Sex pheromone of tomato fruit borer, Neoleucinodes elegantalis

Article abstract of DOI:10.1023/A:1012299021967

Five candidate pheromone components were identified by analyzing pheromone gland extracts by gas chromatography (GC), coupled GC-electro-antennographic detection (EAD), and coupled GC-mass spectrometry (MS): (E)-11-hexadecenol(E11-16: OH), (Z)-11-hexadecenol(Z11-16: OH), (E)-11-hexadecenal, (E)-11-hexadecenyl acetate, and (Z)-3,(Z)-6,(Z)-9-tricosatriene (Z3,Z6,Z9-23: Hy). In electroantennogram (EAG) recordings, synthetic E11-16: OH elicited stronger antennal responses at low doses than other candidate pheromone components. Field tests demonstrated that synthetic E11-16: OH as a trap bait was effective in attracting males, whereas addition of Z11-16: OH inhibited the males' response. Z3,Z6,Z9-23 : Hy strongly enhanced attractiveness of E11-16: OH, but was not attractive by itself. A pheromone blend with synergistic behavioral activity of an alcohol (E11- 16: OH) and hydrocarbon (Z3,Z6,Z9-23: Hy) component is most unusual in the Lepidoptera. The synthetic two-component pheromone is approximately 60 times more attractive than the female-produced blend and might facilitate the control of this pest.

Full text of DOI:10.1023/A:1012299021967

Journal of Chemical Ecology, Vol. 27, No. 10, October 2001 (�c 2001)
SEX PHEROMONE OF TOMATO FRUIT BORER,
Neoleucinodes elegantalis
´
1,2,
3,5
4
AIVLE CABRERA, * ALVARO E. EIRAS, GERHARD GRIES,
4
2
´
1
REGINE GRIES, NEUDO URDANETA, BEATRIZ MIRAS,
5
1
CESAR BADJI, and KLAUS JAFFE
1Laborat o´ ri o` de Comportamiento
2Departamento de Qu ´ı mica
Universidad Sim o´ n Bol ´ı var
Apartado 89000, Caracas 1080-A, Venezuela
3Departamento de Parasitologia, ICB
Universidade Federal de Minas Gerais (UFMG), Belo Horizonte
CEP 31270-901, MG, Brazil
4Centre for Environmental Biology
Department of Biological Sciences
Simon Fraser University
Burnaby, British Columbia V5A 1S6, Canada
5Laborat o´ rio de Prote c¸ a˜ o de Plantas, CCTA
Universidade Estadual do Norte Fluminense
CEP 28045-640, Campos, RJ, Brazil
(Received December 26, 2000; accepted June 12, 2001)
Abstract—Five candidate pheromone components were identified by analyzing
pheromone gland extracts by gas chromatography (GC), coupled GC-electro-
antennographic detection (EAD), and coupled GC-mass spectrometry (MS):
(
E)-11-hexadecenol(E11–16 : OH), (Z)-11-hexadecenol(Z11–16 : OH), (E)-11-
hexadecenal, (E)-11-hexadecenyl acetate, and (Z)-3,(Z)-6,(Z)-9-tricosatriene
Z3,Z6,Z9–23 : Hy). In electroantennogram (EAG) recordings, synthetic E11–
6 : OH elicited stronger antennal responses at low doses than other candidate
(
1
pheromone components. Field tests demonstrated that synthetic E11–16 : OH as
a trap bait was effective in attracting males, whereas addition of Z11–16 : OH
inhibited the males’ response. Z3,Z6,Z9–23 : Hy strongly enhanced attractive-
ness of E11–16 : OH, but was not attractive by itself. A pheromone blend with
synergistic behavioral activity of an alcohol (E11–16 : OH) and hydrocarbon
(Z3,Z6,Z9–23 : Hy) component is most unusual in the Lepidoptera. The syn-
thetic two-component pheromone is approximately 60 times more attractive than
the female-produced blend and might facilitate the control of this pest.
*
To whom correspondence should be addressed; e-mail: acabrera@usb.ve
2
097
0
098-0331/01/1000-2097$19.50/0 �C 2001 Plenum Publishing Corporation

2
098
CABRERA ET AL.
Key Words—Neoleucinodes elegantalis, Lepidoptera, Crambidae, sex
pheromone, (E)-11-hexadecenol, (Z)-3,(Z)-6,(Z)-9-tricosatriene, (Z)-11-
hexadecenol, GC-EAD, tomato.
INTRODUCTION
The tomato fruit borer, Neoleucinodes elegantalis (Lepidoptera: Crambidae), is
one of the most important economic pests of tomato (Lycopersicum esculentum)
crops in tropical South America. Current control tactics include frequent appli-
cations of pesticides (Reis and Souza, 1996) that may lead to the development
of insecticide resistance. Moreover, once larvae have entered the fruit, pesticides
and biological control agents are ineffective. Pheromone-based mating disruption
represents an alternative tactic to manage this pest insect but requires identification
of the female-produced sex pheromone. We know that virgin females attract males
in the field (Mir a´ s et al., 1997), and extracts of abdominal tips with pheromone
glands from virgin females attract males in wind tunnel bioassays (Eiras, 2000).
Here we report identification, electrophysiological studies, and field experiments
of sex pheromone components of N. elegantalis.
METHODS AND MATERIALS
Insects. Tomato fruits infested with N. elegantalis larvae were collected in
commercial tomato crops in the states of Lara and Aragua, Venezuela, and in Rio de
Janeiro, Brazil. Fruits were kept in plastic containers until larvae emerged from the
fruit to pupate. Adult insects were maintained according to Eiras (2000). During
peak calling activity, abdominal tips with pheromone glands of 2-to 3-day-old
virgin female moths were removed and extracted for 10–15 min in hexane. The
�
supernatant was withdrawn and stored in microcapillaries at � 15 C.
Chemical Analyses. Pheromone extracts were subjected to gas chromato-
graphic (GC) and coupled GC-electroantennographic detection (EAD) analyses,
using a Hewlett Packard 5890A gas chromatograph equipped with fused silica
columns (30 m � 0.25 or 0.32 mm ID) coated with DB-5, DB-210, or DB-23 (J&W
Scientific, Folsom, California). Forty female equivalents of pheromone gland ex-
tract were also analyzed by coupled GC-mass spectrometry (MS), employing a
Perkin Elmer QMass-910 attached to a GC-Autosystem 2000, equipped with a
DB-5 column (25 m � 0.18 mm ID, Quadrex, New Haven, Connecticut).
The position and geometry of a double bond in an EAD-active hexade-
cenol were determined by: (1) GC-EAD analyses of synthetic hexadecenols
(E2 to E14 and Z2 to Z14); (2) derivatization of extract with m-chloroperbenzoic
acid (Bierl-Leonhardt et al., 1980), followed by GC-MS analysis of the result-
ing epoxide; and (3) acetylation of pheromone extract with acetic anhydride and

SEX PHEROMONE OF N. elegantalis
2099
pyridine, followed by GC analyses [HP-GC equipped with a SP-1000-coated
column (30 m � 0.25 mm ID; Supelco, Bellefonte, Pennsylvania)] with im-
proved chromatographic resolution of the acetylated alcohol and synthetic acetate
standards.
Effluvia of calling, 1- or 2-day-old female N. elegantalis were adsorbed on a
solid-phase microextraction (SPME) fiber coated with 100 �m polidimethylsilox-
ane (Supelco). The fiber was exposed to a female during the entire calling period
�
and then desorbed at the capillary injection port (270 C) of the GC equipped with
columns (30 m � 0.25 mm ID or 25 m � 0.18 mm ID; Quadrex, New Haven,
Connecticut) coated with methyl–5% phenyl silicone or Carbowax 20 M. Reten-
tioncharacteristicsoffemale-produced, fiber-desorbedcompoundswerecompared
with those of authentic standards.
Electrophysiology. Electroantennogram (EAG) recordings were carried out,
using a Syntech EAG system (NL-1200 BM Hilversum; The Netherlands).
Five compounds (E11–16 : OH, Z11–16 : OH, E11–16 : OAc, E11–16 : Ald, and
6
Z3,Z6,Z9–23 : Hy) at increasing logarithmic doses (10 to 10 ng) were tested with
each of five 48- to 72-hr-old male antennae. An antenna was stimulated by sub-
jecting it to puffs (0.5 sec) of purified and humidified air (1.5 liters/min) delivered
through a Pasteur pipet (15 cm long), containing a filter paper strip (1 � 5 cm)
impregnated with 50 �l of a test solution. Air puffs from an empty pipet, pipet
plus filter paper, or filter paper plus solvent served as control stimuli.
Syntheses. (Z)-9,(Z)-12,(Z)-15-octadecatrien-1-ol (99%) was synthesized
frommethyllinolenateafterreductionwithLiAlH4. Thisalcoholwasoxidizedwith
pyridinium chlorochromate (PCC) to afford (Z)-9,(Z)-12,(Z)-15-octadecatrienal
(98%). n-Pentyl magnesium bromide was added to a solution of this aldehyde in an-
hydrous ether to obtain 6-hydroxy-(Z)-14,(Z)-17,(Z)-20-tricosatriene. A solution
of this compound in anhydrous pyridine was treated with methane sulfonyl chlo-
ride (Kocovsk y´ and Cern y´ , 1978). The obtained mesylate was reduced with zinc
dust and sodium iodide (Kocovsk y´ and Cern y´ , 1978) to obtain (Z)-3,(Z)-6,(Z)-9-
tricosatriene, which was purified (>99%) by flash silica column chromatography
1
using n-hexane as eluent. H NMR (ppm; �): 5.35 (m), 2.80 (t), 2.05 (m), 1.25
(
9
m), 0.95 (t) 0.85 (t); EI-MS [m/z (relative intensity)]: 79 (100); 108 (98); 67 (49);
3 (48); 262 (6); M 318 (2).
+
Sources of Candidate Pheromone Components. (E)-11-Hexadecenol (E11–
6 : OH) was obtained from the Research Institute for Plant Protection (IPO-DLO),
1
Wageningen, The Netherlands; (Z)-11-hexadecenol (Z11–16 : OH) was purchased
from Aldrich Chem. Co. (Milwaukee, Wisconsin); (E)-11-hexadecenyl acetate
(E11–16 : OAc) was prepared by acetylation of E11–16 : OH with acetic anhy-
dride and pyridine; (E)-11-hexadecenal (E11–16 : Al) was prepared by oxidation
of E11-hexadecenol with PCC in dichloromethane; (Z)-3,(Z)-6,(Z)-9-tricosatriene
(Z3,Z6,Z9–23 : Hy) was synthesized as described above. All compounds were
>
98% chemically pure.

2
100
CABRERA ET AL.
Field Experiments. Field experiments were conducted in commercial tomato
plantations in Aragua State, Venezuela, employing a complete randomized block
design. Water traps (Mir a´ s et al., 1997) were suspended � 1 m above ground at
20-m intervalsand baited with red rubber septa (Aldrich Chemical Co., Milwaukee,
Wisconsin, catalog No. Z12,434-6) that were Soxhlet-extracted with ethanol for
4 hr, and impregnated with candidate pheromone components in HPLC-grade
2
dichloromethane. Location, experimental period, and number of replicates for
each experiment are reported in the captions of Figures 3 and 4 below.
Experiment 1 tested traps baited with E11–16 : OH (1 mg) and Z11–16 : OH
(1 mg), singly and in binary combination at ratios of 1 : 0.1 and 1 : 0.01 mg. Solvent-
impregnated rubber septa served as baits in control traps. Both experiments 2 and 3
tested traps baited with two virgin females or E11–16 : OH (1 mg). Experiment 4
tested traps baited with Z3,Z6,Z9–23 : Hy (1 mg) or E11–16 : OH (1 mg) singly, and
E11–16 : OH (1 mg) in binary combinations with Z3,Z6,Z9–23 : Hy (0.025, 0.05,
or 0.1 mg) and in penternary combination with Z11–16 : OH (0.07 mg), E11–
16 : Ald (0.02 mg), E11–16 : OAc (0.02 mg), and Z3,Z6,Z9–23 : Hy (0.04 mg) at
ratios as determined in pheromone gland extract. Experiment 5 tested traps baited
with E11–16 : OH (1 mg) in binary combinations with E11–16 : OAc (0.05 mg),
E11–16 : Ald (0.05 mg), or Z3,Z6,Z9–23 : Hy (0.05 mg), in ternary combination
with E11–16 : OAc (0.05 mg), and E11–16 : Ald (0.05 mg), and in quaternary
combinationwithE11–16 : OAc(0.05mg), E11–16 : Ald(0.05mg), andZ3,Z6,Z9–
23 : Hy (0.05 mg). Experiment 6 tested traps baited with E11–16 : OH (1mg) singly,
and in combination with Z3,Z6,Z9–23 : Hy (0.05 mg).
Statistical Analysis. Multivariate analysis with the using Mann-Whitney test
(Spiegel, 1991) was performed to analyze data from field tests.
RESULTS
Pheromone Identification. GC-EAD analysis of pheromone gland extracts of
female N. elegantalis revealed six components that elicited an antennal response
Figure 1). The most abundant and EAD-active component 2 (Figure 1) was hy-
(
pothesized to be a hexadecenol, based on its retention indices on DB-5 (1865),
DB-210 (2078), and DB-23 (2406) columns. Position and geometry of the double
bond was approximated by comparative GC and GC-EAD analyses of synthetic
hexadecenols. (E)-11- and (E)-12-hexadecenol (E11- and E12–16 : OH) had re-
tention times identical to, and EAD-activity comparable with, female-produced 2.
Epoxidation (Bierl-Leonhardt et al., 1980) of female-produced 2, and GC-MS
analysis of the epoxy alcohol revealed fragmentation ions at m/z 99 and 199 (due
to �-cleavage of the epoxy group), confirming the double bond at C-11. GC-EAD
analyses of pheromone extract on a DB-23 column, which separates the E and
Z isomers, provided tentative evidence also for trace amounts of the Z isomer.

SEX PHEROMONE OF N. elegantalis
2101
FIG. 1. Flame ionization detector (FID) and electroantennographic detector (EAD: male
N. elegantalis antenna) responses to one female equivalent of pheromone gland ex-
�
tract, chromatograph on a DB-5 column; temperature program: 100 C (1 min) then
�
�
1
2
(
0 C/min to 240 C. Compound identity as follows: 1 = (E)-11-hexadecenal (E11–16 : Ald);
= (E)-11-hexadecenol (E11–16 : OH); 3 = unknown; 4 = (E)-11-hexadecenyl acetate
E11–16 : OAc); 5 = unknown; 6 = (Z)-3,(Z)-6,(Z)-9-tricosatriene (Z3,Z6,Z9-23 : Hy).
Note: GC-EAD analyses of pheromone extract on a DB-23 column, which separated E and
Z isomers of candidate pheromone components, provided tentative evidence also for trace
amounts of (Z)-11-hexadecenol (Z11–16 : OH).
EAD-active 1 and 4 (Figure 1) were hypothesized and, through comparative
GC analyses of authentic standards, confirmed to be E11–16 : Ald (1) and E11–
16 : OAc (4). Mass spectrum and retention indices of EAD-active 6 were indica-
tive of an triunsaturated C23 hydrocarbon, such as Z3,Z6,Z9–23 : Hy (Bell and
Meinwald, 1986). Identical retention and mass spectrometric characteristics of an
authentic standard and female-produced 6 confirmed this structural assignment.
EAD-active compounds 3 and 5 occurred below detection threshold of the flame
ionization detector and are yet to be identified.
In GC analyses of SPME-desorbed volatiles, small amounts of E11–16 : OH
and Z3,Z6,Z9–23 : Hy could be detected.
Electrophysiology. Synthetic equivalents of candidate pheromone compo-
nents evoked significant EAG responses. E11–16OH was most EAG-active and,
unlike other test stimuli, elicited >5 mV EAG responses from male antennae
already at a 10 ng dose (Figure 2). Z11–16 : OH, E11–16 : OAc, E11–16 : Ald,

2
102
CABRERA ET AL.
FIG. 2. ResponsesbymaleN. elegantalisantennaeinelectroantennogram(EAG)recordings
to increasing amounts of five candidate pheromone components; compound nomenclature
as in Figure 1.
4
3
2
5
and Z3,Z6,Z9–23 : Hy required doses of 10 , 10 , 10 , and 10 ng, respectively, to
induce EAG voltages greater than those of control stimuli (Figure 2).
Field Tests. Traps baited with E11–16 : OH captured significant numbers of
male moths (Figure 3, experiment 1). Z11–16 : OH, in contrast, was not attractive
and, when added to E11–16 : OH, strongly reduced captures of males. Traps baited

SEX PHEROMONE OF N. elegantalis
2103
FIG. 3. Captures of male N. elegantalis in experiments 1–3 in water traps baited with two
virgin female N. elegantalis or candidate pheromone components singly and in various
combinations. Experiment 1: Casablanca, Aragua State; October 16–December 1, 1996;
four replicates. Experiment 2: Casablanca, Aragua State; December 5–19, 1996; 7 repli-
cates. Experiment 3: M u´ cura, Aragua State; February 15–20, 1997; 4 replicates. In each
experiment, bars with the same letter superscript are not significantly different,P > 0.05.
with E11–16 : OH at 1 mg captured more males than those baited with two virgin
female moths (Figure 3, experiments 2, and 3). Z3,Z6,Z9–23 : Hy enhanced attrac-
tiveness of E11–11 : OH, but was not attractive by itself (Figure 4, experiment
4
). E11–16 : OH plus Z3,Z6,Z9–23 : Hy was the most attractive blend among sev-
eral two-, three-, or four-component blends of candidate pheromone components
Figure 4, experiment 5). Synergistic activity of Z3,Z6,Z9–23 : Hy was confirmed
in experiment 6 (Figure 4).
(
DISCUSSION
Our data show that E11–16 : OH and Z3,Z6,Z9–23 : Hy are sex pheromone
components in N. elegantalis. Evidence in support of this conclusion includes
the following: (1) both compounds were present and EAD-active in pheromone

2
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CABRERA ET AL.
FIG. 4. Captures of male N. elegantalis in experiments 4–6 in water traps baited with
candidate pheromone components singly and in various combinations. Experiment 4: Pao
de Z a´ rate, Aragua State; June 23–August 3, 1998; 5 replicates. Experiment 5: Valle de
Tucutunemo, Aragua State; February 5–March 26, 1999; 5 replicates. Experiment 6: Pao
de Z a´ rate, Aragua State; July 3–25, 2000, 10 replicates. In each experiment, bars with the
same letter superscript are not significantly different, P > 0.05.

SEX PHEROMONE OF N. elegantalis
2105
gland extracts and effluviums of female moths (Figure 1); (2) identifications were
based on comparative GC, GC-MS, and GC-EAD analyses of female-produced
compounds and authentic standards; (3) both compounds elicited stronger anten-
nal responses from male antennae in EAG recordings than did control stimuli
(Figure 2); and (4) blends of E11–16 : OH and Z3,Z6,Z9–23 : Hy attracted signif-
icant numbers of males (Figure 4). E11–16 : OH is the major pheromone com-
ponent, because it was most abundant in gland extracts (Figure 1), elicited the
strongest EAG response at low doses (10 or 100 ng) (Figure 2), and, unlike
Z3,Z6,Z9–23 : Hy, was attractive by itself as a bait (Figures 3 and 4).
E11–16 : OH has also been identified as a sex pheromone component in other
moths. It is the major pheromone component of the pod worm, Leucinodes or-
bonalis(Zhuetal., 1987;Attygalleetal., 1988), andaminorpheromonecomponent
of female pickleworms Diaphania hyalinata and D. nitidalis (Raina et al., 1986;
Klun et al., 1986). It was also present in pheromone gland extracts of eggfruit
caterpillar, Sceliodes cordalis (Clearwater et al., 1986) and of sugarcane borer,
Sesamia grisescens (Whittle et al., 1995), but behavioral activity of E11–16 : OH
in these two species is yet to be determined.
Z3,Z6,Z9–23 : Hy is reported here for the first time as a sex pheromone compo-
nent in the Lepidoptera. While it was identified as a trace component in pheromone
glands of the arctiid moth Creatonotos gangis (Bell and Meinwald, 1986), be-
havioral activity was not determined. A sex pheromone blend with synergistic
behavioral activity between a triene hydrocarbon (Z3,Z6,Z9–23 : Hy) and an al-
cohol (E11–16 : OH), as demonstrated in our study, is most unusual. Most triene
hydrocarbons as pheromone components in the Lepidoptera are either attractive
by themselves (Millar et al., 1992) or serve as synergists of corresponding diene
epoxides (T o´ th et al., 1994).
Further compounds (Z11–16 : OH, E11–16 : Ald, E11–16 : OAc) in pher-
omone gland extracts of female N. elegantalis seem to have no pheromonal activity.
This conclusion is based on their low EAG activity at low doses (Figure 2) and/or
lack of attractiveness in field experiments. The role of Z11–16 : OH is interesting.
Its low EAG activity but strong behavioral inhibition at a low dose (Figures 2 and 3)
suggests that mate-seeking males may select calling females based on the pres-
ence or absence of Z11–16 : OH in the pheromone effluvium, providing a basis
for a highly efficient system of sexual selection (Jaffe, 1996, 1998, 1999), as only
very fit females would produce E11–16 : OH with small amounts of Z11–16 : OH.
Alternatively, Z11–16 : OH may serve as a bifunctional pheromone component
(Linn and Roelofs, 1995) in a sympatric species to enhance the specificity of the
communication signal, although no such sympatric species is known.
The two-component pheromone blend of E11–16 : OH and Z3,Z6,Z9–23 : Hy
seems to work as a supernormal stimulus (Tinbergen and Perdeck, 1950), as it is
approximately 60 times more attractive than the natural pheromone blend emitted
by the females (Experiments 3 and 4, in Figures 3 and 4, respectively).

2
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CABRERA ET AL.
A synthetic mixture of E11–16 : OH and Z3,Z6,Z9–23 : Hy as a trap bait is
currently used by Brazilian and Venezuelan tomato growers to reduce infestations
of N. elegantalis, promising efficient control in pheromone-based mass trapping
programs.
Acknowledgments—This research was financial supported by BID-CONICIT QF-36, Venezuela
and FAPERJ (Brazil). We thank L. Caraballo, C. C a´ rdenas, and J. Vel a´ squez for field assistance. Grants
from Venezuela (CONICIT and USB-Decanato de Postgrado) and Brazil (CNPq-300698/91-7 and IFS-
F/2478-1) are acknowledged. A.E.E. acknowledges Dr. J. van der Peers for his collaboration with EAG
recordings. The pheromone-lure system is protected by patent application 002488, Venezuela.
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