Response of female Cydia molesta (Lepidoptera: Tortricidae) to plant derived volatiles

Bulletin of Entomological Research (2003) 93, 335–342 DOI: 10.1079/BER2003250

Response of female Cydia molesta

(Lepidoptera: Tortricidae) to plant

derived volatiles

D. Natale

, L. Mattiacci

, A. Hern

, E. Pasqualini

and

S. Dorn

Institute of Plant Sciences, Applied Entomology, Swiss Federal Institute of

Technology (ETH), Clausiusstrasse 25/NW, CH-8092 Zurich, Switzerland:

DiSTA, University of Bologna, Via Filippo Re 6, I-40126 Bologna, Italy

Abstract

Peach shoot volatiles were attractive to mated female oriental fruit moth, Cydia

molesta (Busck), in a dual choice arena. No preference was observed between leaf

odours from the principle host plant, peach, and the secondary host plant, apple.

Twenty-two compounds were identiﬁed in headspace volatiles of peach shoots

using gas chromatography–mass spectrometry. Green leaf volatiles accounted for

more than 50% of the total emitted volatiles. A bioassay-assisted fractionation

using different sorbent polymers indicated an attractant effect of compounds with

a chain length of 6–8 carbon atoms. The major compounds of this fraction were

tested either singly or in combinations for behavioural response of females.

Signiﬁcant bioactivity was found for a three-component mixture of (Z)-3-hexen-1-

yl acetate, (Z)-3-hexen-1-ol and benzaldehyde in a 4:1:1 ratio. This synthetic

mixture elicited a similar attractant effect as the full natural blend from peach

shoots as well as the bioactive fraction.

Introduction

The primary sensory modality involved in host plant

ﬁnding of female lepidopteran insects is considered to be

chemical (reviewed by Honda, 1995; Hern & Dorn, 2002). A

possible strategy for monitoring female herbivores could

thus rely on chemical stimuli derived from host plant (Dorn

et al., 2001). The semiochemicals mediating the host location

behaviour of the oriental fruit moth, Cydia molesta (Busck)

(Lepidoptera: Tortricidae), are unknown (e.g. Natale et al.,

1999; Dorn et al., 2001). This species is an important pest of

stone fruits, particularly peach, where it infests the growing

shoots at the beginning of the season (Rothschild & Vickers,

1991). As the season progresses, it also damages fruits and is

also found in apple orchards (Pollini & Bariselli, 1993). This

is surprising as this species was considered to be

oligophagous, and damage to apples was considered rare in

western Europe until the late 1970s (Bovey, 1979). In recent

years, however, considerable levels of C. molesta damage to

apple orchards have been widely observed in several fruit

growing regions including Latin America, Asia and Europe

(Popovich, 1982; Reis et al., 1988; Zhao et al., 1989; Hickel &

Ducroquet, 1998; Bradlwarter et al., 1999).

Monitoring of C. molesta predominantly relies on

pheromone trapping of male moths (Vickers et al., 1985).

However, the ﬂight performance of this species exhibits

marked sexual differences, and gravid females can be

considered to be the main colonists (Dorn et al., 2001;

Hughes & Dorn, 2002). In the ﬁeld, female C. molesta are

capable of making inter-orchard ﬂights (Yetter & Steiner,

1932; Steiner & Yetter, 1933). This can pose a serious threat to

apple cultivation in orchards in the vicinity of peach crops.

Development of a semiochemical-based monitoring strategy

is thus desirable (Dorn et al., 2001). In addition, host plant

odours which are attractive to female C. molesta could also

be used in an attract and kill deployment.

The goal of this study was to identify compounds

derived from host plant or mixtures of compounds which

are attractive to C. molesta females. First, the response of

females to peach and apple shoots was characterized. As

*Author for correspondence

Fax: +41 1 632 11 71

E-mail: silvia.dorn@ipw.agrl.ethz.ch

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both odour sources were similarly attractive, the study

focused on peach as the main host plant. The headspace

volatiles of peach shoots were analysed using combined gas

chromatography–mass spectrometry. Subsequently, they

were fractionated using different sorbent polymers which

are able to trap compounds based on their volatility range.

As bioassays indicated an attractant effect of compounds of

a distinct range of carbon atoms, the hypothesis was tested

that the major compounds of this chain length, either singly

or in combination, would elicit the desired behavioural

effect in C. molesta females.

Materials and methods

Insects and plants

Pupae of C. molesta were purchased from a commercial

station in Italy (BioTechnologie B.T., Todi-Perugia, Italy). The

colony originated from individuals collected in peach

orchards in Emilia-Romagna (northern Italy). Moths were

bred in culture for approximately 68 generations. Larvae

were reared on an artiﬁcial diet based on corn semolina,

wheat germ and brewers yeast as described by Ivaldi-Sender

(1974). On arrival, pupae were placed inside a plastic box (30

× 30 × 30 cm) and supplied with a honey solution. Pupae

and emerging adults were maintained at 24 ± 1°C, 60 ± 10%

relative humidity, and a photoperiod of 16L: 8D. A previous

study indicated that mated females responded better than

virgins when exposed to peach shoots in a dual choice arena

(Natale et al., in press). For the bioassays, 3- to 5-day-old

mated females were chosen without a conscious bias from

the cage. Before the test started, adults of the two sexes were

singled out based on the slight sexual dimorphism, the

females being larger-sized. The mated status of female

moths was checked by dissection of the bursa copulatrix for

the presence of a spermatophore at the end of each test. All

female individuals used in the tests were found to have

successfully mated. Moths were used only once and were

not exposed to odour sources before the bioassay.

Three-year-old potted plants of peach Prunus persica L.

Batsch cv. Redhaven and apple Malus domestica L. Borkh

cv. Golden Delicious were used for bioassays and

collection of volatiles. Plants were maintained outdoors at

20 ± 5°C.

Chemical analysis

Volatile collection

Volatiles from excised shoots of peach were sampled

using a dynamic headspace sampling system similar to that

described by Boevé et al. (1996). A dilated glass cylinder with

a glass joint (500-ml) was used as a collection chamber in

which airﬂow was generated using a vacuum pump.

Incoming air was ﬁltered with an activated-charcoal ﬁlter

(Supelco, Mounting Clip for S-Trap, Buchs SG Switzerland)

connected by Teﬂon tubing. A cylindrical trap (Supelco)

ﬁlled with 300 mg of Tenax-GR 60/80 was plugged to the

chamber by means of a PTFE-lined cap. The sorbent trap

was connected to the vacuum using Teﬂon tubing. The ﬂow

rate was set at 100 ml min

1

by a ﬂow meter connected

between the pump and the trap. The ﬂow meter was set and

adjusted during the early phase of the collections to ensure

that the correct ﬂow was obtained. A moisture-removing

ﬁlter (Supelco, 400 cc 1/4 ) for the adsorption of

condensation, observed to form within the tubing, was

connected between the Tenax trap and the ﬂow meter.

Headspace collections lasted 3 h and were performed at

22°C, 60 ± 10% relative humidity.

Identiﬁcation with coupled gas chromatography–mass

spectrometry

Samples were analysed using a Hewlett Packard GC-MS

instrument (GC 6890 mass selective 5973) equipped with a

HP1, polydimethyl siloxane column with nominal ﬁlm

thickness 1 µm, diameter 0.25 mm, and length 30 m. The

initial oven temperature was 40°C. The oven was heated up

to 220°C at a rate of 8°C min

1

. A post run of 10 min at 300°C

was applied to remove impurities from the column.

Analyses were carried out using a thermal-desorption

system (Unity, Markes Int. Ltd

, Rhondda Cynon Taff, UK),

in which the desorbed headspace volatiles were transferred

to the GC-column without use of a solvent. Volatiles were

desorbed from the Tenax trap with helium (99.99%) for 5

min, starting at 50°C and then up to 300°C at approx. 20°C

min

1

, and transferred to the cold trap (10°C) which was

packed with a bed of Tenax GR and Carbopak B of 4 cm and

2 cm in length, respectively. The cold trap was subsequently

heated up to 300°C at approx. 60°C s

1

for 3 min. The

desorption ﬂow was kept at 30 ml min

1

for all analyses.

The thermal desorber was operated with a double split, i.e.

the split was operational during both the sample tube and

the cold trap desorption, and the GC was operated splitless.

As the desorption ﬂow was kept at 30 ml min

1

for all

analyses and the split ﬂow was 10 ml min

1

, the total split

ﬂow ratio during the thermal desorption was 7.7:1. This split

operation was used to prevent overloading of the GC

column. It enhances the chromatographic separation of the

components. A second Tenax trap recollected part of the

sample. The transfer line to the GC was kept at 200°C. The

identiﬁcation of chromatographically separated compounds

was carried out using a NIST98 spectral library, a user

created library, and matching GC retention time and mass

spectra with authentic standards. Quantiﬁcation of volatiles

was based on the response factors of the MS detector to the

components, and carried out using a calibration standard

containing 50 µl of each component and 50 µl of internal

standard (hexylbenzene, Fluka, purity > 99.8%) (Raffa &

Steffeck, 1988). Fifteen headspace collections of peach shoot

volatiles were carried out.

Bioassays

Bioassay arena

All behavioural tests were performed in a dual choice

arena (Natale et al., 2003). The arena consisted of a test-

chamber, where insects were released, and two odour

chambers, where insects were captured. Based on

preliminary observations, the test chamber was a bottomless

cylindrical glass bottle (10 l volume; 41 cm long; 22 cm

diameter) covered at the two ends with ﬁne nylon mesh. The

mesh at the large-sized end was pierced with two 2 cm

diameter holes, 18 cm apart. The two odour chambers,

300 ml ﬂasks each with a tubing at the top, were connected

to the test chamber by the two holes described above. Air

was ﬁltered through an activated-charcoal ﬁlter, regulated

by a ﬂoat ﬂow meter and moistened through a glass

chamber containing water. The airﬂow was pumped into the

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odour chambers connected to the olfactometers at a rate of

700 ± 10 ml min

–1

at the entrance. The arena was placed on a

workbench, 130 cm below 7 Lux line plus 36W ‘cool white’

(Sylvania

, Mississauga, Ontario Canada) lamps which

provided a uniform light intensity of 2400 lux. Bioassays

were conducted at 24 ± 1°C, 60 ± 5% relative humidity. For

each replicate, the position of the odour chambers was

exchanged in order to avoid positional bias. The arena was

cleaned before every trial session using a laboratory

glassware liquid cleaner (Sigmaclean®, Buchs SG

Switzerland), acetone (purity > 90%), hexane (purity > 90%)

and heat treatment (250°C, approx. 8 h). The bioassays were

carried out over a number of days. They started always 3 h

before the onset of the scotophase. A group of 30 females per

replicate was released into the test chamber from the smaller

end of the arena and allowed to move upwards and to

choose one odour source. After 15 ± 1 h, moths captured in

the two odour chambers were recorded as responders. All

other moths were removed and classed as non-responders.

A pilot study using a fruit volatile ester in small disposable

capillary pipettes, showed a positive olfactory response of

females, as the chamber containing the odour was entered

statistically more frequently compared to the blank (D.

Natale et al., unpublished).

Female response to plant volatiles

Peach and apple shoots were used as odour sources in

experiments investigating a possible preference of female C.

molesta for one of the two host plants. Female C. molesta lay

eggs on the foliage of top shoots, on the lower leaf surface in

peaches and on the upper surface in apples (Rothschild &

Vickers 1991). Top shoots of both peach and apple with 6 ± 1

leaves were used for the bioassay. The shoots were excised

within 10 min before experiments, at the phenological stage

deﬁned as full leaf unfolding (Dierschke, 1970). The

response of mated females to host plant volatiles was tested

in three experiments in order to elucidate possible

preference for: (i) peach shoot volatiles tested versus blank;

(ii) apple shoot volatiles versus blank; and (iii) apple shoot

volatiles versus peach shoot volatiles.

Female response to fractions

Fractions of volatiles emitted by excised peach shoots

were used as odour sources in experiments investigating the

attraction of mated female C. molesta to candidate active

compounds. For this purpose, a bioassay-assisted

fractionation was developed. Fractions were obtained from

volatiles of excised peach shoots using selective tube

sorbents. Based on previous observations, stainless steel

cylinders (1/4 × 30 cm), c. 10 ml volume, ﬁlled with sorbent

polymers of differing adsorbing strength were used as tubes.

The range adsorbed by each sorbent depends upon the

volatility of compounds and therefore their number of

carbons in length (in ‘Guidelines for sorbent selection’,

Markes Int. Ltd

). A tube sorbent was plugged between a

‘plant chamber’ and an ‘odour chamber’ of a dual choice

arena. The plant chamber was a ﬂask with the same size as

the odour chamber deﬁned above. The goal was to adsorb

all the volatiles in the range of a given sorbent from the

headspace of the peach shoot in the ‘plant chamber’, and

only allow the remaining fraction to enter the ‘odour

chamber’. Four fractions of volatiles were tested using the

following selective sorbents:

1. Porapak N

, 190 mg/tube, of medium sorbent strength

with an approximate compound volatility range of n-C5 to

n-C8, thus eluting compounds with more than 8 carbons;

2. Carboxen

, 250 mg/tube, of strong sorbent strength with

an approximate compound volatility range of n-C5 to n-C30,

eluting compounds with more than 30 carbons;

3. Carbopak F

, 40 mg/tube, of medium / weak sorbent

strength with an approximate compound volatility range of

n-C9 to n-C30, eluting compounds with 5–8 carbons.

4. Tenax TA

, 120 mg/tube, of weak sorbent strength with

an approximate compound volatility range of n-C7 to n-C30,

eluting compounds with 5–6 carbons.

A ﬁfth empty tube, eluting all compounds from the

headspace of peach shoot, was used as a control. The

response of mated females to the fractions of peach shoot

volatiles as explained above was tested with ﬁve

experiments in which the effect of each single odour source

was compared to a blank.

Female response to synthetic chemicals

As the fraction bioassays indicated the attractant effect of

compounds of n-C6 to n-C8, major compounds in this range

were used either singly or in combination as odour sources

in experiments investigating the attraction of mated female

C. molesta to artiﬁcial chemicals. Chemicals used for the

bioassay were (Z)-3-hexen-1-ol (Fluka, purity > 99.5%), (Z)-

3-hexen-1-yl acetate (Avocado, purity > 99%) and

benzaldehyde (Aldrich, purity > 99%) as potentially

attractive compounds, cis-

-ocimene (Robertet, purity >

99%) and (Z)-3-hexen-1-yl butyrate (Aldrich, purity > 99%)

as compounds representative of the behaviourally inactive

fractions. Based on preliminary observations, disposable 0.5

µl Hirschmann microcapillary pipettes (Hirschmann®,

Eberstadt, Germany) were baited with pure chemicals and

immediately placed in the odour chamber. To obtain the

required ratios between individual compounds, each

compound was applied using an appropriate number of 0.5

µl microcapillaries. Quantities released over the trial period

of 14 ± 1 h were determined gravimetrically using a

microbalance set with a readability of 1 µg (Mettler-Toledo

model MT5, San Juan, Puerto Rico). This microbalance has

the electronic control unit separated from the mechanical

components of the balances minimizing any effects on

weight. An automatic vibration damper and a built-in

calibration feature eliminate effects of unstable ambient

conditions. The response of mated females to chemicals was

tested with four experiments in which the activity of

compounds, singly or in combination, was compared to a

blank.

Data analysis

Numbers of captured moths from all bioassays were

analysed with generalized linear model using a Poisson

distribution and a log link (Crawley, 1993).

Results

Chemical analysis

Twenty-two compounds were found in the headspace of

excised peach shoots (table 1). Compounds ranged from n-

C6 to n-C16. The major classes of compounds were

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terpenoids and esters, which accounted for more than 27%,

hydrocarbons for more than 18%, while aldehydes and

alcohols accounted for 9% of the total emitted volatiles.

From a functional point of view, the green leaf volatiles, (E)-

2-hexenal, (Z)-3-hexen-1-ol, 1-hexanol, (Z)-3-hexen-1-yl

acetate and (Z)-3-hexenyl butyrate, were the major group

accounting for more than 50% of total emitted volatiles.

Major compounds identiﬁed were an acetate, (Z)-3-hexen-1-

yl acetate, an aromatic aldehyde, benzaldehyde, and an

alcohol, (Z)-3-hexen-1-ol with a short-chain (n-C6 to n-C8),

as well as a monoterpene, cis-

-ocimene, and an ester, (Z)-3-

hexen-1-yl butyrate with a medium-chain (n-C10).

(Z)-3-hexen-1-yl acetate, benzaldehyde, cis-

-ocimene, (Z)-3-

hexen-1-yl butyrate, and

-farnesene were consistently

detected in all 15 headspace samples analysed.

Female response to plant volatiles

Volatiles from peach and apple shoots attracted mated

female C. molesta in the dual choice arena (F-value = 11.77;

DF = 3, 8; P < 0.001; F-value = 25.28; DF = 3, 8; P < 0.001

respectively) (ﬁg. 1). There was no signiﬁcant preference of

female moths for peach shoot volatiles versus apple shoot

volatiles (F-value = 1.07; DF = 1, 10; P = 0.33) (ﬁg. 1).

Movements of female C. molesta, from the test chamber to

the two odour chambers of the dual choice arena, occurred

only in the few hours before the onset of scotophase and

after the onset of photophase (ﬁg. 2).

Female response to fractions

There was a signiﬁcant difference between the four

fractions of volatiles tested (F-value = 4.28; DF = 9, 29; P <

0.05) (ﬁg. 3). The two fractions eluted with Tenax TA

and

the Carbopak F

, expected to contain the short-chain

compounds n-C5 to n-C6 and n-C5 to n-C8, respectively,

attracted female C. molesta in the dual choice arena (F-value

= 4.28; DF = 9, 29; P < 0.01; F-value = 4.28; DF = 9, 29; P <

0.01). The fraction eluted with the empty tube, containing all

the peach shoot volatiles and used as a control, also attracted

female moths (F-value = 4.28; DF = 9, 29; P < 0.01). In

contrast, the two fractions eluted with Porapak N

and

Carboxen

, expected to contain medium- and long-chain

compounds with more than n-C8, and more than n-C30,

respectively, did not attract female C. molesta (F-value = 4.28;

DF = 9, 29; P < 0.01; F-value = 4.28; DF = 9, 29; P = 0.165). The

short-chain compounds identiﬁed from peach shoot volatiles

(table 1) with (1) n-C5 to n-C6: comprise (E)-2-hexenal, (Z)-3-

hexen-1-ol, 1-hexanol, and valeric acid and (2) n-C5 to n-C8:

the same four compounds plus benzaldehyde, benzonitrile,

(Z)-3-hexen-1-yl acetate, methyl benzoate, benzylnitrile and

methyl salicilate. The subsequent bioassays were straight-

338 D. Natale et al.

Table 1. Quantities and percentages of major volatile compounds released by peach shoots as observed in the analysis.

Peaks Compounds Carbons Rt (min) n ng ± se % ± se

1 valeric acid 5 5.90 14 0.46 ± 0.04 0.03 ± 0.1

2(E)-2-hexenal

6 5.15 12 19.8 ± 0.3

0.42 ± 0.05

3(Z)-3-hexen-1-ol

6 5.30 13 128.5 ± 4

6.67 ± 0.47

4 1-hexanol

6 5.60 13 1.93 ± 0.15 0.37 ± 0.02

5 benzaldehyde 7 7.54 15 116.4 ± 3

8.08 ± 2.57

6 benzonitrile 7 8.09 10 1.29 ± 0.24 0.08 ± 0.01

7(Z)-3-hexen-1-yl acetate

8 8.74 15 613 ± 8 40.62 ± 1.25

8 methyl benzoate 8 10.53 15 0.71 ± 0.10 0.04 ± 0.01

9 benzylnitrile 8 11.44 11 0.79 ± 0.34 0.36 ± 0.08

10 methyl salicylate 8 12.55 15 2.00 ± 0.50 0.49 ± 0.04

11 nonatriene 9 11.02 13 1.21 ± 2.13 0.8 ± 0.64

12 cis-

-ocimene 10 9.57 15 3.61 ± 1.50

1.96 ± 0.12

13 3-carene 10 10.20 10 0.16 ± 0.03 0.01 ± 0.00

14 (Z)-3-hexen-1-yl butyrate

10 12.42 15 9.93 ± 1.13

2.25 ± 0.70

15 dodecane 12 12.69 13 1.12 ± 0.14 0.08 ± 0.01

16 (Z)-3-hexen-1-yl hexanoate 12 16.03 15 0.54 ± 0.32 0.12 ± 0.01

17 (Z)-3-hexen-3-yl benzoate 13 19.16 15 1.48 ± 0.50 0.40 ± 0.03

18 tetradecane 14 16.36 15 0.78 ± 0.10 0.17 ± 0.02

19 trans-caryophyllene 15 16.78 15 1.14 ± 0.13 0.34 ± 0.04

20 (E,E)-

-farnesene 15 17.26 14 0.19 ± 0.11 0.07 ± 0.01

-farnesene 15 17.36 15 1.08 ± 0.28 0.32 ± 0.01

22 pentadecane 15 18.02 15 1.02 ± 0.64 0.20 ± 0.03

Rt, retention time;

green leaf volatile;

compounds quantiﬁed with relative response of synthetic standard in comparison to internal

standard; n, frequency of detection of the compounds in a total of 15 samples.

Mean no. of captured female moths

***

Fig. 1. Olfactory preference of mated female Cydia molesta

exposed in a dual choice arena to excised peach shoot ( ) vs.

blank ( ); excised apple shoot ( ) vs. blank ( ); excised peach

shoot ( ) vs. excised apple shoot ( ). N = 3, 30 moths per

replicate. (***P < 0.001; generalized linear model).

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forward, the approach based on the simple assumption that

major components of these fractions might be bioactive as

has previously been shown for a major constituent of apple

fruit volatiles, (E,E)--farnesene (Hern & Dorn, 1999). For

comparison, bioassays were carried out with the two major

constituents of the long-chain carbons (n-C10) identiﬁed

from peach shoot volatiles (table 2), cis-

-ocimene and (Z)-3-

hexen-1-yl butyrate which were assumed to be

behaviourally ineffective.

Female response to synthetic chemicals

In the search for bioactive single constituents of peach

shoot volatiles, the prevailing short-chain compounds in the

natural blend (table 2) were tested as synthetic chemicals,

singly or in combination, and compared to the two

prevailing longer-chain compounds, (Z)-3-hexen-1-yl

butyrate and cis-

-ocimene. There was a signiﬁcant

difference between the ﬁve combinations or single

constituents tested (F-value = 2.43; DF = 9, 49; P < 0.05)

(ﬁg. 4). Neither of the two long-chain compounds attracted

female moths (EST F-value = 0.54; DF = 1, 9; P = 0.486; TRP

F-value = 0.67; DF = 1, 9; P = 0.439). In contrast, a mixture of

the two major green leaf volatiles, (Z)-3-hexen-1-yl acetate

and (Z)-3-hexen-1-ol, plus benzaldehyde attracted female C.

molesta (MIX F-value = 22.75; DF = 1, 9; P < 0.01). However, a

mixture of only these two major green leaf volatiles, or the

aldehyde tested singly, did not attract female moths (GLV F-

value = 0.46; DF = 1, 9; P = 0.517; ALD F-value = 0.22; DF = 1,

9; P = 0.649).

Discussion

Olfaction appears to be involved in host habitat location

behaviour of mated female C. molesta. Female moths, known

to oviposit on shoots or foliage of peach and apple trees

(Rothschild & Vickers, 1991), were attracted to volatiles from

shoots of both host plants. No discrimination between the

volatiles from peach and apple foliage was found in this

oligophagous herbivore. The signiﬁcance of olfactory stimuli

in lepidopteran species is assumed to reﬂect their level of host

specialization. Highly specialized species are expected to be

dependent on olfaction, while this sensory modality is

considered to be of low importance in highly polyphagous

species (Ramaswamy, 1988). The host range of C. molesta is

conﬁned to plant species in the family Rosaceae, mostly in the

genera Prunus and Pyrus, and to one shrub from the family

Myrtaceae, reﬂecting an intermediate level of specialization.

Analysis of the volatile blend emitted by the main host

plant, peach, revealed the presence of 22 compounds. A

previous investigation (Horvat & Chapman, 1990) on leaf

volatiles found only two compounds in sizable amounts

which is possibly due to methodological differences (see

below). In the current study the major constituents were (Z)-

3-hexen-1-yl acetate and (Z)-3-hexen-1-ol, accounting for 41

and 7% of the total quantity of volatiles in the shoot

Female C. molesta response to plant volatiles 339

Cumulative captures of female moths

9 20 21 22 23 24 1 2 3 4 5 6 7 8 9

Photophase Scotophase Photophase

Time (h)

Fig. 2. Time course of cumulative captures of mated female

Cydia molesta (dual choice arena bioassay) in the odour chamber

containing a peach shoot (—

•

) compared to a blank (— ).

Mean no. of captured female moths

>n-C8

>n-C30 n-C5 to n-C8

n-C5 to n-C6

natural blend

Fig. 3. Bioactivity of fractions of compounds ( ) with a different number of carbons compared to a blank ( ). Attraction of mated

female Cydia molesta in a dual choice arena to a fraction of compounds with >8 carbons; fraction of compounds with >30 carbons;

fraction of compounds of 5–8 carbons; fraction of compounds of 5–6 carbons; total emitted peach shoot volatiles ( ). N = 3; 30 moths

per replicate. (**P < 0.01; generalized linear model.)

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headspace, respectively. Both compounds have also been

reported from volatile collection of apple foliage (Bengtsson

et al., 2001). They are categorized as green leaf volatiles and

consist of a number of compounds of saturated or mono-

unsaturated aldehydes, alcohols and acetates, which occur

in all plants, but in very varying proportions depending on

species (Hansson et al., 1999). Antennal receptors of different

lepidopteran species were stimulated in response to green

leaf volatiles which included (Z)-3-hexen-1-yl acetate

(reviewed by Visser, 1986; Bengtsson et al., 2001). This class

of compounds, possibly in combination with further

constituents of plant odours, is assumed to be involved in

herbivore orientation to its host plant (van Tol & Visser,

2002).

An attractive effect on C. molesta females was found for

these two major green leaf volatiles in combination with

benzaldehyde. This aromatic aldehyde has also been

detected during certain periods of the season in volatile

blends from an apple tree (Bengtsson et al., 2001; A. Vallat &

S. Dorn, unpublished). This is, to our knowledge, the ﬁrst

time that host-plant-derived attractants for female C. molesta

have been reported. The ratio of these three compounds

tested for the behavioural response of the moth reﬂects their

ratio in the natural blend. Two major compounds from a

non-bioactive fraction did not elicit any response in C.

molesta females. A combination of a minor constituent of the

green leaf volatiles, (E)-2-hexenal with (Z)-3-hexen-1-ol, was

behaviourally inactive as well (data not shown). This does

not exclude that further combinations may exhibit an

attractant effect on the moths. In the apple maggot fruit ﬂy,

Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) a seven-

component mix was ﬁrst reported to be attractive to sexually

mature adults (Fein et al., 1982), while a later study identiﬁed

an even higher effect for a ﬁve-component blend (Zhang et

al., 1999). Further studies will be needed to analyse to what

degree the bioactivity found for the mixture characterized

above is sex-speciﬁc in C. molesta.

New methods were used for the fractionation of peach

volatiles and for the bioassay. In a previous study, peach

leaves were frozen, ground to a ﬁne powder and then

solvent extracted (Horvat & Chapman, 1990). The current

study beneﬁted from the technology of direct thermal

desorption of headspace volatiles from intact shoots. This

yielded a larger number of quantiﬁable compounds without

the risk of including artefacts caused by oxidation in leaf

homogenates. The method of fractionation based on sorbent

polymers that trap different compounds within a given

volatility range proved to be appropriate for the purpose of

this study. An artiﬁcial mixture prepared to mimic

components of the bioactive fraction was behaviourally

340 D. Natale et al.

Table 2. Artiﬁcial combinations or single constituents used in bioassays with chemicals.

Constituents Ratio Release (µg)

GLV (Z)-3-hexen-1-yl acetate : (Z)-3-hexen-1-ol 4 : 1 1750 ± 0.32 : 175 ± 0.15

ALD benzaldehyde 1 161 ± 0.07

MIX (Z)-3-hexen-1-yl acetate : (Z)-3-hexen-1-ol : benzaldehyde 4 : 1 : 1 1750 ± 0.37: 175 ± 0.11 : 161 ± 0.08

EST (Z)-3-hexen-1-yl butyrate 1 326 ± 0.27

TRP cis-

-ocimene 1 221 ± 0.4

Ratios used reﬂect approximate ratios released from the peach shoots. To obtain the required initial ratios (µl : µl) between individual

compounds, an appropriate number of 0.5 µl microcapillaries was used. Quantities released over the trial period were determined

gravimetrically (mean ± standard error).

GLV ALD MIX EST TRP

Mean no. of captured female moths

Fig. 4. Bioactivity of single or combinations of compounds ( , bioactive fractions; , inactive fractions) as compared to a blank ( ).

Attraction of mated female Cydia molesta in a dual choice arena to (Z)-3-hexen-1-yl acetate plus (Z)-3-hexen-1-ol (GLV), benzaldehyde

(ALD), (Z)-3-hexen-1-yl acetate plus (Z)-3-hexen-1-ol plus benzaldehyde (MIX), (Z)-3-hexen-1-yl butyrate (EST), cis-

-ocimene (TRP). N

= 5; 30 moths per replicate. (**P < 0.01; generalized linear model.)

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effective. This indicates the usefulness of this procedure. It

is related to the so-called subtractive combination method

deﬁned as ‘subtracting fractions from the whole blend of

compounds for bioassays’ (Byers, 1992). However, the

current study started with headspace volatiles instead of

crude extracts and subtracted all but one fraction instead of

subtracting a single fraction from the total blend. The

bioassay was carried out in a dual choice arena under light

conditions simulating a diurnal cycle. Major movements of

the moths into the odour chambers were recorded before

the onset and after the termination of the period without

light. This coincides with a previous laboratory study

reporting ﬂight activity in C. molesta during dusk and dawn,

and minimal movement during dark (Hughes & Dorn,

2002). As this photoperiodicity is identical to that observed

for this species in the ﬁeld (Dustan, 1964; Roerich, 1961;

Rothschild & Minks, 1974), it is concluded that the bioassay

used offers favourable conditions for the assessment of

adult behaviour.

In addition to olfaction, vision might also be an

important sensory modality for host habitat location as it is

in other lepidopteran species (Ramaswamy, 1988). Further

work should evaluate the three-compound mixture for

trapping C. molesta females on a larger scale, paying

attention also to the design of the trap. Such investigations

might lead to an effective tool for attracting female moths,

and to a better understanding of their host plant selection

process.

Acknowledgements

This work was supported by the Centro Ricerche

Produzioni Vegetali, CRPV, Diegaro di Cesena (FO) Italy.

The authors thank Dr Kathrin Tschudi-Rein, Dr Anja Rott

and the two anonymous referees for useful comments on

this manuscript.

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