+ 95% confidence
interval). The dienic aldehyde, Z9,E11-14 :Ald, also elicited high-amplitude
EAG ( 1.5 _+ 0.5 mV). Responses to other compounds assayed, and in particular
to the two acetates identified in the gland extracts, were all inferior to 0.7 inV.
The activity of 14 sensilla trichodea was recorded from six different males
in response to 10 pheromone compounds and pheromone analogs. Sensilla tri-
chodea could be separated into four different groups based on the response
profiles of their sensory neurons (Figure 2). Sensilla 1-6 presented a firing
 Spikes / ls. -mY

nt _ _

at _ _

Zg,E11-14:AId o.

Z9,E11,13-14:AId
5°~m~m~[~~o ~ o2

Z9-14:OH~1. . . . . . nt-] _! nt
Zg,E11,13o14:OHo - - _ _ m . . . . [ - - ] r E ] o
Sensillumnumberll2 3 4 5 6 7 ~ 91011121314 EAG
Sensillumgroup I V
FIG. 2. Electroantennograms (EAG) and single sensillum recordings obtained from male
Stenoma cecropia antennae stimulated by synthetic compounds. EAG are expressed as
the mean (in millivolts) of 5-10 replicates. The vertical bars represent the 95 % confidence
interval of the mean. The single sensillum recordings were obtained from 14 sensilla
trichodea belonging to six different male moths. Black bars: A cell activity; open bars:
B cell activity (groups Ill and IV). Name of compounds were abridged as follows:
Z9-, geometry and position of the double bond; Ynel 1-, triple bond; -14, chain length;
OH, Aid, Ac, alcohol, aldehyde, and acetate, respectively; nt: compound not tested.
1116 P. ZAGATTI ET AL.

activity consisting of a single amplitude class of action potentials. Their firing

activity was stimulated by Z9,E 11,13-14 :Ald. Responses to other compounds
were not significant when compared to the air control. A second group, sensilla
7-9, presented a single class of action potentials and responded to the other
EAG-active compound, Z9,E 11-14:Aid. These sensilla were less specific than
group I sensilla since they also weakly responded to Z 9 , E l l , 1 3 - 1 4 : A c and to
Z 9 , Y n e l l - 1 4 : A l d . The firing activity of group III (sensilla 10 and 11) and
group IV (sensilla 12-14) clearly consisted of two amplitude classes of action
potentials, indicating the presence of at least two receptor neurons. In groups
III and IV, the cells firing high-amplitude action potentials, up to 3 mV, are
referred to as the A cells. Action potentials emitted by the other cells, referred
to as B cells, were much smaller (50% of the amplitude of A cell action poten-
tials). In group III sensilla, A cells clearly responded to Z9,E11-14: Aid and B
cells to Z9,E11,13-14: Aid. The response profiles of sensilla from group IV
were characterized by strong responses from B cells to Z9,E 11-14:Ald and to
a lesser exent to Z9,E 11-14 : Ac, Z9,Yne 11-14 : Ac, Z9-14 :Ald and Z9,Yne 11-
14:Aid. In two out three of these sensilla, A cells responded weakly to
Z9,E11,13-14 : Aid.

Otfactometric Tests
The results are shown in Table 1. It appeared that two glands of virgin
females were able to evoke wing fanning and orientation toward the odor source
in mate moths. The percentage of moths making contact with the source was
very low (18.8%). Two blends of synthetic compounds evoked the same
responses by virgin males as did female glands: a blend of Z9,E11-14: Aid,
Z 9 , E l l , 1 3 - 1 4 : A I d , Z 9 , E I I - 1 4 : A c , and Z 9 , E l l , 1 3 - 1 4 : A c and a blend of
Z 9 , E l l - 1 4 : A I d and Z9,EI 1,13-14: Ald.

Field Trapping
The results of the first trapping experiment (Figure 3) showed that the
population levels were very high. The number of living pupae per leaf reached
30 at the beginning of the experiment. The emergence of adults, as shown by
the decrease of pupa counts, occurred during the first days of September. By
this time the number of unhatched eggs had increased and reached a maximum
on September 8. These data suggest that the population of sexually active females
was at its highest between September 3 and September 15, 1989.
The attractiveness of virgin females as bait was not correlated with these
data. The number of males caught in traps was very small during the first two
weeks of September, started increasing as late as September 18, and reached a
maximum of 37 males/day/trap on September 20. The three female traps also
SEX PHEROMONEOF Stenoma cecropia 1117

Number of pupae found on 1 leaf

Number of eggs found on 10 leaflets
-e- Mean number of males caught in virgin female traps
40

30

2O

10

0
08124 08•29 09•03 09108 09/13 09/18 09•23 09128

FIG. 3. Chronological catches of Stenoma cecropia males obtained with traps baited with
two virgin females (mean of three traps) from September 25 to October 27, 1989 (San
Alberto, central Colombia). The population levels were estimated by counting living
pupae on leaf number 17 and counting unhatched eggs on 10 leaflets (of leaf number 17)
on the trees from which the traps were hung (mean of three counts). The scale of the y
axis is the same for the three curves.

caught females regularly throughout the month the experiment lasted, with an
average of 0.25 females/day/trap.
The other part of this experiment was carried out at the same time and in
the same habitat as virgin female trapping. It consisted of a comparison between
different synthetic pheromone blends. The results (Table 2) showed that all traps
caught female moths (average of 0.2 female/trap/day), with no significant dif-
ferences between blends. Male and female catches in the same trap were sig-
nificantly different when the traps were baited with blend SC5 (Z9,E 11-14 : Ald,
50/zg, and Z9,E 11,13- t 4 :Ald, 250/zg) or virgin females (P = 0.031). Blend
SC5 caught significantly more males than the other blends but catches with this
blend were five times lower than those obtained using the virgin females as bait.
The second trapping campaign was conducted in 1992 with a low S. cec-
ropia population level that did not exceed one larva per leaf. Mono- or polyun-
saturated aldehydes were tested along with the best blend of the 1989 experiment
(SC5). The results (Table 3) showed that only males were caught. Blend SC11
caught four times more males than SC5 (Table 3). It consisted of the same blend
of Z 9 , E l l - 1 4 : A I d and Z 9 , E l l , 1 3 - 1 4 : A l d at a load four times higher than
1118 P. ZAGATTIET AL.

SC5. When tested separately (SC7 and SC8, Table 3) these aldehydes were not
attractive.

DISCUSSION

Sex Pheromone of Stenoma cecropia

Four compounds were identified in the sex pheromone gland extracts of
Stenoma cecropia females: a dienic and a trienic aldehyde, and the correspond-
ing acetates. The electrophysiological study gave evidence for specific receptors
to the aldehydes only. The olfactometric tests showed a similar level of response
to female glands and to a blend containing the four synthetic compounds in a
ratio close to that of female extracts. However, removal of the two acetates did
not alter the response of males. In field-trapping experiments, blends containing
the dienic and trienic aldehydes attracted males, whereas a four-component
blend, which included the acetates, did not.
From these biological data it can be concluded that (Z,E)-9,11,13-tetra-
decatrienal and (Z,E)-9,11-tetmdecadienal are components of the sex pheromone
of Stenoma cecropia. The corresponding acetates do not seem to be involved
in the pheromone communication system. They are probably precursors of the
aldehydes in a way similar to the biosynthetic pathway described in Choristo-
neura fumiferana. In this tortricid moth, the pheromone components (E- and
Z-ll-tetradecenal) are biosynthesized de novo via A-11-tetradecen-l-ol and
A-11-tetradecen-t-ol acetate. The amounts of z~-I 1-tetradecen-l-ol acetate in
female glands of C. fumiferana vary during the daily cycle as this acetate is
used as a storage precursor of the pheromone and converted into the correspond-
ing aldehyde just before pheromone emission (Morse and Meighen, 1984, 1986).
Traps baited with the best attractants in the present work caught fewer
insects than traps baited with virgin females. This may mean that at least one
component of the pheromone of S. cecropia remains unknown.
(Z,E)-9,11,13-Tetradecatrienal has been identified as a pheromone com-
ponent of the carob moth Ectomyelois ceratoniae (Baker et al., 1989), which
belongs to the family Pyralidae, a family not closely related to Elachistidae.
This compound was also identified in another Stenoma species, Stenoma decora,
a major pest of cocoa in Brazil (D. R. Hall, personal communication).
The phylogenetic relationships between families of the Gelechioidea remain
puzzling. Minet (1989) included in the Elachistidae several genera formerly
thought to belong to the Oecophoridae. Some of them are listed in a recent
review of sex attractants (Am et al., 1992): Ethmia spp. (Elachistidae: Ethmi-
inae), Depressaria olerelta and Agonopterix spp. (Elachistidae: Depressariinae).
None of them use aldehydes as sex attractants. Instead they use monoenic ace-
tates or alcohols with chain lengths of 10-14 carbons. Thus, the chemotaxonomy
SEX PHEROMONEOF Stenoma cecropia 1119

of sex pheromones does not seem to be promising for clarifying the status of
this group.

Influence o f Insect Density

The results of the field-trapping experiments raised questions about the
influence of the population density on the effectiveness of sexual trapping. Dur-
ing the 1989 season, the population levels were very high. It can be seen in
Figure 3 that the maximum number of sexually active females occurred between
September 3 and September 15. The number o f males caught during this period
by the virgin female traps was very small (33 out o f a total catch of 458), It
began to increase after September 18, at a time when few females were likely
to be unmated and calling for males.
It is generally admitted that catches in traps baited with synthetic attractants
are not correlated with insect population levels at the highest levels of density
(Cardr, 1979). To our knowledge, this has not been reported for traps baited
with virgin females. These are expected to be very attractive, because the calling
period of the caged females lasts 1 or 2 hr whereas the " f r e e " females stop
calling as soon as they mate. The lack o f male catches during the early part of
our experiment with S. cecropia suggests that the insects used other mate-
searching systems than pheromone orientation when density was high. The
movements of many moths in the vicinity of the traps may explain the number
of drowned females (Table 2). It may also result in sexual encounters and
subsequent matings.
The monitoring of pest populations with pheromones has now become a
routine technique for plant protection in temperate countries. It is not yet wide-
spread in the tropics (Zagatti, 1993), where different pest characteristics may
result in a different approach to pest management programs.

Acknowledgments--This work was accomplished through the financial and technical support
of lndupalma S.A. We are pleased to thank Dr. E. Andrade, general manager, and Ing. Manoloin
Avila for field assistance. Palmas de Tumaco Ltda. helped us finish the work, and we are indebted
to Drs. F. Bernal and F. Rodriguez, D. Mariau (CIRAD-CP) provided financial support and per-
manent contact between French and Colombian partners. We also thank Drs. D. R. Hall (NRI,
Chatham) for results on Stenoma decora, J, Minet (Museum National d'Histoire Naturelle. Paris)
lor his help on systematics, and B. Frrrot, M, Lettere, F. Ramiandmsoa, and M. Renou (INRA)
for their help during the work and discussions on the manuscript. We are indebted to Mr. Ph. Butler
(INRA, Traductions) for his complete revision of the English text.

REFERENCES

ANDREIN1,B, P,, BENE'Vn,M,, CARPITA,A., and Rossl, R. 1987. Highly stereo- and regioselective
palladium catalyzed syntheses of (3E,5Z)-, (3E,5E)-, and (3Z,5E)-l,3,5-andecatriene. Tetra-
hedron 43:4591-4600.
1120 P. ZAGA'r'rt E'r AL.

ARN, H., TrTH, M., and PRIESNER, E. 1992. List of sex pheromones of Lepidoptera and related
attractants. 2rid ed. OILB-SROP, Monffavet, France. 179 pp.
BAKER, T. C., FRANCKE,W., LOFSTEDT,C., HANSSON,B. S., Du, J. W., PHELAN, P. L., VEr'rER,
R. S., and YOUNGMAN, R. 1989. Isolation, identification and synthesis of sex pheromone
components of the carob moth, Ectomyetois ceratoniae. Tetrahedron Lett. 30:2901-2902.
CARDE, R. T. 1979. Behavioral responses of moths to female-produced pheromones and the utili-
zation of attractant-baited traps for population monitoring, pp. 286-315, in R. L. Rabb and
G. G. Kennedy (eds.). Movements of Highly Mobile Insects; Concepts and Methodology in
Research. North Carolina State University, Raleigh.
DAGNI~LIE,P. 1975. Throrie et Mrthodes Statistiques, Vol. 2. Les Presses Agronomiques de Gem-
bloux, Gembloux, Belgium. 453 pp.
EINHOaN, J., VIRELIZIER, H., GEMAL, A, L., and TABET, J. C. 1985. Direct determination of
double-bond position in tong chain conjugated dienes by t-Call9 + chemical ionization mass
spectrometry. Tetrahedron Lett. 26:1445-1448.
GENTY, P. 1978. Morphologie et biologie d'un Lrpidopt~re drfoliateur du palmier h huile en
Amdrique latine, Stenoma cecropia Meyrick, Oleagineux 33:421-427.
GENTY, P., DESMmRDE CHENON, R., and MOalN, J. P. 1978. Les ravageurs du palmier h huile en
Amrrique tropicale. Oleagineu.r 33:325-419.
HODGES, R. W. 1974. Gelechioidea: Oecophoridae, pp. 1-145, in R. B. Dominick et al. (eds.).
The Moths of America North of Mexico Fasc. 6-2, E. W. Classey Ltd., London.
KAISSLING, K. E., and THORSON, J. 1980. Insect olfactory sensilla: Structural, chemical and elec-
trical aspects of the functional organization, pp. 261-282, in D. B. Sattelle, L. M. Hall, and
J. G. Hildebrand (eds.). Receptors for Neurotransmitters, Hormones and Pheromones in Insects.
Elsevier/North Holland Biomedical press, Amsterdam.
LUCAS, P., and RENOU, M. 1989. Responses to pheromone compounds in Mamestra suasa (Lepi-
doptera: Noctuidae) olfactory neurones. J. Insect Physiol. 35:837-845.
MmET, J. 1989. Remaniement partiet de la classification des Gelechioidea, essentiellement en
fonction de caractbres prd-imaginaux. Alexanor 16:239-255.
MORSE, D., and ME1OHEN, E. 1984. Aldehyde pheromones in Lepidoptera: Evidence tbr an acetate
ester precursor in Choristoneurafum(ferana. Science 226:1434-1436.
MORSE, D., and MEmHEN, E. 1986. Pheromone biosynthesis and role of functional groups in
pheromone specificity. J. Chem. Ecol. 12:335-351.
NAE. F., DECORZAN'r,R., THOMMEN,W.. WlUmAt,M, B., and Ore.OFF, G. 1975. The four isomeric
1,3.5-undecatrienes. Synthesis and configuvational assignments. Heh'. Chim. Acta 58:1016-
t037.
RAMIArqDRASOA,F., and TELLIER. F. 1990. Stereoselective synthesis of a potential sex pheromone
of Stenoma cecropia. Synth. Commun. 20:333-344.
RATOVELOMANANA,V.. and LINSTRUMELLE, G. 1987. Synth~se des undrcatri~nes-l,3,5 naturels.
Bull. Soc. Chim. Fr. 1987:174-176.
S~EGEL, S. 1956. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York.
312 pp.
SOWER, L. L., KAAE, R. S., and SHOaEY, H. H. 1973. Sex pheromones of Lepidoptera: XLI.
Factors limiting potential distance of sex pheromone communication in Trichoplusia ni. Attn.
Entomol. Soc. Ant. 66:1121-1122.
TELLmR, F. 1991. Stereospecific synthesis of (E,E)-9,11,13-tetradecatrienyl acetate and aldehyde
(geometrical isomers of the major components of two sex pheromones of Lepidoptera). Synth.
Commun. 21:2287-2294.
TELUER, F., and DESCOmS, C. 1990. Stereospecific synthesis of (Z,E)-9,11,13-tetradecatrien-l-yl
acetate and aldehyde, sex pheromone components of Stenoma cecropia and Ectomyelois cer-
atoniae. Tetrahedron Lett. 31:2295-2298.
SEX PHEROMONE OF Stenoma cecropia 1121

TELLIER. F,. DESCOINS, C., and SAUVETRE, R. 1991. Stereospecific synthesis of 1,5-dien-3-ynes
and 1,3~5-trienes. Application to the stereochemical identification of trienic sex pheromones.
Tetrahedron 47:7767 -7774.
ZAGAT'n, P. 1993. Pheromones in developing countries: The 15-year experience of French research
organizations (INRA-CIRAD). IOBC- WPRS Bull. 16:131-135,
ZAGATTI, P., KUNESCH, G., RAMIANDRASOA,P., MALOSSE, C., HALL, D. R., LESTER, R., and
NESBITT, B. F, 1987. Sex pheromones of rice moth, Cor~3,ra cephalonica Stainton. I. Identi-
fication of male pheromone. J. Chem. EcoL 13: 1561-1573.