DOI: 10.1111/j.1570-7458.2007.00611.x
Blackwell Publishing Ltd
Electrophysiological responses of the blue willow leaf
beetle, Phratora vulgatissima, to volatiles of different
Salix viminalis genotypes
Patricia C. Fernandez1*, Torsten Meiners1, Christer Björkman2 & Monika Hilker1
1
Institute of Biology, Freie Universität Berlin, Haderslebener Straße 9, D-12163 Berlin, Germany, 2Department of Ecology
Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden
Accepted: 1 July 2007
Key words: host-plant selection, plant volatiles, Coleoptera, Chrysomelidae, electroantennogram, GC-MS
Abstract
Among numerous other factors, host-plant volatiles may affect selection of food plants by herbivorous
insects. The blue willow leaf beetle, Phratora vulgatissima (L.) (Coleoptera: Chrysomelidae), is known
to differentiate between willow species and genotypes. However, so far no knowledge is available on
the physiological abilities of this leaf beetle to respond to willow volatiles. In this study, we recorded
electroantennograms of male and female P. vulgatissima to volatiles from two Salix viminalis L.
(Salicaceae) genotypes: Jorr and 78021. The headspace of these genotypes were analysed by coupled
gas chromatography–mass spectrometry. In addition to known green leaf volatiles (GLV), several
terpenoid components were found. Both males and females of P. vulgatissima showed strong
responses to the GLVs (Z)-3-hexenol and (Z)-3-hexenyl acetate, and moderate responses to (E)-ocimene
and β-caryophyllene. Females, but not males, also responded to R-(+)-limonene. This work represents
a further step to identify substances relevant for the orientation of P. vulgatissima to host plants.
Introduction
The blue willow beetle, Phratora vulgatissima (L.)
(Coleoptera: Chrysomelidae), is a serious pest insect on
willows (Sage & Tucker, 1998; Björkman et al., 2000,
2004). Salix viminalis L. (Salicaceae), a species with low
concentrations of phenolglycosides, is preferred among
various species of willows by P. vulgatissima (Pasteels &
Rowell-Rahier, 1992). This species is predominantly
planted for short-rotation coppice (Tabbush & Parfit, 1999).
The susceptibility of willows to P. vulgatissima varies not
only among different species, but also among genotypes of
the same species (Peacock et al., 2002, 2004).
Damage of a plant by conspecifics or heterospecifics is
well known to affect host-plant selection by chrysomelids
and other herbivorous insects (Schindek & Hilker, 1996;
Schoonhoven et al., 2006; Fernandez & Hilker, 2007). The
blue willow leaf beetle shows aggregated distribution on
preferred hosts in willow plantations (Peacock et al.,
1999). Studies by Peacock et al. (2001b) suggest that these
*Correspondence and present address: Patricia C. Fernandez, School
of Life Sciences, Arizona State University, Tempe, AZ 85287-4501,
USA. E-mail: [email protected]
aggregations are due to a synergistic effect of plant
cues induced by feeding and attractants released by
P. vulgatissima feeding on the plant. When feeding damage
was mimicked by artificial damage, willows increased
the release of (Z)-3-hexenol and (Z)-3-hexenyl acetate
(Peacock et al., 2001a,b), as is known for several other
plant species (Karban & Baldwin, 1997). The ratio of these
two green leaf volatiles (GLV) has been suggested to play a
significant role for host-plant selection by P. vulgatissima
(Peacock et al., 2001a). Green leaf volatiles are ubiquitous
plant volatiles and their presence and quantities might
play a role for host-plant selection in admixture with other
volatiles, as was shown for several chrysomelid species
(Visser & Avé, 1978; Schütz et al., 1997; Dickens, 2000;
Müller & Hilker, 2000).
In contrast to the broad information on the behavioral
ecology of P. vulgatissima, nothing is known on the
physiological abilities of P. vulgatissima to respond to
host-plant volatiles. Therefore, a major aim of this
study was to determine which host-plant volatiles this
leaf beetle species is physiologically able to perceive. We
recorded electroantennograms (EAG) of male and female
P. vulgatissima to volatiles from S. viminalis, a preferred
host-plant species.
© 2007 The Authors Entomologia Experimentalis et Applicata 125: 157–164, 2007
Journal compilation © 2007 The Netherlands Entomological Society
157
158
Fernandez et al.
Prior to the EAGs, we analysed volatiles released from
two S. viminalis genotypes by coupled gas chromatography–
mass spectrometry (GC-MS). We studied volatiles released
from both non-damaged plants and plants having been
damaged by feeding and egg-laying beetles to check
whether plants already colonized by P. vulgatissima release
a different blend of volatiles compared to plants not yet
attacked by this leaf beetle (Fernandez & Hilker, 2007).
Volatiles from artificially damaged S. viminalis have already
been studied by Peacock et al. (2001a). However, feeding
damage can induce another blend of volatiles compared to
artificial damage, even though similarities occur (Takabayashi
et al., 1994; Turlings et al., 1995; Schütz et al., 1997). To
elucidate the leaf beetle’s response specifically to host-plant
odour, we excluded beetle odour by collecting systemically
released plant volatiles from feeding-damaged willows,
that is, volatiles released from an undamaged part of the
plant adjacent to the damaged part.
Materials and methods
Plants and insects
Adults of P. vulgatissima overwintering in the hollow stems
of reed, Phragmites australis, were collected in April 2004
and 2005 in the surroundings of Uppsala, Sweden. Several
willow species were growing close to these overwintering
sites including S. viminalis, Salix dasyclados clones,
and Salix cinerea. The beetles were reared in the laboratory
in Berlin, Germany, in transparent plastic boxes (20 ×
20 × 6 cm) under an L18:D6 photoperiod at 20 °C on cut
twigs of Salix fragilis. Sixteen cuttings (25 cm in length)
each of two S. viminalis genotypes (eight S. viminalis
Jorr and eight S. viminalis 78021) from Uppsala were
transported to Berlin and were planted individually in
small pots in March. During the first month, they were
maintained in a greenhouse; afterwards, they were placed
outside until used. After 3-month growth, plants were
subjected to treatments of feeding and egg deposition as
described below.
Plant treatments
Treatment of saplings (60 cm in length) started in the
morning (about 09:00 hours) in the summer (June–July)
and lasted for a period of 72 h in a greenhouse, in order
to obtain plant material for volatile collection. Two
treatments (intact and damaged) were assigned at each of
two S. viminalis genotypes, resulting in four different
groups with n = 4 saplings each: (i) S. viminalis Jorr intact,
(ii) S. viminalis 78021 intact, (iii) S. viminalis Jorr damaged,
and (iv) S. viminalis 78021 damaged. To obtain damaged
plants, 15 P. vulgatissima adults (seven males and eight
females) were allowed to feed and oviposit on the lower
half of a potted sapling for a period of 72 h. Because of the
headspace sampling method used (see below), beetles were
confined to the lower half of the plant by a net (mesh
width: approximately 1.5 mm2), while the upper part was
kept free from beetles. The lower half of the control plants
was wrapped with a net in the same way, but no beetles
were added. During treatment, each sapling was placed in
a cage that was built of a wooden frame (12 × 80 × 60 cm)
and a plastic mesh.
Headspace samples
Headspace samples of the two genotypes (each n = 4) were
taken from the saplings before and immediately after
treatment in a climate chamber at 25 °C under daylight
conditions (approximately 10 000 lux). To collect volatile
compounds from a treated twig, the upper part of a twig
was placed into a bag made of polyethylene (PET) foil.
As beetles were confined to the lower part of the plant by
a net, this upper part was free of beetles, feeding damage,
or feces. Thus, this procedure allowed sampling systemically
treatment-induced plant volatiles while avoiding collection
of volatiles released from the beetles. Headspace samples
from untreated control twigs of the two genotypes
(each n = 4) were also only taken from their upper
parts. Headspace samples of the two genotypes (treated
and control twigs) were taken at the same time (9:00–
16:00 hours).
Charcoal-filtered air was pumped into the PET bags
(120 ml min–1) containing the twig parts. Air leaving the
bag through an outlet passed a small charcoal filter (5 mg,
precision charcoal filter; Trott, Kriftel, Germany), where
volatiles were collected for analysis (closed–loop–stripping
analysis). After a sampling period of 7 h, the filter was
eluted with 20 ml dichloromethane containing tridecane
(50 ng μl–1) as internal standard. All tubing necessary for
air in- and outlet were made of Teflon.
Identification and quantification
Headspace samples were analysed by coupled GC-MS on
a Fisons GC Model 8060 and Fisons MD 800 quadrupole
MS with EI ionisation (70 eV) (Fisons Instruments, MainzKastel, Germany). A J&W 30-m DB5-MS capillary column
was used (0.32-mm i.d.; film thickness: 0.25 μm). Eluted
samples (1 μl) were injected at 240 °C in a splitless mode.
Helium was used as carrier gas (inlet pressure: 10 kPa).
The temperature programme started at 40 °C (4-min
hold) and rose 10 °C per min to 280 °C. Compounds were
identified by comparing mass spectra and linear retention
indices of detected volatiles with those of authentic
reference compounds or with those provided by the NIST
(National Institute of Standards and Technology) library
(MassFinder 2.2) or our own library. Retention indices
Electrophysiological responses of willow leaf beetles 159
were calculated for each compound according to van den
Dool & Kratz (1963) and compared to tabulated data
(Adams, 1995). Confirmation of the identity by comparison
of retention times with those of authentic standards
were obtained for (Z)-3-hexenol (Sigma Aldrich, St.
Louis, MO, USA; 98% purity), β-myrcene (Sigma
Aldrich; technical purity), (+)-2-carene (Fluka, Basel,
Switzerland; 96% purity), (Z)-3-hexenyl acetate (Sigma
Aldrich; 98% purity), R-(+)-limonene (Sigma Aldrich;
96% purity), (E/Z)-β-ocimene (courtesy of Dr. Stefan
Schulz, Braunschweig, Germany), (E)-4,8-dimethyl-1,3,7nonatriene (courtesy of Dr. Stefan Schulz, E form >95%),
α-copaene (Fluka; 97% purity), β-caryophyllene (Sigma
Aldrich; 80% purity), α-humulene (Carl Roth GmbH,
Karlsruhe, Germany), and (E)-nerolidol (Carl Roth GmbH;
90% purity). In a previous study, α-pinene (Fluka) has
been analysed with the same GC-MS set-up, and its mass
spectrum and retention index were included in the library
of the laboratory. The compound α-farnesene was not
available as a synthetic component. Therefore, it has only
tentatively been identified by comparison of retention
index and mass spectrum of the detected component with
those of the NIST library.
Compounds detected in the headspace of at least three
out of four samples in at least one of the treatments were
quantified. Even though we detected (Z)-3-hexenyl acetate
only in a few of our samples, we quantified this component
based on its previous detection in Salix volatile collections
(Peacock et al., 2001a) and its potential behavioural
activity (Peacock et al., 2001a,b). Relative quantities of
components were calculated by relating their peak areas
with those of the internal standard and dry weight of the
twig part from which volatiles were collected. Dry weight
was estimated by drying and weighing the experimental
twigs after they were used for headspace collection and
later in bioassays (see below). Data were analysed by means
of Statistica 4.5 (StatSoft Inc., Hamburg, Germany).
Repeated measures analysis of variance (ANOVA) was
performed, with the genotype as independent factor
and the treatment as repeated measure factor. Limonene
data were transformed by log (x + 1) to fulfill ANOVA
requirements (Sokal & Rohlf, 1995).
EAGs
The EAGs of P. vulgatissima males and females were
recorded by offering to the beetles volatiles frequently
present in the headspace of the willow genotypes studied
(compare Tables 1 and 2). Most of the components
were the same we used as authentic standards for the
confirmation of identity by comparison of retention times
(see above). We also used S-(-)-limonene (Sigma Aldrich;
97% purity) for testing and 1-hexenol (Sigma Aldrich;
98% purity) as a positive standard. The component
α-farnesene was not tested because of the lack of an
authentic standard. Electroantennograms were made using
a commercially available electroantennographic system
(Syntech, Hilversum, The Netherlands). A 10-μl aliquot of
the test components (1 μg μl–1 in hexane) was pipetted
onto a strip of filter paper and inserted into a glass Pasteur
pipette after 10 s. The pipette was connected to a tube that
was connected to a Syntech stimulus controller (CS-05).
Excised beetle antennae were stimulated with pulses of
odour by injecting the vapour phase of the Pasteur pipette
150 mm upstream from the antenna into the continuous
humid air stream (pulse time 0.5 s, continuous flow
25 ml s–1). At least 60 s were allowed between stimuli to
provide time for recovery of antennal responsiveness.
For analyses of the EAGs, the mean solvent signal
was subtracted from each mean stimulus signal. Each
compound was tested on at least three (3-upper value)
different antennae of both sexes. Additionally, dose–
response studies for EAG-active volatiles were conducted
using antennae from males and females. Dilutions of
synthetic standards were made in hexane at decadic
steps, from 10 μg μl–1 to 10–2 μg μl–1. The GLV (Z)-3hexenol (1 μg μl–1) was used as positive standard for
β-caryophyllene, (E/Z)-β-ocimene, and (Z)-3-hexenyl
acetate measurements. When recording the response to
(Z)-3-hexenol, 1-hexanol (1 μg μl–1) was used as a positive
standard. Data were analysed using Statistica 4.5 (StatSoft
Inc.). Repeated measures ANOVA was performed with
sex as the independent factor and dose as the repeated
measures factor. The data of response to β-caryophyllene
were log transformed to fulfill ANOVA requirements.
Following ANOVA analysis, Newman–Keuls comparisons
were performed in order to detect specific differences
among doses.
Results
Chemical analysis of closed loop extracts
When comparing the genotypes, the total amount of
volatiles collected during the experiment was more than
three times higher in S. viminalis 78021 than in Jorr for
both intact and damaged plants (see Table 1). However,
damage did not induce a significant change of the total
amount of volatiles, neither in Jorr nor in 78021. All
components detected in the headspace of S. viminalis
78021 (intact and damaged) were found in Jorr (intact
and damaged), except for α-humulene, rarely found in
Jorr. The willow trees emitted predominantly monoand sesquiterpenes, regardless of their treatment. Major
compounds detected were β-caryophyllene, α-farnesene,
(E)-nerolidol, (E)-4,8-dimethyl-1,3,7-nonatriene, and
160
Fernandez et al.
Table 1 List of volatiles from Salix viminalis twigs following various treatments. Only compounds that were identified in at least three out
of four samples of at least one of the treatments are given [except for (Z)-3-hexenyl acetate, see text]
Compound
Intact plants
Damaged plants
ANOVA results
Jorr
Jorr
Source
78021
1 (Z)-3-hexenol
0.8 ± 0.3
0.8 ± 0.4
0.3 ± 0.2
2 α-pinene
0.3 ± 0.2
1.1 ± 0.5
0.4 ± 0.3
3 β-myrcene
1.0 ± 0.5
2.3 ± 1.5
0.4 ± 0.1
4 (Z)-3-hexenyl acetate
1.1 ± 1.0
5 2-carene
1.0 ± 0.9
3.4 ± 1.8
1.7 ± 1.4
6 limonene
0.6 ± 0.1
2.1 ± 0.9
0.3 ± 0.2
7 (E)-β-ocimene
0.9 ± 0.5
2.0 ± 1.4
1.6 ± 1.0
8 (E)-4,8-dimethyl1,3,7-nonatriene
14.4 ± 5.5
0
0.3 ± 0.2
63.9 ± 18.8 12.7 ± 2.7
9 α-copaene
0.1 ± 0.1
0.1 ± 0.0
0.1 ± 0.0
10 β-caryophyllene
0.5 ± 0.1
4.7 ± 1.0
0.5 ± 0.1
11 α-humulene
0.0
1.0 ± 0.3
0.0
12 α-farnesene
9.5 ± 3.6
12.9 ± 4.0
3.1 ± 1.0
13 (E)-nerolidol
5.5 ± 2.7
7.0 ± 3.9
5.1 ± 1.7
Total relative amount volatiles 35.9 ± 9.0 101.1 ± 0.1
25.8 ± 6.0
78021
0.3 ± 0.2
Clone
Treatment
Clone*treatment
1.4 ± 0.6 Clone
Treatment
Clone*treatment
1.2 ± 0.6 Clone
Treatment
Clone*treatment
0.0 ± 0.0 Clone
Treatment
Clone*treatment
3.8 ± 3.0 Clone
Treatment
Clone*treatment
0.9 ± 0.4 Clone
Treatment
Clone*treatment
10.8 ± 5.4 Clone
Treatment
Clone*treatment
61.0 ± 16.7 Clone
Treatment
Clone*treatment
0.2 ± 0.1 Clone
Treatment
Clone*treatment
4.7 ± 0.9 Clone
Treatment
Clone* treatment
1.0 ± 0.2 Clone
Treatment
Clone* treatment
4.0 ± 2.0 Clone
Treatment
Clone*treatment
4.4 ± 1.6 Clone
Treatment
Clone*treatment
92.0 ± 19.3 Clone
Treatment
Clone*treatment
d.f. Mean square
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.000002
0.00042
0.000004
0.0014
0.000037
0.000007
0.0018
0.0012
0.00006
–
–
–
0.0081
0.0005
0.00001
0.00027
0.00015
0.00005
0.041
0.036
0.026
3.822
0.0083
0.0005
0.000002
0.0000002
0.00001
0.0285
0.000002
0.000001
0.0015
0.0000007
0.0000006
0.0072
0.0930
0.0027
0.0003
0.0034
0.0019
6.7444
0.0535
0.0174
F
P
0.01 0.92
5.38 0.06
0.05 0.83
2.75 0.15
0.29 0.60
0.06 0.82
1.26 0.30
1.13 0.33
0.06 0.82
–
–
–
–
–
–
0.82 0.40
0.23 0.65
0.01 0.94
2.82 0.15
2.62 0.16
0.89 0.38
2.91 0.14
3.28 0.12
2.37 0.17
7.75 0.03
0.20 0.67
0.01 0.91
0.20 0.67
0.08 0.78
3.97 0.09
325
<0.001
0.001 0.97
0.001 0.98
80.5 <0.001
0.01 0.92
0.01 0.93
0.35 0.57
13.8
0.01
0.40 0.55
0.02 0.90
0.50 0.51
0.27 0.61
14.2
0.01
0.45 0.53
0.15 0.71
Values are given in ng/μl/sampling unit ± SE; number of replicates for each treatment: n = 4.
Repeated measures analysis of variance (ANOVA) of volatile emission were performed: ‘treatment’ was set as ‘within-subject’ factor and
‘clone’ as ‘between-subject’ factor.
Electrophysiological responses of willow leaf beetles 161
Table 2 Electroantennograms (EAG) activity of synthetic plant
volatiles detected in the headspace of Salix viminalis on male and
female antennae of Phratora vulgatissima
EAG activity
Compound
Male
Female
(Z)-3-hexenol
β-myrcene
(Z)-3-hexenyl acetate
(+)-2-carene
(S)-(-)-limonene
(R)-(+)-limonene
(E/Z)-β-ocimene
(E)-4,8-dimethyl-1,3,7-nonatriene
α-copaene
β-caryophyllene
α-humulene
(E)-nerolidol
++
–
++
–
–
–
+
–
–
+
–
–
++
–
++
–
–
+
+
–
–
+
–
–
++, clear response (>0.4 mV); +, weak response (0.2– 0.4 mV);
–, no response.
Source load = 10 μl of 1 μg μl–1 synthetic standards.
(E)-β-ocimene. Both intact and damaged twigs of 78021
released significantly more β-caryophyllene and (E)-4,8dimethyl-1,3,7-nonatriene than those of Jorr. After
damage, the release of α-farnesene was significantly
reduced in both willow genotypes (Table 1).
EAGs
Five compounds were found to be EAG active: (Z)-3hexenol, (Z)-3-hexenyl acetate, R-(+)-limonene, (E/Z)-βocimene, and β-caryophyllene (Table 2). A comparison of
responses from antennae of both sexes revealed that,
except for R-(+)-limonene, males responded to the same
set of volatiles as females (Table 2). An analysis of the
absolute (non-normalized) EAG values showed highly
significant dose–response curves for the EAG-active
volatiles detected by both males and females (repeated
measures dose factor: P<0.0001 in all cases; Figure 1).
Males and females showed the same sensitivity to all of the
compounds (two-way ANOVA; sex factor: non-significant
in any case). Interaction factors were not significant in any
case.
Discussion
The genotypes of S. viminalis studied here (Jorr and 78021)
released several terpenoids in addition to the expected
GLVs detected in previous studies of S. viminalis
headspace (Peacock et al., 2001a). Qualitative similarities
and quantitative differences in the volatile blends were
found when comparing the two S. viminalis clones.
Significant differences in amounts were found for
total volatiles, (E)-4,8-dimethyl-1,3,7-nonatriene, βcaryophyllene, and α-humulene. Adult P. vulgatissima
have antennal receptors that respond to GLVs and
terpenoids present in volatile collections from this
host-plant species.
Only very small quantitative differences were detected
between the volatiles emitted by control and induced
plants (significantly different only for α-farnesene; P = 0.01,
factor treatment; Table 1). The similarity of the volatile
blends of non-damaged and damaged plants may be due to
the fact that volatiles were not collected locally at the site of
feeding damage, but systemically at a non-damaged site
adjacent to the damaged one (Materials and methods). This
also could explain the absence of (Z)-3-hexenyl acetate in
the headspace of S. viminalis 78021 and the low quantities
of (Z)-3-hexenol detected, as release of GLVs may be
considerably reduced at sites distant from the point of
damage (Schütz et al., 1997). Peacock et al. (2001a) found
several GLVs by collecting volatiles locally at the site of
damage. Our finding that P. vulgatissima strongly responds
physiologically to the GLVs (Z)-3-hexenol and (Z)-3hexenyl acetate indicates that these compounds may play
a role in host-plant finding as was previously suggested by
Peacock et al. (2001a).
Both the EAG-active GLVs and terpenoids detected
in the headspace of S. viminalis are widespread plant
volatiles. (E)-β-Ocimene is an acyclic terpene known to be
present in non-damaged and insect-damaged plant species
(e.g., Loughrin et al., 1994; Paré & Tumlinson, 1999;
Cardoza et al., 2002; Hern & Dorn, 2002). Some insects were
found to respond electrophysiologically to (E)-β-ocimene
(Bichao et al., 2005). It also functions as an attractant of
natural enemies (Shimoda & Dicke, 1999). In P. vulgatissima,
(E)-β-ocimene evoked a moderate EAG-positive response,
slightly lower than that of GLVs. Nevertheless, even
unspecific compounds can be used for the detection of
host plants when an insect is capable of evaluating the
relative amounts of components within a complex odour
mixture.
β-Caryophyllene is a common sesquiterpenoid found as
a major compound in the headspace of numerous plant
species. Its emission is known to be enhanced after insect
feeding in several plants (Wegener et al., 2001; Blackmer
et al., 2004; Colazza et al., 2004; Finlay-Doney & Walter,
2005; Szafranek et al., 2005). Moreover, numerous insect
species respond electrophysiologically to β-caryophyllene
(e.g., Soares et al., 2003; Asaro et al., 2004; Wei & Kang,
2006). Some herbivores are attracted behaviourally to
162
Fernandez et al.
Figure 1 Dose-dependent responses (mean + SE) of male (white bars) and female (black bars) Phratora vulgatissima antennae to synthetic
plant volatiles in electroantennographic experiments. The responses for all doses were reduced by the mean response to equivalent
amounts of the solvent hexane. Absolute (non-normalized data) are shown. Repeated measures analysis of variance (ANOVA) was
performed. In all cases, only the factor dose was significant at P<0.0001. Different letters indicate significant differences (P<0.05,
Newman–Keuls comparisons).
blends enriched in β-caryophyllene. For example, in
Colorado potato beetles, the most significant attraction
was achieved to potato plants when β-caryophyllene was
added (Schütz et al., 1997). In field traps for western
corn rootworms, β-caryophyllene, which was only weakly
attractive by itself, synergistically raised the number of
female captures when added to a linalool–methyl salicylate
blend (Hammack, 2001). These examples indicate an
important role of β-caryophyllene in host-plant recognition
by herbivorous insects.
Females of P. vulgatissima, but not males, moderately
responded to R-(+)-limonene, which might suggest a
sexual role of this component. Interestingly, Tiberi et al.
(1999) demonstrated that R-(+)-limonene deters
oviposition by pine processionary caterpillar females, even
more effectively than the naturally occurring enantiomer
S-(-)-limonene.
This work represents a further step to identify substances
relevant for the orientation of a willow leaf beetle to
host plants. Our finding of the positive EAG response
of P. vulgatissima to GLVs and terpenoids released by a
preferred willow species will need future behavioural
studies to further elucidate the role of these volatiles for
host-plant finding in this leaf beetle species.
Acknowledgements
Many thanks are due to Frank Mueller for his assistance in
volatile collection and GC-MS. We are also grateful to
Zainulabedin Syed and Nina Stahl, who helped with the
EAGs, and Fernando Locatelli, who helped to set up the
plants in the chamber for volatile collection. Patricia C.
Fernandez was supported by Fundación Antorchas,
Argentina. All authors thank the two anonymous reviewers
for their highly valuable comments that contributed a lot
to improve and focus our manuscript.
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