![Mean number ( ± SEM) of P. pubescens males (black bars) and females (grey bars) caught in traps containing 2.5 µL of (+)-trans-pityol [(+)tP)] or ( ± )-trans-pityol [( ± ) tP] and different release rates of (E, E)-α-farnesene (A), (R)-(+)-limonene (B) and (S)-(−)-verbenone (C). Means with the same letter are not significantly different at P < 0.05 (Tukey's multiple comparison test).](https://www.researchgate.net/profile/Angel-Guerrero-6/publication/228325001/figure/fig1/AS:267495118471223@1440787214767/Mean-number-SEM-of-P-pubescens-males-black-bars-and-females-grey-bars-caught-in_Q320.jpg)
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40
Research Article
Received: 22 September 2011 Revised: 14 March 2012 Accepted article published: 10 May 2012 Published online in Wiley Online Library: 5 July 2012
(wileyonlinelibrary.com) DOI 10.1002/ps.3359
Electrophysiological and behavioural
responses of Pityophthorus pubescens
(Coleoptera: Scolytinae) to (E,E)-α-farnesene,
(R)-(+)-limonene and (S)-(−)-verbenone in
Pinus radiata (Pinaceae) stands in northern
Spain
Sergio L ´
opez,aCarmen Quero,bJuan Carlos Iturrondobeitia,c
´
Angel Guerreroband Arturo Goldarazenaa∗
Abstract
BACKGROUND: Some twig beetles in the genus Pityophthorus (Coleoptera: Scolytinae) may vector pitch canker disease Fusarium
circinatum (Niremberg & O’Donnell) of Pinus spp. (Pinaceae). Because Pityophthorus pubescens (Marsh.) has been found to be
associated with F. circinatum in the Basque Country (northern Spain), various experiments were conducted to assess the beetle’s
behavioural responses to (E,E)-α-farnesene, (R)-(+)-limonene and (S)-(−)-verbenone to develop a potential inhibitor to host
attraction. These experiments comprise electroantennographic and double-choice olfactometer tests, as well as field assays in
Pinus radiata D. Don stands.
RESULTS: Both sexes of P. pubescens showed similar electroantennographic responses to different doses (from 1 ng to 1 µgin
decadic steps) of each individual compound, with depolarisations to (S)-(-)-verbenone (100 ng) being similar to those of the
aggregation pheromone (+)-trans-pityol. In olfactometer assays, both sexes were significantly attracted to (+)-trans-pityol,
but the attraction was reduced when increasing amounts of the chemicals were added to the pheromone. Particularly relevant
was the repellent effect induced by (S)-(−)-verbenone at 1 ng dose and higher. In the field, (E,E)-α-farnesene, (R)-(+)-limonene
and (S)-(−)-verbenone reduced significantly the number of beetles attracted to (+)-trans-pityol and racemic trans-pityol, with
(S)-(−)-verbenone being the most effective.
CONCLUSIONS: (S)-(−)-Verbenone showed an interesting potential for use in the protection of P. radiata stands. A potentially
effective strategy, which could be implemented in further, more in-depth studies, could involve the use of this semiochemical
as repellent and (+)-trans-pityol-baited traps as attractant in a ‘push-pull’ strategy.
c
2012 Society of Chemical Industry
Keywords: Pityophthorus pubescens; Scolytinae; behaviour; olfaction; inhibitor; management; push-pull strategy
1 INTRODUCTION
Several species of bark beetles (Coleoptera: Scolytinae), including
twig beetles of the genus Pityophthorus Eichh., have been shown
to be phoretically associated with Fusarium circinatum (Niremberg
& O’Donell) (Hypocreales: Nectriaceae), the causal agent of pitch
canker disease. The insects may vector the pathogen or create
wounds through which the pathogen can enter the tree.1–4Beetles
of the Pityophthorus spp. complex rarely cause mortality of trees,
but attack weakened or dying trees, where they breed under the
bark or in the pith of small twigs.5Indeed, Pityophthorus carmeli
Swaine, P. setosus Black. and P. nitidulus (Mann.) have been shown
to be associated with pitch canker fungus, and to infest Monterey
pine Pinus radiata D. Don (Pinaceae) in California.2,3 In addition, an
association has recently been reported4between Pityophthorus
pubescens (Marsh.) and F. circinatum in Monterey pine stands
of this pine species in the Basque Country (northern Spain),
∗Correspondence to: Arturo Goldarazena, NEIKER-TECNALIA, Department of
Plant Production and Protection, Basque Institute of Agricultural Research and
Development, Arkaute, 46, E-01080 Vitoria, Spain.
E-mail: agoldarazena@neiker.net
aNEIKER-TECNALIA, Department of Plant Production and Protection, Basque
Institute of Agricultural Research and Development, Vitoria, Spain
bDepartment of Biological Chemistry and Molecular Modelling, IQAC-CSIC,
Barcelona, Spain
cDepartment of Zoology and Animal Cell Biology, University of Basque Country,
Leioa, Spain
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Response of P. pubescens to various host attraction inhibitors in P. radiata stands www.soci.org
where about 163 000 ha have been planted. The existence of
P. radiata monocultures and the association between P. pubescens
and F. circinatum make the study of the epidemiology of pitch
canker disease and its relationship to P. pubescens, as well as the
development of possible management options, highly desirable.
Knowledge regarding the chemical ecology of beetles of P.
pubescens is limited. Francke et al.6identified (2R,5S)-2-(1-hydroxy-
1-methylethyl)-5-methyltetrahydrofuran [(+)-trans-pityol] and
cis-1-(2-hydroxyethyl)-1-methyl-2-(1-methylethenyl)cyclobutane
[(±) -grandisol] from males of P. pityographus Ratz., and showed
that both compounds were active in the field. (+)-Trans-pityol
was later found in males of P. carmeli and females of P. nitidulus
and P. setosus.7Regarding P. pubescens, trans-pityol has been
recently reported as a component of the aggregation pheromone
of this species.8In addition to trans-pityol, the spiroacetal
(5S,7S)-(−)-7-methyl-1,6-dioxaspiro[4.5]decane (conophthorin)
has been identified as a component of the aggregation
pheromone released by P. carmeli males.7It is well known that
host monoterpenes of conifers, e.g. from Pinus spp. (Pinaceae),
play an important role in the behaviour of many Scolytinae,
including host seeking and selection.9,10 However, unlike most
conifer-attacking scolytids, Pityophthorus spp. appear not to be
attracted to volatiles from cut branches, and it is hypothesised
that they may recognise a suitable host through a process of
random searching and ‘exploratory tasting’.11,12
(4S)-(+)-Limonene [(4S)-4-isopropenyl-1-methyl-cyclohexene,
hereafter (+)-limonene] is considered to be one of the most
toxic monoterpenes for the bark beetle species Dendroctonus bre-
vicomis Lec.,13 D. frontalis Zimm.14 and Scolytus ventralis Lec.,15 and
the (-)-enantiomer inhibits the attraction of Pityophthorus puberu-
lus (Lec.) to racemic trans-pityol.16 However, Miller17 reported later
(in 2007) that both enantiomers act as attractant kairomone in
Conophthorus coniperda (Schwarz).17 As the genus Conophthorus
is closely related to Pityophthorus spp., the present authors con-
sidered it worth testing the effect of (+)-limonene on P. pubescens.
(1S,4S)-(−)-Verbenone {4,6,6-trimethylbicyclo-[3.1.1]hept-3-en-2-
one, hereafter (−)-verbenone}has been shown to inhibit the
response of several species of bark beetles to pheromone-baited
traps and to deter attacks in conifer stands when used alone4,18 – 22
or in combination with non-host volatiles.23 – 25 In addition, in pre-
liminary assays, (−)-verbenone reduced catches of P. pubescens
and Ips sexdentatus (Boern.) but not of other beetles according
to host-age characteristics.4(E,E)- α-Farnesene (3,7,11-trimethyl-
1,3,6,10-dodecatetraene, hereafter α-farnesene) is a sesquiterpene
produced especially when conifers are infested by acarid species of
the genus Nalepella Keifer (Acari: Eriophyoidea: Nalepellidae)26 or
attacked by the white pine weevil Pissodes strobi Peck (Coleoptera:
Curculionidae).27 However, its possible activity on insects has
not yet been demonstrated. Therefore, α-farnesene was included
along with (+)-limonene and (−)-verbenone in this study to
evaluate their potential as possible aggregation inhibitors of P.
pubescens in the laboratory and in the field. This work comple-
ments and enlarges the authors’ previous study on the effects of
(−)-verbenone on bark beetles associated with the pitch canker
fungus, Fusarium circinatum, among them P. pubescens.4
2 MATERIALS AND METHODS
2.1 Insects
For the laboratory bioassays, P. pubescens adults were collected
weekly from infested branches of P. radiata in a mature (nearly
30 years) stand located at Gorosika (43◦16N, 2◦43W) (Basque
Country, northern Spain). After identification, the insects were
maintained in an incubator at 25 ◦C and 65% RH under a 10 :14
L : D photoperiod until their use 2 days later.
2.2 Chemicals
(E,E)-α-Farnesene (67%) in closed polyethylene centrifuge tubes
(release rate 0.8 mg day−1), (R)-(+)-limonene (99%) in polyethy-
lene screw-cap bottles (release rate 5.0 mg day−1)and(S)-(−)-
verbenone (98%) in polyvinyl bubble caps (release rate 2.0 mg
day−1) were purchased from Contech Inc. (Delta British Columbia,
Canada). (+)-Trans-pityol (99%) and the racemic trans-pityol (99%)
were kindly provided by Prof. Dr Wittko Francke (University of
Hamburg, Germany). Propylene glycol (99%) was purchased from
Panreac (Barcelona, Spain). Serial dilutions tested in electrophys-
iological and walking bioassays were obtained from all of these
compounds, as described below.
2.3 Electrophysiological assays
Owing to the very small size of the beetle P. pubescens (1.0– 1.2 mm
long),28 the entire EAG preparation and recordings were done
under a stereomicroscope. Recordings were achieved using
Ag–AgCl glass microcapillaries filled with Ringer’s solution. To
hold the microelectrodes, glass micropipettes were pulled from
2.0 mm capillary glass (World Precision Instruments, Sarasota,
FL) on a PE-2 pipette puller (Narishige, Tokyo, Japan), and tips
of the micropipettes were broken to 30 µm diameter. Each
beetle (male or female) was fixed upside down on a double-
sided sticky tape, and the head was excised using a microscalpel.
Subsequently, the recording electrode was put into contact with
the tip of the antenna (ca 0.30 mm long), and the reference
electrode was inserted into the insect club (ca 120 µm long, 95 µm
wide) using MP15 micromanipulators (Syntech, Hilversum, The
Netherlands). Only one antenna of each insect was used. In each
assay, (+)-limonene, (−)-verbenone, and α-farnesene were puffed
individually over the antenna (n=7 for each compound) at four
different doses (from 1 ng to 1 µg in decadic steps). For comparison
purposes, 100 ng of each compound and (+)-trans-pityol were
also applied to a similar filter paper and then ‘puffed’ to male and
female antennae (n=6). The required amounts of the compounds
were obtained by dilution of 10 mg of the commercial compound
[synthetic in the case of (+)-trans-pityol] in 10 mL of hexane,
followed by a log dilution series in hexane to obtain solutions
of every compound at 0.1, 1, 10 and 100 ng µL−1. A quantity of
10 µL of each solution was applied to a round filter paper (2.5 cm
diameter), the solvent was allowed to evaporate and the filter
paper was placed inside a Pasteur pipette with the tip at 2 cm from
the head of the beetle. Each dose was delivered as 400 ms puffs
three consecutive times over the antenna, and two puffs of hexane
were applied just before and after the test compound. The net
electroantennographic responses were calculated by subtracting
the mean response of hexane before and after each stimulus from
the mean response of each dose of the test compound. Stimuli
were delivered in increasing order of doses to avoid saturation
of the antennal receptors. The recovery time for the antenna
between two consecutive stimuli was 1–1.5 min. The EAG system
was enclosed in a Faraday cage (70 ×65 ×60 cm) connected to
the ground to prevent extraneous electrical signals.
2.4 Behavioural response
Walking responses of P. pubescens to α-farnesene, (+)-limonene
and (−)-verbenone were recorded using a Y-tube olfactometer
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opez et al.
adapted to the very small size of the beetle. The main tube was
5 cm long ×5 mm ID, the arms were 4 cm long ×5mmID
and the angle between arms was 90◦. The terminal tubes of the
arms were connected to two glass chambers, which contained
the odour source. One of the arms held 10 µLofhexaneona
2.5 cm diameter filter paper as control. The other arm contained
another filter paper of the same size with 1 ng of (+)-trans-
pityol either alone (reference) or with one of the five different
doses of the test compounds. The doses consisted of 0.01–100 ng
in decadic steps and were obtained from 10 µL solutions of
the required concentrations in hexane. The insect response was
evaluated versus that of 1 ng of (+)-trans-pityol alone. Filter
papers were renewed in each arm after every second insect was
tested. Incoming air was filtered through activated charcoal, and
the airflow was maintained at 70 cm s−1in the olfactometer.
All tests were conducted in a room at 23 ±1◦Cand50±9%
RH, and, before the assays, insects were allowed to acclimatise in
a petri dish for 15 min. Insects were allowed to respond for 5 min;
if there was no response after this time, the insect was discarded.
A response was considered positive if the beetle walked at least
3 cm into one arm. After testing five insects, arms were switched
over to avoid unidirectionality, and, after ten insects, the entire
olfactometer was washed, first with soap and water, then with
absolute ethanol, and left to dry. A total of 35–40 insects were
used for each sex and dose.
2.5 Field assays
During May–June 2007, 2008 and 2009, three field experiments
were conducted to evaluate the effect of the terpenoids as
potential disruptants of P. pubescens aggregation. Only a single
compound at five different release rates was tested each year,
as follows: α-farnesene in 2007, (+)-limonene in 2008 and (−)-
verbenone in 2009. Field assays were implemented based on
previous research conducted to find the flight period of the
species in the Basque Country.4,29,30 The experiments were done
in four mature Monterey pine stands at the following localities:
Llodio (43◦08N, 2◦59W), Amurrio (43◦03N, 2◦57W), Gorosika
(43◦16N, 2◦43W) and Olabarrieta (43◦01N, 2◦22W). At
each locality, two blocks (with two replicates each) of six 12-unit
Lindgren multiple-funnel traps were set out (Table 1). One trap in
each block, baited only with the attractant [either with (+)-trans-
pityol or racemic trans-pityol], was considered as positive control,
whereas the other traps were baited with attractant plus the test
compound at different release rates (0.8– 4.0 mg day−1for α-
farnesene, 5.0 – 25.0 mg day−1for (+)-limonene and 2.0–10.0 mg
day−1for (−)-verbenone) (Table 1). Release rates higher than
the initial 0.8, 5.0 and 2.0 mg day−1for the three compounds,
respectively, were obtained by adding new commercial devices as
needed; for instance, to achieved a release rate of 15.0 mg day−1
of (+)-limonene, 3 ×5.0mgday
−1of polyethylene bottles was
required. Traps were spaced 20 m apart in a 3 ×2gridwithineach
block, and the distance between blocks was greater than 100 m.
Each trap was hung between two adjacent trees 2 m above ground.
Because traps were not rerandomised during the trapping period,
all traps were placed unbaited in the field 2 weeks before the assay
to discard the hypothesis that the possibly obtained catches would
be due to a spatial effect of traps. Control attractants (2.5µLof
racemic and chiral trans-pityol) were dispensed in 1.5 mL capped
plastic microcentrifuge vials that were attached to the outside of
the middle funnel. After sealing them, the caps were pierced with
a pin to allow evaporation of the chemical. The vials were refilled
every week. The dispensers of α-farnesene allowed a release rate
of 0.8 mg day−1,(+)-limonene was dispensed at 5.0 mg day−1
and (−)-verbenone was dispensed at 2.0 mg day−1. Propylene
glycol was added to each trap-containing jar to preserve captured
insects. The experiments lasted 2 months.
Insects were collected weekly and identified on the basis of their
characteristic anatomical features of the elytra and elytral declivity
using a LEICA MZ9.5 stereomicroscope (Leica Microsystems GMBh,
Wetzlar, Germany).28,31 Insects were separated by sex on the
basis of the yellowish hairs on the frons, an exclusive feature of
females. For identification purposes, reference specimens were
obtained from the British Natural History Museum (London, UK)
and the Museo Nacional de Ciencias Naturales (Madrid, Spain).
Voucher specimens have been deposited at the Entomology
Collection of the NEIKER-Basque Institute of Agricultural Research
and Development (Basque Country, Spain).
Table 1. Parameters for each field experiment performed in 2007– 2009
Experiment Date BlockaAttractant Volume (µL) Tested compound Release rate (mg day−1) of test compound
1 1 May– 30 June 2007 1 (+)-Trans-pityol 2.5 (E,E)-α-Farnesene 0 (control)
0.8– 4b
2(±)-Trans-pityol 2.5 (E,E)-α-Farnesene 0 (control)
0.8– 4b
2 1 May– 30 June 2008 1 (+)-Trans-pityol 2.5 (R)-(+)-Limonene 0 (control)
5–25c
2(±)-Trans-pityol 2.5 (R)-(+)-Limonene 0 (control)
5–25c
3 1 May– 30 June 2009 1 (+)-Trans-pityol 2.5 (S)-(−)-Verbenone 0 (control)
2–10d
2(±)-Trans-pityol 2.5 (S)-(−)-Verbenone 0 (control)
2–10d
a In duplicate experiments.
bIn dispensers releasing 0.8, 1.6, 2.4, 3.2 and 4 mg day−1.
cIn dispensers releasing 5, 10, 15, 20 and 25 mg day−1.
dIn dispensers releasing 2, 4, 6, 8 and 10 mg day−1.
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Response of P. pubescens to various host attraction inhibitors in P. radiata stands www.soci.org
2.6 Statistical analysis
Electroantennographic responses and field catches were analysed
by analysis of variance (ANOVA) at a significance level of
α=0.05, followed by Tukey’s post hoc test for separation
of means (SPSS v.13.0 for Windows). For field trapping assays,
several transformations (ln, square root) of trap catches and the
independent variable were tested, as required from examinations
of residuals, to correct heteroscedasticity and non-linearity. In
olfactometer tests, the results were analysed by the chi-square
test (χ2) at a significance level of α=0.05. Males and females
showed no preference for either olfactometer arm (a response
equal to 50 : 50) in the absence of any stimuli.
3 RESULTS
3.1 Electroantennographic assays
Antennae of both sexes of P. pubescens responded slightly to all
doses of the three compounds tested, although no significant
differences were found between different concentrations within
sex and compound (α-farnesene: males F3,28 =1.119, P=0.360;
females F3,28 =0.581, P=0.633; (+)-limonene: males F3,28 =
0.772,P=0.772; females F3,28 =0.053,P=0.984; (−)-verbenone:
males F3,28 =0.474, P=0.860; females F3,28 =0.474, P=0.860)
(Fig. 1). Depolarisations elicited by 100 ng of (+)-trans-pityol were
higher than those induced by the other compounds in males
and females, the differences being significantly different to those
elicited by (+)-limonene (males F3,24 =0.642, P=0.772; females
F3,24 =0.053, P=0.984) and α-farnesene (males F3,24 =11.683,
P<0.001; females F3,24 =12.391, P<0.001) (Fig. 2). (−)-
Verbenone elicited an intermediate response, not significant
relative to (+)-trans-pityol or the other two semiochemicals.
3.2 Behavioural response
As expected, males and females were significantly attracted to
1ngof(+)-trans-pityol when tested alone. However, attraction of
both sexes towards the (+)-trans pityol arm was reduced when
increasing amounts of α-farnesene were added to the pheromone
(Fig. 3). Neither males nor females walked preferentially to any arm
in the presence of mixtures (+)-trans-pityol:α-farnesene 1 : 1 and
higher. A similar effect was observed with (+)-limonene. Males
and females still preferred the arm containing (+)-trans-pityol plus
(+)-limonene when the doses of the monoterpene were 0.1 ng or
lower, but higher doses (1–10 ng) reduced the attraction to the
synthetic attractant (Fig. 4). The effect of (−)-verbenone was more
notable. Attraction of males to the arm containing 0.01–0.1 ng of
(−)-verbenone became practically null, and insects significantly
preferred the control arm when the amount of the chemical at the
attraction source was 1 ng and higher (Fig. 5). Females behaved
similarly, but preference for the control arm was already noticed
at0.1ngof(−)-verbenone.
3.3 Field assays
In 2007, a total of 50 212 beetles (39 903 males and 10 309 females)
were caught. α-Farnesene significantly inhibited the attraction of
P. pubescens to (+)-trans-pityol (males F5,186 =2.689, P=0.023;
females F5,186 =2.651, P=0.024) and (±)-trans-pityol (males
F5,186 =2.354, P=0.042; females F5,186 =2.727, P=0.021)
(Fig. 6A). Catches of both sexes were significantly lower in traps
baited with (+)-trans-pityol plus α-farnesene at a release rate of
2.4 mg day−1and higher. With (±)-trans-pityol, the effect of α-
farnesene was more pronounced, 1.6 mg day−1being sufficient to
Figure 1. Absolute net EAG (mV ±SEM) responses of P. pubescens males
(black bars) and females (grey bars) to different doses (1 ng to 1 µgin
decadic steps) of (E,E)-α-farnesene (A), (R)-(+)-limonene (B) and (S)-(−)-
verbenone (C). The compounds were deposited on a filter paper and
puffed. Bar means followed by different letters within sex are significantly
different by ANOVA followed by Tukey’s multiple test (α=0.05).
promote partial but significant inhibition of captures. Maximum
reduction in male catches relative to control traps containing
pheromone alone was 44.8 and 51.4% for the (+)-enantiomer
and (±)-trans-pityol respectively, whereas female catches were
reduced by 49.7 and 58% respectively (Fig. 6A).
In experiment 2, 16 371 beetles (11 765 males and 4606 females)
were trapped. (+)-Limonene significantly reduced the number of
catches to (+)-trans-pityol when released at 10 mg day−1(males
F5,186 =8.909, P<0.001; females F5,186 =5.716, P<0.001) and
higherand to (±)-trans-pityolwhen released at15 mg day−1(males
F5,186 =5.538, P<0.001; females F5,186 =2.775, P=0.018) and
higher (Fig. 6B). The maximum reduction in catches for both sexes
was 68% with regard to the (+)-trans-pityol-baited traps alone and
59% relative to the (±)-trans-pityol-containing traps.
In 2009, 22 159 P. pubescens (18 706 males and 3453 females)
were caught. (−)-Verbenone also significantly inhibited attraction
of beetles to traps baited with chiral trans-pityol when released
at 2 mg day−1(males F5,186 =9.716, P<0.001; females
F5,186 =7.620, P<0.001) or racemic trans-pityol when released
at the same dose (males F5,186 =10.562, P<0.001; females
F5,186 =8.313, P<0.001) (Fig. 6C). Higher doses did not further
increase the inhibitory effect. The maximum decrease in catches
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opez et al.
Figure 2. Absolute net EAG (mV ±SEM) responses of P. pubescens
males and females to 100 ng of (±)-trans-pityol, (E,E)-α-farnesene, (R)-
(+)-limonene and (S)-(−)-verbenone. The compounds were deposited on
a filter paper and puffed. Bar means followed by different letters within
sex are significantly different by ANOVA followed by Tukey’s multiple test
(α=0.05). Legend: (±)tP=(±)-trans-pityol; αF=(E,E)-α-farnesene; (+)L
=(R)-(+)-limonene; (−)V =(S)-(−)-verbenone.
was 66.6% in males and 75% in females at 10 mg day−1of (−)-
verbenone in racemic and chiral pheromone traps respectively
(Fig. 6C).
4 DISCUSSION
Electroantennography is a useful technique for isolating volatiles
that insects use to locate suitable host plants.32 – 34 In the
first electroantennographic recordings performed on the genus
Pityophthorus, α-farnesene, (+)-limonene and (−)-verbenone
elicited slight antennal responses in both sexes of P. pubescens
(Figs 1 and 2), in line with the small number of olfactory sensilla
(300–800 antenna−1in scolytids)35 with regard to Lepidoptera
[105sensilla in Manduca sexta (L.)].36 Slight responses have
also been previously reported in other Scolytinae species, e.g.
the depolarisations elicited on Tomicus destruens (Woll.) by
different pine shoot and bark volatiles ranged from 0.06 to
0.12 mV.37 In the present case, the depolarisations were smaller
than those elicited by racemic trans-pityol, one component
of the aggregation pheromone, although with regard to (−)-
verbenone the differences were not significant at 100 ng dose.
Females responded better than males, but the difference was
not significant, and neither sex responded in a dose-dependent
fashion, in contrast to the electrophysiological responses of female
moths to host odours.38
In dual-choice bioassays, α-farnesene, (+)-limonene and (−)-
verbenone inhibit the attraction of pityol (racemic or chiral) to
males and females. The effect of (−)-verbenone is particularly
noticeable, with doses of 1% relative to (+)-trans-pityol being
sufficient to abrogate the attraction of the synthetic attractant to
either sex. Moreover, doses of 10% and higher of (−)-verbenone
completely switch attraction of females to the control arm. On
males, the repulsive effect is evoked when placing the same
amount of the inhibitor [1 ng of (−)-verbenone with 1 ng of chiral
pityol] in the corresponding arm.
Field experiments confirmed the results obtained in the
laboratory bioassays. The three terpenoid compounds caused
a reduction in the number of P. pubescens caught, with (−)-
verbenone and (+)-limonene having a stronger effect. Previous
research has demonstrated that (−)-limonene inhibited the
attraction of P. puberulus to traps baited with (−)-α-pinene,
(−)-β-pinene or mixtures of both.16 The inhibitory effect of (−)-
Figure 3. Response of P. pubescens males ( ) and females ( )to
different mixtures of (E,E)-α-farnesene and 1 ng of (+)-trans-pityol in Y-
tube olfactometer trials. Hexane was used as control. One and two asterisks
indicate significant differences at P<0.05 and P<0.01 (chi-square test)
respectively. Number in parentheses represents number of beetles not
responding. Legend: (+)tP =(+)-trans-pityol; αF=(E,E)-α-farnesene.
limonene was not evident, however, when racemic trans-pityol
was added to various mixtures of monoterpenes, except when
the pheromone was combined with (−)-α-pinene. The inhibition
followed a dose-dependent trend.16 Because (−)-α-pinene and
(−)-β-pinene appear to be attractive to P. puberulus, the inhibition
elicited by (−)-limonene suggests that this species is able to
discriminate among terpenes to locate suitable hosts. It should be
noted that the (−)-limonene release rates used by Brauner and
De Groot16 (34–234 mg day−1) were considerably higher than the
rates used in the present tests with (+)-limonene. In the present
case, there was no need of (−)-α-pinene for the (+)-enantiomer
to elicit a disruptant effect of the pheromone action.
(−)-Verbenone shows promise in reducing P. pubescens aggre-
gation in the field. The present results partially agree with those
of a previous study in which a negative linear relationship be-
tween (−)-verbenone dosage and the number of insects caught
was found.4However, higher release rates were tested than those
used by Rom ´
on et al.4(2 –10 mg day−1versus 0.01 – 3.1 mg day−1),
and it is known that different release rates may greatly influence the
level of catches.21 This is the case, for instance, with Dendroctonus
ponderosae Hop., catches of which were unaffected relative to the
control [no (−)-verbenone] at release rates of 0.2 mg day−1or less,
but were significantly reduced at 1.8 mg day−1of (−)-verbenone or
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2012 Society of Chemical Industry Pest Manag Sci 2013; 69:40–47
45
Response of P. pubescens to various host attraction inhibitors in P. radiata stands www.soci.org
Figure 4. Response of P. pubescens males ( ) and females ( )to
different mixtures of (R)-(+)-limonene and 1 ng of (+)-trans-pityol in Y-
tube olfactometer trials. Hexane was used as control. One and two asterisks
indicate significant differences at P<0.05 and P<0.01 (chi-square test)
respectively. Number in parentheses represents number of beetles not
responding. Legend: (+)tP =(trans)-(+)-pityol; (+)L =(R)-(+)-limonene.
higher.21 In the present case, the results suggest a threshold-type
response rather than a dose-dependent relationship,4as there are
no significant differences in catches among traps when baited
with (−)-verbenone at any release rate tested. If it is assumed
that (−)-verbenone is a good indicator of the nutritive quality of
plant material owing to its generation via microbial activity,17,39
this chemical could be expected to have a strong inhibitory ef-
fect on species primarily feeding on fresh host tissue,21 whereas
species inhabiting aged tissues would have higher tolerance to
(−)-verbenone and/or a more efficient detoxifying system. In light
of the present results, it can be hypothesised that P. pubescens
might avoid colonisation of hosts with aged tissue, as previously
suggested for other bark beetles, such as D. ponderosae Ips pini
(Say) and I. latidens (Lec.), but not Hylurgops porosus (Lec.) and
Hylastes longicollis Swaine, two species that usually feed on aged
phloem tissue below or at ground level.21 (−)-Verbenone effec-
tively reduced catches of D. ponderosae in a threshold-type pattern
and catches of Ips spp. in a negative dose-dependent relationship,
but did not affect catches of the two latter species.21
On the other hand, although (−)-verbenone is an antiaggre-
gation pheromone for a number of scolytid species,10 it did
not reduce catches of the white pine cone beetle Conophtho-
Figure 5. Response of P. pubescens males ( ) and females ( )to
different mixtures of (S)-(−)-verbenone and 1 ng of (+)-trans-pityol in Y-
tube olfactometer trials. Hexane was used as control. One and two asterisks
indicate significant differences at P<0.05 and P<0.01 (chi-square test)
respectively. Number in parentheses represents number of beetles not
responding. Legend: (+)tP =(+)-trans-pityol; (−)V =(S)-(−)-verbenone.
rus coniperda when added at 0.6 mg day−1to a (±)-trans-pityol
(0.2 mg day−1)andα-pinene (12 mg day−1) attractive blend.40
α-Farnesene appears to be the least disruptive of the three
compounds tested. In spite of the fact that the three highest
release rates used caused a reduction of almost 50% in catches, it
is unclear whether the use of even higher release rates would have
a stronger repellent effect, or whether its disruptive potential is
limited, as indicated by the present results.
5 CONCLUSIONS
The results provide basic knowledge for the possible development
of IPM strategies to protect P. radiata stands from pitch canker
disease. A ‘push-pull’ strategy is proposed as potentially effective
against P. pubescens,using(−)-verbenone as a repellent (‘push
tactic’) within a landscape, and either (±)-trans-pityol- or (+)-
trans-pityol-baited traps (‘pull tactic’) on the perimeter to capture
repelled beetles. Future research could be focused on testing
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2012 Society of Chemical Industry wileyonlinelibrary.com/journal/ps
46
www.soci.org S L ´
opez et al.
Figure 6. Mean number (±SEM) of P. pubescens males (black bars) and females (grey bars) caught in traps containing 2.5µLof(+)-trans-pityol [(+)tP)]
or (±)-trans-pityol [(±) tP] and different release rates of (E,E)-α-farnesene (A), (R)-(+)-limonene (B) and (S)-(−)-verbenone (C). Means with the same letter
are not significantly different at P<0.05 (Tukey’s multiple comparison test).
this strategy in greater depth, as already proposed against
D. ponderosae.41
ACKNOWLEDGEMENTS
Thanks are due to the Department of Education, Universities and
Research of the Basque Country, for granting a PhD fellowship to
Sergio L ´
opez, and to the Department of Agriculture and Fisheries
of the Basque Country for financial support. Thanks also to the
members of NEIKER, Biscay Government, and of the Chemical
Ecology Unit (IQAC-CSIC) for the time and technical assistance
dedicated to this work. Prof. Dr Wittko Francke (Institute of Organic
Chemistry, University of Hamburg, Germany) kindly provided pure
chiral and racemic trans-pityol. The authors are very grateful to
Prof. Dr Lawrence Mound and Prof. Dr B Staffan Lindgren for
critical comments that improved significantly an early version of
this paper.
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