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Talanta 52 (2000) 525–532

Analytical studies of Spodoptera littoralis sex pheromonecomponents by electroantennography and coupled gas

chromatography–electroantennographic detection

Edi A. Malo a, Michel Renou b, Angel Guerrero c,*a El Colegio de la Frontera Sur ( ECOSUR ) , Apdo. Postal 36 , Tapachula, 30700 Chiapas, Mexico

b INRA, Unite de Phytopharmacie et des Mediateurs Chimiques, Rte. St. Cyr, 78026 Versailles Cedex, Francec

Department of Biological Organic Chemistry, IIQAB ( CSIC ) , Jordi Girona 18 -26 . 08034 Barcelona, SpainReceived 15 December 1999; received in revised form 17 March 2000; accepted 31 March 2000

Abstract

In this paper we present analytical studies of the sex pheromone components of the Egyptian armyworm

Spodoptera littoralis (Lepidoptera, Noctuidae) by electroantennography (EAG) and coupled gas chromatography-

electroantennographic detection (GC-EAD). EAG responses in three different preparations, using an insect’s head, an

excised antenna and a live insect, have been recorded. EAG depolarizations of live insects were significantly higher

than those elicited by the insect’s head or the excised antenna. The responses were dose-dependent. Live insects also

allowed regular pheromone stimulations for 40 min with only 38% decrease of the EAG initial depolarization. Thesynthetic pheromone blend elicited the highest EAG activity (2.090.3 mV), followed by the major compound

(Z ,E )-9,11-tetradecadienyl acetate (I) (1.5490.1mV), and the minor components (Z )-9-tetradecenyl acetate (II),

(E )-11-tetradecenyl acetate (III), tetradecyl acetate (IV) and (Z )-11-tetradecenyl acetate (V) (1.21-1.32 mV range).

(Z ,E )-9,12-tetradecadienyl acetate (VI), although not present in the pheromone blend of our strain, also showed an

EAG activity (1.3290.09 mV) similar to that of the monoenic components. GC-EAD responses confirmed the

composition of the sex pheromone blend, the major response being elicited by the main component I followed by the

other minor compounds II-V. The new dienic compound found in the female pheromone gland, (E ,E )-10,12-te-

tradecadienyl acetate (VII), was not electrophysiologically active. Regarding sensitivity, the minimum amount

detectable to elicit an antennal response in our GC-EAD system was 15 pg of the major component. In our system,

which was built with cheap and easily available materials, no cooling of the effluent at the outlet of the

chromatographic column is required. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Electroantennographic detector; Egyptian armyworm; Spodoptera littoralis; Sex pheromone

www.elsevier.com /locate/talanta

1. Introduction

Since development of the electroantennography

technique (EAG) by Schneider [1] 40 years ago,

* Corresponding author. Tel.: +34-93-4006120; fax: +34-

93-2045904.

E -mail address: [email protected] (A. Guerrero)

0039-9140/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

PII: S 0 0 3 9 - 9 1 4 0 ( 0 0 ) 0 0 4 0 1 - X

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E .A. Malo et al . / Talanta 52 (2000) 525–532 526

the technique has been used to study olfactory

mechanisms in insects [2 –7], for structural charac-

terization of sex pheromone components and at-

tractants [8] and for measurement of pheromone

concentration in the field [9]. The EAG represents

the summation of bioelectrical potentials gener-

ated by many antennal olfactory receptors re-

sponding almost simultaneously [10]. The EAG

requires purified samples and, when used for iden-tification purposes, transfer of GC-purified com-

ponents of the pheromone blend is also necessary.

This can result in important losses of essential

secondary pheromone compounds. To overcome

this problem, an insect antenna, positioned at the

exit of a gas chromatograph, has been used as a

very sensitive detector of pheromone components

(electroantennographic detector, EAD). Coupling

GC-EAD takes full advantage of the great separa-

tion and detection capability of the two tech-

niques, being therefore almost indispensable to

tackle identification studies of active compounds

[11–14]. In this regard, development of capillary

columns and high impedance amplifiers (1012

ohms) [15] greatly improved the level of detection

and the signal to noise ratio. Recently, Soares-

Leal et al. [16] applied a GC-EAD system with a

GC chiral column to the determination of the

stereochemistry of the brownbanded cockroach

Supella longipalpa sex pheromone, and

Weibbecker [17] described analysis of volatilesemitted by potato plants by an electroantenno-

graphic detector of the Colorado beetle.

The Egyptian armyworm Spodoptera littoralis is

an important pest of cotton and vegetable crops

in Europe, Asia and Africa. The sex pheromone

of the pest was identified by Nesbitt et al. [18] as

a mixture of (Z ,E )-9,11-tetradecadienyl acetate

(I), (Z )-9-tetradecenyl acetate (II), (E )-11-tetrade-

cenyl acetate (III) and tetradecyl acetate (IV).

However, the pheromone composition varies with

the origin of the strain [19], and, thus, othercompounds have been found in abdominal tip

extracts, like (Z )-11-tetradecenyl acetate (V) and

(Z ,E )-9,12-tetradecadienyl acetate (VI) in Israel

[20]. This latter compound has also been found in

abdominal tip extracts of insects from Kenya [21],

and is an important component of the sex

pheromone of Sp. littura [22]. Previous studies on

our laboratory strain have shown the presence in

the pheromone gland of compounds I-V in

66:12:11:2:9 ratio [23], and later on (E ,E )-10,12-

tetradecadienyl acetate (VII) was also identified[24]. Structures of compounds I-V and VII are

listed in Scheme 1. The behavioral activity in a

wind tunnel induced by each component alone

and in blends with the major compound I has

been recently described by us [19]. In this paper,

we report a cheap GC-EAD system, easy to build

from readily available materials, which we have

applied to the analysis of the sex pheromone

complex of the Egyptian armyworm. We have

previously established the electrophysiological ac-

tivity of the individual pheromone components incomparison to the pheromone blend, and tested

three different EAG preparations using the in-

sect’s head, an excised antenna fixed between two

glass electrodes, and intact live insects.

2. Experimental

2 .1. Insects

Insects were obtained from a laboratory colonyon a slightly modified artificial diet compared

with the one previously reported [25]. Au-

reomycine hydrochloride was not included in the

diet and Wesson salt mixture (1%, Sigma) and

formaldehyde (0.08 of a 35% aqueous solution)

were added instead. Pupae were sexed, placed inScheme 1. List of the pheromone components (I-V and VII) of

our strain of S . littoralis.

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groups of 20–25 into 20×20 cm plastic boxes

and maintained in a climatic chamber on a

16L:8D regime at 2591°C with 60–70% relative

humidity until emergence. Adults were separated

daily by age and provided with 10% sucrose solu-

tion. Only males 1–2 day old were considered.

2 .2 . Chemicals

Compounds I-III were obtained from Sigma (St

Louis, MO), and their purity (\95%) was

checked by GC on a SPB-5 (30 m×0.32 mm i.d.,

Supelco) fused silica capillary column. Com-

pounds IV and V were prepared by acetylation of

tetradecyl alcohol and (Z )-11-tetradecenol

(Sigma), respectively, with acetyl chloride 99%+

(Aldrich Chemie, Steinheim, Germany), followed

by column chromatography purification to assess

a ]98% purity by GLC analysis.

2 .3 . EAG preparations

S . littoralis male antenna is 10 mm long and

0.22 mm wide at the base. The EAG apparatus

was based on that already reported by us [26] and

to optimize the responses three different antennal

preparations were tested:

1. Use of the insect’s head (preparation A). The

insect’s head was cut off carefully, and the

indifferent electrode inserted into the base.

After removing the last 1-2 antennal segments,the distal end of the antenna was inserted into

the tip of the recording electrode. Both silver

electrodes had been previously chloridized and

filled with Ringer solution (mixture of sodium

chloride 7.5 g, calcium chloride 0.21 g, potas-

sium chloride 0.35 g and sodium bicarbonate

0.20 g dissolved in 1 l of water). Short coaxial

cables connected the electrodes to a high

impedance (1012 ohm) DC amplifier, the am-

plified signal (100×) was filtered and fed into

an oscilloscope (Tektronix 5111).2. In this preparation (B), the antenna was

severed at the base and one or two segments

from the tip were also cut off. The excised

antenna was then fixed between two glass elec-

trodes filled with Ringer solution. The refer-

ence electrode was slipped over the base and

the recording electrode contacted the cut tip.

The remaining preparation was the same as in

(A).

3. In this preparation (C), live insects were used.

Males were immobilised with CO2 for 30 s and

secured in a Styrofoam block. Antennal seg-

ments (1– 2) were cut off and the distal end

inserted into the recording electrode. The in-

different electrode was implanted into the in-sect’s neck. The remaining preparation was the

same as in (A)).

The different preparations were protected from

extraneous electrical signals by an aluminum

Faraday cage of 76×70×60 cm. In each system

a minimum of five puffs over six insects were

applied (Fig. 2).

2 .4 . Time response of S . littoralis males in EAG

To determine the time response of S . littoralis

males in EAG, the major component of the sex

pheromone I was used as a stimulus. Two assays

were performed using preparations A and C. Ten

mg of I were applied to a small filter paper (ca. 0.8

cm2), which was placed into a Pasteur pipette.

The tests were carried out by making five puffs of

pheromone at 5 min intervals during 40 min and

the corresponding EAG depolarizations were

recorded. The puffs were delivered by an elec-

trovalve-driven membrane pump, which openedfor 400 ms, and the air fed into another indepen-

dent stream of air that flowed continuously over

the antenna. This airline was supplied by a second

pump which could be regulated to give 0.1– 2 l

min−1. A total of ten insects were evaluated and

pure air was used as blank (Fig. 3).

2 .5 . EAG acti 6ity of the pheromone components

I -VI

Evaluation of the EAG responses to the iso-lated pheromone components in comparison to

that of the pheromone blend was carried out by

preparing, just prior to the experiments, solutions

of the required concentrations of compounds I-VI

in nanograde hexane, so that application of 100 ml

of each one deposited 10 mg of the compound

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Fig. 1. Components of the GC-EAD system: (a) injector; (b)

column; (c) split valve; (d-I) make up I; (d-II) make-up II; (e)

FID; (f) heating system; (g) EAD system; (h) amplifier; (i)

oscilloscope; (j) recorders.

preparation (Fig. 1). Both connection tubings

were made of deactivated fused silica and had the

same length and diameter to allow simultaneous

monitoring of the FID and EAD responses. The

split ratio to both detectors was modulated by the

split valve from the outside of the gas chro-

matograph. At the entrance of the valve a second

inlet of make-up gas (make-up II) was used to

reduce residence time in the transfer lines andbroadening of the peaks. To avoid condensation

problems, the transfer line to the EAD was placed

inside a copper tubing (25 cm×1 mm i.d.), which

in turn was also introduced into another copper

tubing (25 cm×4 mm i.d.), that was heated

through an electric coil. To insulate the system the

coil was covered with a glass wool jacket, and the

internal temperature controlled by a thermostat to

a maximum temperature of 230°C (Fig. 1). At the

outlet of the system, the capillary column to the

EAD was introduced into a clean small glass tube(5 cm×0.5 mm i.d.) directed to the insect an-

tenna, which was placed with the aid of two

micromanipulators (Fine Science Tools, Heidel-

berg, Germany) in perpendicular position at ca. 2

cm from the tube end, as in preparation A. The

GC program used was the following: injection at

100°C and hold for 1 min, increase at 5°C min−1

up to 240°C and hold at this temperature for 10

min more. The EAD amplified signal was sent to

a fast-response recorder and synchronized with

that from the FID, which was sent to a recorder-integrator. To assess the activity of the antenna

along the runs, a single puff of air through the

pipette containing 10 ng of compound I was

ocassionally insuflated over the antenna. A depo-

larization value of 0.7– 1 mV indicated that the

antenna remained in good condition.

2 .7 . Gland extracts

Pheromone glands, from a total of ten virgin

females, were extruded by gently pressing female’sabdomen. They were immediately dissected and

extracted in a vial containing nanograde hexane

(10 ml gland−1) for 30 min at room temperature.

One-tenth of the extract was subjected to GC-

EAD analysis using the same chromatographic

conditions as cited in Section 2.6 (Fig. 5).

onto a small piece of filter paper (ca. 0.8 cm2).

The pheromone blend was prepared by mixing 10

mg of the main component I with the correspond-

ing amounts of the minor compounds II-V in

66:12:11:2:9 ratio. The solvent was allowed toevaporate and the filter paper placed inside a

Pasteur pipette. Responses of male antennae were

determined following preparation A. Five puffs of

every compound were applied and a minimum of

ten insects were considered. A blank of pure air

was insuflated before and after every puff. Presen-

tation of the different doses of a specific com-

pound was randomized, the data subjected to

ANOVA and mean responses to the same doses

of the different compounds were compared using

Tukey test (PB0.05) (Fig. 4).

2 .6 . GC -EAD

The GC-EAD system used a gas chro-

matograph (Vega series 6000, Carlo Erba, Ro-

dano, Italy) equipped with a capillary column

(BP-20, 25 m×0.22 mm i.d., SGE), a flame ion-

ization detector (FID) and a split/splitless injec-

tor. The injector was operated in the splitless

mode. Hydrogen was used as carrier gas at 3.0 ml

min−1

and nitrogen as make-up gas. From themain line of nitrogen a ‘T’ was inserted and one

arm directed to the FID (make-up I) and the

other to the EAD (make-up II). At the end of the

capillary column a split ‘T’ valve (OSS-2, SGE)

distributed the effluent from the column to the

FID and to the transfer line towards the EAG

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3. Results and discussion

High depolarization values to the major com-

ponent I were obtained (up to 3.8 mV) using

preparation C, with an average of 2.3 mV. Prepa-

rations A and B rarely afforded responses higher

than 1.8 mV, the mean values ranging 0.5–1.4 mV

at doses 102 –104 ng. As could be expected, the

responses of the different EAG preparations in-

creased with the dose being preparation C clearly

superior to the other two (Fig. 2). At all doses,

the responses with live insects were significantly

higher (Tukey test, PB0.05), the detection limit

being the amount of stimulant delivered by one

puff on 1 ng of stimulant. In turn, in preparations

A and B puffs on 10 ng of the attractant were

required to elicit a significantly different response

from that of a puff of pure air. The greatest

increases in response amplitude occurred between

102 and 104 ng, with the highest differences among

treatments at 103 and 104 ng. Comparison of the

other two preparations showed that the decapi-

tated head (A) generally induced higher EAG

responses than the isolated antenna (B), the dif-

ferences being statistically significant at 102 and

104

ng doses (Tukey test, PB0.05) (Fig. 2). Asexpected, by cutting off the antenna a number of

sensory neurons are lost inducing therefore a sub-

sequent decrease in EAG depolarization. This is

consistent with the work of Nagai [27], in which

the EAG amplitude was approximately propor-

tional to the distance between the recording and

the indifferent electrodes placed along the an-

tenna, and Moore [7] who described an amplifica-

tion of the EAG signal when several antennae

were connected in series. Our results indicate that,

whenever possible, it is recommended to use liveinsects in ordinary EAG preparations, not only

because they last much longer than those using

the antenna or head of the insect, allowing there-

fore more treatments, but also because they elicit

stronger EAG depolarizations. However and in

spite of these results, utilization of live insects was

not suitable for our GC-EAD system since they

provided a remarkable EAG background.

The decrease of the EAG response with time

was faster in preparation A than in C, the diminu-

tion being steadily constant in the cut head prepa-ration (Fig. 3). Thus, the antennal responses in

preparation A decreased in average from 2.03 mV

in the first puff to 1.45 mV (28%) after 20 min and

to 0.57 mV (72%) after 40 min from the first

stimulus, while with live insects EAG amplitudes

diminished in average from 2.67 to 1.8 mV (32%)

after 20 min and to 1.64 mV (38%) after 40 min.

The differences from the first puff between both

preparations were statistically significant at PB

0.05 (Student-t test). Other insects can also re-

spond to repeated stimuli of pheromone for longperiods of time, like the corn borer Ostrinia nubi -

lalis (Lepidoptera: Pyralidae), which responded to

20 mg of (Z )- and (E )-11-tetradecenyl acetates, the

two sex pheromone components, for 2 h, after

which period the depolarization had decreased by

only 50% [28]. In other cases, antennal prepara-

Fig. 2. EAG mean responses of S . littoralis males to increasing

doses of the main pheromone component I on the threepreparations A, B and C. Each value is the mean 9SE of 30

puffs.

Fig. 3. Time response of males of S . littoralis to the main

pheromone component I in EAG using preparations A and C.

Each value is the mean9SE of five puffs over ten insects.

Differences between means were statistically significant (Stu-

dent t-test, PB0.05).

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Fig. 4. EAG activity of S . littoralis males to 10 mg of the

individual pheromone components I-VI in comparison to the

pheromone blend (P). The pheromone blend was prepared by

mixing 10 mg of the main component I with the corresponding

amounts of the minor compounds II-V in 66:12:11:2:9 ratio.

Bars with different letters are statistically significant (Tukey

test, PB0.05). Each value is the mean9SE of five puffs over

a minimum of ten males.

to obtain an optimum 1:1.6 (FID:EAD) split ra-

tio. The threshold level of detection of the major

component I in our system was determined by

injecting sequentially decreasing amounts of this

compound in the 10 pg–5 ng range. We obtained

reliable and reproducible results with 15 pg of I

(ca. 1 mV depolarization), which was considered

the lowest level of detection in our preparation

[32]. Analysis of an extract of the equivalent of one tenth of the pheromone gland extract showed

a major EAD response corresponding to the main

component I, followed by smaller depolarizations

corresponding to the monoene acetates II, III+

IV and V (Fig. 5). In the calling period the

calculated amount of the major component in the

pheromone gland extract was 10–12 ng gland−1.

Under the chromatographic conditions used,

compounds III and IV could not be fully sepa-

rated, so we assumed that the unique EAD re-

sponse elicited was the sum of the depolarizationsinduced by both compounds, taking into account

the similar intrinsic responses elicited by each

individual component (see Fig. 4). Moreover, the

EAD response elicited by these two compounds

correlates well with the total amount of the two

minor components III and IV (13%) present in the

gland in comparison with the other two com-

pounds II and V, which are present in 11 and 9%,

respectively, as cited above. The new dienic com-

pound found in the gland (E ,E )-10,12-tetradecadi-

enyl acetate (VII) was not EAG active, as shownin the EAD trace (Fig. 5).

In summary, we have shown that for S . lit-

toralis EAG responses elicited by live insects were

significantly higher than those shown using the

insect’s head and the excised antenna. The highest

EAG depolarizations displayed by the pheromone

components followed the order: pheromone

blend\major component\minor components.

(Z ,E )-9,12-tetradecadienyl acetate, although ab-

sent in the pheromone complex of our strain,

elicited similar EAG activity to that of the mo-noenic components. An interesting feature of the

GC-EAD described herein is that no cooling for

the effluent at the outlet of the GC capillary

column is required, being sufficient to hold it at

ca. 2 cm from the antenna to get it cold enough

for not disturbing antennal receptors. This dis-

tions did not last long, as in Epiphyas post6ittana

(Lepidoptera: Pyralidae) whose duration in a field

EAG ranged from 20–60 min [29].The EAG responses to each individual compo-

nent I-V were lower than those to the correspond-

ing pheromone blend. Responses to the major

component I were significantly higher than those

to the other secondary components II-V (Fig. 4).

Compound VI, although not present in our strain,

also showed an EAG activity similar to the other

monoene derivatives, in a similar way to the

behavioral activity in wind tunnel elicited by this

compound in comparison to the other minor com-

ponents [19]. Our results mostly agree with thosereported by Ljunberg et al. [30], except with re-

gard to the activity of saturated acetate IV, which

showed practically no EAG response. To us, ac-

etate IV has a significant electrophysiological ac-

tivity and may play an important behavioral role,

because we have found receptor neurons tuned to

this component [19] and it is a remarkable in-

hibitor of the males’ arrestment to the source and

other behaviors as well, depending on the concen-

tration of compound V in the attractant blend

[19]. This type of effect is similar to the onedisplayed by dodecyl acetate in Trichoplusia ni

[31].

For our GC-EAD system, we have determined

that a flow rate of 3.0 ml min−1 of hydrogen as

carrier gas, 35 ml min−1 of nitrogen for make-up

I and 25 ml min−1 for make-up II were required

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Fig. 5. Analysis of the equivalent of one female gland extract of S . littoralis by GC-EAD (only trace of the active fraction of the

extract is shown).

tance represents an approximate value to elicit

good depolarizarions from the antenna without a

significant loss of effluent components after leav-

ing the capillary column. No condensation prob-

lems were detected. This is one of the critical parts

of other GC-EAD preparations, which require an

additional system to cool down the chromato-graphic effluent in the proximity of the antenna.

Our set-up, which has been built using cheap and

easily available materials, offers reliable electro-

physiological responses which closely correlates

with the relative amounts of the pheromone com-

ponents of the Egyptian armyworm found by GC

or GC-MS. Therefore, its utilization for the anal-

ysis of other natural extracts, plant volatiles, etc.

can be inferred.

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