El-Sayed, A.M. and Byers, J.A. 2000.
INHIBITORY EFFECT OF MONOTERPENES ON RESPONSE OF Pityogenes
bidentatus TO AGGREGATION PHEROMONE RELEASED BY PIEZOELECTRIC SPRAYER
FOR PRECISION RELEASE OF SEMIOCHEMICALS Journal of Chemical Ecology
Rotating trap pair in background, collection of host or
nonhost volatiles on Porapak Q in foreground, in Scotch pine forest near Sjöbo,
Abstract-- A piezoelectric sprayer for
dispensing semiochemicals was developed and used for a field test of bark beetle
semiochemicals. The sprayer consists of a geared pump that pushes a syringe
slowly to dispense semiochemicals in solvents through a microtube to a glass
micropipet fixed to a piezoelectric high-frequency vibrator. The frequency is
adjusted via a function generator to about 120 kHz until the harmonic properties
of the glass micropipet, drawn by an electrophysiological pipette puller, cause
vibrations that atomize the solvent from the micropipet tip. The sprayer,
syringe, pump, function generator, and power supply were hung on one arm of a
rotating trap pair (traps 6 m apart) that was slowly rotated at 2 revolutions
per hour (rph) to even out the position effects on trap catches. The aggregation
pheromone components of Pityogenes bidentatus, grandisol and
cis-verbenol, were released by standard tube dispensers in one trap and
compared to the release of similar amounts by the sprayer in the other trap. No
significant differences in catch were observed. No effect of the solvent hexane
on aggregation could be observed. The trap pair also caught approximately equal
numbers of bark beetles when the baits were identical. The release of (+)- and
(-)-alpha-pinene, (+)-3-carene, and terpinolene, monoterpenes of host Scotch
pine, Pinus sylvestris, at increasing rates from 0.01 to 10
log-equivalents in decadic steps (each at 0.1-100 µg/min) resulted in decreasing
responses to aggregation pheromone (only 9% at highest rate). Inhibition by the
individual monoterpenes tested at the 100 µg/min rate was significant for (+)-
and (-)-alpha-pinene and terpinolene (12, 13, and 15% of control, respectively).
The inhibition by the host Scotch pine monoterpenes may allow P.
bidentatus to avoid resistant trees that release large amounts of toxic
monoterpenes in their resin and instead colonize dying and diseased limbs or
slash, the usual host substrate. The piezoelectric sprayer should prove
generally useful to dispense precise nmounts of semiochcmicals in field and
Key Words--Host selection, dispenser, release
rates, Coleoptera, Scolytidae, Pityogenes bidentatus, Pinus
sylvestris, Scotch pine, conifers.
Bark beetles (Coleoptera:
Scolytidae) attack trees by boring through the bark and tunneling in the phloem
and cambium layers surrounding the sapwood. Conifers such as pines and spruce
usually produce copius amounts of resin to defend against the penetration by
bark beetles (Raffa and Berryman, 1987). These resins have long been known to
contain monoterpenes with toxic properties as well as being viscous and sticky.
causing entrapment and suffocation of beetles (Webb,1906; Smith, 1961, 1965;
Hodges et al., 1979; Raffa and Berryman, 1982, 1987; Byers, 1989; Werner, 1995;
Klepzig et al., 1996). Therefore, it could be expected that bark beetles have
evolved olfactory mechanisms and behaviors for the avoidance of specific
volatile monoterpenes in tree resins. Pitman et al. (1966) reported that gas
chromatographic effluents of frass from male, pine bark beetles, Ips
paraconfusus Lanier, containing the monoterpenes alpha-pinene, myrcene,
(B-pinene, 3-carene, and limonene, elicited "strong negative klinotactic
movements" by walking beetles. However, most other earlier studies found that
certain monoterpenes enhance the attraction to pheromone components in some of
the most "aggressive" bark beetles that kill living trees (Bedard et al., 1969;
Werner, 1972; Rudinsky et al.. 1972).
The role of monoterpenes in the
ecology of bark beetles is further complicated since some host monoterpenes
(alpha-pinene and myrcene) have been implicated or proven as precursors of
aggregation pheromone components of several bark beetle species (Hughes, 1974;
Renwick et al., 1976; Hendry et al., 1980; Klimetzek and Francke, 1980; Byers,
1981, 1989). However. more recent studies have indicated that the pheromone
components that can be synthesized by the beetles from myrcene, e.g., ipsenol,
ipsdienol, and (E)-myrcenol, are mostly made de novo (Byers and
Birgersson, 1990; Ivarsson et al., 1993; Seybold et al., 1995). In addition,
monoterpenes appear to aid in host selection since certain host monoterpenes
increase the proportion of beetles entering holes or attractive to flying
beetles (Byers et al., 1985, 1988; Phillips et al., 1988; Byers, 1989, 1992).
The roles of host monoterpenes in the chemical ecology of even the most studied
bark beetle pests are not fully understood, while in many other species little
Progress in elucidating the functions and interactions of
insect semiochemicals has been hindered by the lack of a dispenser that can be
easily adjusted to release semiochemicals at practically any rate from among the
wide range of rates desired in the laboratory and field. The first objective of
this study was to modify the piezoelectric sprayer designed for laboratory wind
tunnels (El-Sayed et al., 1999a, b) to be portable for field use. The second
objective was to release exact amounts of aggregation pheromone components of
the bark beetle P. bidentatus from the sprayer in a rotating trap pair
and compare the catches to similar releases of the neat components. In addition,
the release of host Scotch pine P. sylvesfris monoterpenes at various rates
would indicate whether they increased or decreased attraction to pheromone
components of P. bidentatus when compared to baits with only the neat
METHODS AND MATERIALS
Piezoelectric Sprayer for Field Use. A custom-made gear
pump delivers a specific amount of semiochemicals through a microtube (0.12 mm
ID, CMA/ 100, Carnegie Medicine AB, Stockholm, Sweden) to a glass capillary of
1.4 mm OD and 0.62 mm ID (ABS, Zurich, Switzerland), which were drawn out and
broken to tip diameters of about 55 um OD and 40 um ID. The capillary tube was
fixed to a piezo disk (Type Nr 4322020, Valvo, Hamburg, Germany) of 10-25 mm OD
and ca. 1-2 mm thickness by a U-shaped wire. The piezo disk was driven at its
vibration mode resonance by a sine or square wave of about 12 V peak to peak
(see below). The U-shaped wire clip transfers the oscillations from the piezo
disk to the microtubing and the glass capillary tip that oscillate at about 120
kHz. This produces an aerosol of the semiochemical solution that disperses and
immediately evaporates. Due to their small size, the droplets evaporate
completely within a small distance from the capillary tip. Smooth tips that are
created by a micropipet pulling and cutting device tend to release one droplet
at each half-oscillation. Normally we used tips that were pulled manually with
the ignition flame (disposable micropipets from ABS). This yielded an irregular
tip that tended to release one droplet at only one of the extreme positions of
the oscillating tip. For example, a flow of 1 ml/hr is dispersed into droplets
of ca. 4.3 pl (corresponding to a droplet diameter of ca. 25 um) that are
sprayed into the ambient air. A sprayer kit was adapted for field use by using a
hand-wired circuit for generating sine or square waveforms and a portable
syringe pump with low power consumption.
The sine or the square waveform signals used to drive the piezo disk were taken
from hand-wired circuits (Figure 1).
FIG. 1. Circuit used to generate a sine or square waveform with minimum harmonic
distortion for driving the piezoelectric disk.
The main element of the circuit is a frequency-tunable oscillator chip XR-2206
monolithic IC (Exar Corp.). This circuit provides two basic waveforms: sine and
square waves. There are four overlapping ranges of 100 Hz to 200 kHz, and the
desired range was obtained by changing the capacitor (C) connected between pins
5 and 6 or by changing the value of resistor (R1). In this set-up. the frequency
is inversely proportion to the value of the capacitor connected between pins 5
and 6, (F) = l/RC, where C is the capacitance in farads, and R = R1 + R2 in
ohms. C was set to 0.001 uF in our design. which produces a frequency range of
90-160 kHz. The amplitude of the waveform output can be varied from 0 to 12 V
(peak to peakj. The distortion in the output waveform was minimized by changing
the value of R3 and R4 and observing the sinusoidal waveform in an oscilloscope.
This circuit is desiUned to operate with a power supply of 15 mA at 12 V DC,
which makes it ideal for battery-based field work due to its lower power
Syringe Pump. We constructed a
bear syringe pump to deliver the semiochemicals in this study (Figure 2).
FIG 2. Schematic diagram of a portable gear pump with low power
consumption. The pump was used to deliver a specific amount of the aggregation
pheromone of P. bidentatus or host Scotch pine monoterpenes via
microtubing to a glass capillary tube fixed to a piezoelectric disk.
The syringe pump was actuated by means of a 12-V DC gearmotor with a low power
consumption (10 rpm, 40 mA; Japan Servo Co. Ltd., No. 4301). The motor armature
is indirectly connected to a piston via two gears and a threaded-screw guide.
The motor armature rotates gear 1 (2 cm OD, Gl) at 10 rpm. The rotation of axial
motion is translated from gear 1 to gear 2 (4.5 cm OD, G2) which is tightly
fixed in the threaded-screw guide (25 cm long). This rotation is converted to
linear motion through a piston soldered between two nuts attached along the
guide (Figure 2). As the motor armature turns, the threaded guide slides,
depending upon the direction of rotation, forward or backward and displaces the
syringe piston causing fluid to move through the microtubing to the glass
capillary tube. The flow rate of the fluid is determined by: (1) the speed of
the motor, (2) the diameter of the syringe, and (3) the ratio of G1:G2. In our
set-up, (1) and (2) were constant; accordingly, the rate of the displaced fluid
is determined by the ratio of G1 to G2 and is inversely proportional to the
diameter of G2 and directly proportional to G1. The release rate of odorant was
controlled by changing the concentration of the semiochemicals in solution or by
changing the ratio of G1 to G2. In all experiments, the pump was set to deliver
approximately 10 ± 3% µl/min by a gas-tight 1-ml syringe (Hamilton Bonaduz AG).
Leakage and contamination are prevented by using tubing adapters (CMA/l00) that
connect the microtubing to the syringe tip and glass micropipet. One milliliter
of semiochemicals when expelled at a rate of 10 µl/min typically took about 100
Release of Semiochemicals in the Field. Test
solutions of semiochemicals were prepared by dissolving the appropriate
quantities of the synthetic semiochemicals in HPLC grade hexane. Solutions were
pressed from a 1-ml syringe at 10 µl/min, through a microtube 1 m long, to the
glass capillary tube. The glass micropipet was fixed and hung centered in one
trap of a rotor trap pair (Figure 3).
FIG. 3. Photograph of the active sprayer installed in a rotating trap for
investigating the effect of Scotch pine monoterpenes on the attraction of P.
bidentatus to its aggregation pheromone components in the field. (A) tubing,
(B) capillary tube, (C) piezo disk, (D) aerosol of solvent containing
semiochemicals, (E) standard polyethylene and glass dispenser tubes (F), and
edge of plastic funnel in background.
The traps in a pair were kept 6 m apart by two tubular-steel poles horizontally
suspended by guy-wires from an upright center pole slowly rotated at 2
revolutions per hour (rph) by a 12-V regulated gearmotor (Byers et al., 1990,
1998). Each trap consisted of two panes of polycarbonate plastic (20 cm high x
32 cm wide) forming a cross-barrier trap. Wire from the cross-barrier suspended
(15 cm below) a 32-cm-diem. plastic collecting funnel and bottle. The micropipet
and piezoelectric vibrator, as well as the glass/plastic tubes with neat
semiochemicals, were centered between the barrier trap and collecting funnel by
a 1-mm wire (Figure 3).
Tests were performed to determine possible
effects of hexane solvent, equality of trap pairs, and the relative attraction
rates of beetles to components released by the sprayer versus the standard tube
dispenser. (4S)-(-)-cis-Verbenol (99%, Borregaard) and grandisol,
(1R,2S)-2-propenyl-1-methyl-cyclobutaneethanol (>98%, from G. Birgersson),
both pheromone components of P. bidentatus, served as the attractive
baits. In the standard dispensers, cis-verbenol was placed as a powder to
cover the bottom of a 30-mm-long polyethylene tube (6 mm ID) while about 20 µl
of grandisol was placed neat in the bottom of a 32-mm-long glass tube (3.5 mm
ID). The release rates were estimated at 20°C to be 500 µg/day for
cis-verbenol and 100 µg/day for grandisol. For the sprayer, the pheromone
component test solution contained 17.5 ng each of grandisol and
cis-verbenol per microliter hexane solvent. Since the pump and sprayer
released about 10 µl/min, this was about 175 ng of each component per min or
about half the rate for cis-verbenol and 2.5 times the rate for grandisol
from the standard dispensers. However, in the trap catch comparison of the
sprayer versus the standard dispensers, two such standard dispensers were used,
so that the sprayer released 25% of the cis-verbenol and equivalent
amounts of grandisol as the standard dispensers.
In the second series of
tests to determine the effects of monoterpenes on attraction responses, only one
tube for each pheromone component was used in each trap of the pair. One of
these traps also used the sprayer to release a mixture of Scotch pine
monoterpenes (-)-alpha-pinene ([a]20D = -50°, >99.5% pure, Fluka),
(+)-alpha-pinene ([a]20D = 46.5°, >99%, Aldrich), (+)-3-carene
([a]20D = 17°, >99%, Fluka), and terpinolene (>97.3%, Carl
Roth). The concentrations of each monoterpene in the mixtures ranged in decadic
steps, 0.01, 0.1, 1, and 10 µg/µl, again released at 10 µl solution/min (or 14.4
mg of each monoterpene/day) from the sprayer (Figure 4). These release rates are
similar to what freshly cut Scotch pine logs (30 cm long x 15 cm diem.) emit at
0.01, 0.1, 1, and 10 log equivalents, respectively. The quantities of
alpha-pinene and 3-carene from Scotch pine logs were mistakenly reported in
Byers et al. (1985) as 13 or 14 µg/hr; they should have been micrograms per
minute to give the measured amounts (20 mg/day). The monoterpenes also were
tested for inhibition individually at the 10 log equivalent rate. Usually, one
test was performed for a given semiochemical comparison, with a test usually
conducted from 30 min to 1 hr or until the sprayer syringe was spent (ca. 100
min). Because of the continuous trap rotation, the population density of flying
beetles is expected to be homogeneous for both treatments. Thus, the paired
control and treatment were compared with a chi-square goodness of fit test to an
expected catch if there were no differences based on the average for both traps.
FIG. 4. Inhibition of P. bidentatus response to pheromone components
(cis-verbenol and grandisol) by increasing release rates of a mixture of
Scotch pine monoterpenes [(-)-alpha-pinene. (+)-alpha-pinene. (+)-3-carene, and
terpinolene] each released at 10 µg/min in hexane with the piezoelectric sprayer
(1.0 log-equivalent rate). The pheromone components were released from
glass/plastic tubes in both traps of the pair rotated at 2 revolutions per hour.
Error bars represent 95% binomial confidence limits for the
monoterpene-releasing trap proportion based on the total paired catch of the
rotor traps (chi square).
The piezoelectric sprayer in one
trap and the standard glass/plastic dispensers in the other trap of the rotating
pair, releasing comparable amounts of pheromone components, caught similar
numbers of P. bidentatus (male-female, 34:94 vs. 33:76, respectively, P =
0.22, chi square). The catching ability of both traps in the pair appeared
balanced since placement of standard dispensers in both traps resulted in
similar numbers caught (Figure 5; P = 0.83, not significantly different).
FIG. 5. Reduction of attraction of P. bidentatus to aggregation pheromone
components (cV = cis-verbenol and G1 = grandisol) by host tree
monoterpenes released in hexane by the piezoelectric sprayer in a trap rotating
at 2 revolutions per hour. The pheromone components were released from
glass/plastic tubes in both traps of the pair while the monoterpenes were
released from the sprayer in one trap. Asterisks indicate a significant
difference between trap baits of a pair at P < 0.001 (chi square). Release
rates and details given in text.
A comparison of one unbaited trap and one with standard dispensers releasing
aggregation pheromone showed that beetles oriented toward the pheromone trap
with little interference by the unbaited trap (13:24 vs. 0; P < 0.001 ).
Hexane atomized from the sprayer in one trap apparently had no effect on the
response to pheromone from standard dispensers as the catches were similar
(Figure 5; P = 0.58, not significantly different). The sprayer was used to
increase the release rate of a mixture of monoterpenes (+)- and
(-)-alpha-pinene, (+)-3-carene, and terpinolene from 0.1 to 100 µg/min. which is
equivalent to natural rates of release from Scotch pine logs from 0.01 to 10
log-equivalents, respectively. A significant decrease in attraction to
aggregation pheromone components was found beginning at the 0. 1 log-equivalent,
or 1 µg/min, release of each of the monoterpenes (Figure 4). Individual
monoterpenes were also tested at 100 µg/min release (10 log-equivalents) to see
if they inhibited attraction of P. bidentatus to the standard dispensers
with aggregation pheromone (Figure 5). All of the tested monoterpenes reduced
responses (Figure 5); however, the reduction by (+)-3-carene was not
statistically significant (P = 0.1) with the numbers caught (Figure 5).
The piezoelectric sprayer
dispensed aggregation pheromone components at a constant rate that attracted
P. bidentatus males and females in numbers that were comparable to the
same components released neat at equivalent rates. Although the rates were not
identical, it would be expected that under uniform conditions the trap catches
would not significantly differ unless releases from the traps in a paired
differed significantly (2-5 times). This is because significant differences in
trap catches or behavioral responses are usually not observed unless there is a
difference in release rates over an order of magnitude (Byers et al., 1988;
El-Sayed, unpublished data), as is found in the release rates of monoterpenes
over several orders of magnitude in our study (Figure 4). Browne et al. ( 1974)
devised a delivery system for releasing semiochemicals based on a spring-powered
chart drive motor that depressed a plunger through a microliter syringe. The
amount of material released could be changed by simply diluting the stock
solutions. The major problem with this device, however, was that globules of
solvent with semiochemicals might build up at the syringe tip. Furthermore, the
solvent evaporates faster than the less volatile semiochemicals in a mixture,
thereby concentrating them at different rates over time and causing increasing
release rates of each during an experiment. Still another problem is that
mixtures of semiochemicals compete for the vapor pressure, thus producing
complicated effects on their release rates (Byers, 1988). Temperature and its
potential changes during a field experiment will affect the volatilities of
various semiochemicals and solvents differently, further confounding the even
and precise release rates desired. These problems are avoided with the
piezoelectric sprayer because each semiochemical is expressed to the atmosphere
in the exact ratio of its concentration in solution.
discussed dispenser technologies that use wicks, rubber septa, plastic bags, and
"test-tubes." Rubber septa, zeolites, and other absorbent materials have release
curves of semiochemicals that decline exponentially with time. Wicks have
inexact surface areas, and there is the problem of differences in the elution of
the solvent and the semiochemicals. Semipermeable plastic bags give constant
rates that ought to vary with the dilution, but the release probably varies with
the polarity of the solvent as well as the type of plastic used. The differences
in elution of the solvent and semiochemicals would tend to change the ratios and
release rates over time. Test-tube-type dispensers with dilutions of
semiochemicals based on the diffusion-dilution method (Byers, 1988) give nearly
constant rates for fairly long time periods, but eventually these tubes also
show differences in elution of solvent and semiochemicals that result in a
change in release rates of semiochemicals with time (usually an increase). With
the piezoelectric sprayer, the ratio of semiochemical to solvent remains
constant, and there is no difference in elution at the spray tip if the solvent
is properly atomized. The piezoelectric sprayer can be made to dispense
semiochemicals over longer periods by means of larger reservoirs and continuous
The release of either enantiomer of alpha-pinene, and
terpinolene, alone or in combination at 100 µg/min, or 10-log equivalents,
strongly inhibited attraction of P. bidentatus to its aggregation
pheromone components, grandisol and cis-verbenol. Beetles could be seen
in the evening orienting toward the aggregation pheromone but then becoming
disoriented when within about 0.5-1 m from the monoterpene release. It might be
surprising that host Scotch pine monoterpenes are strongly inhibitory at rates
similar to those from natural substrates. However, this bark beetle does not
attack living trees that can produce significant amounts resin, or even healthy
limbs, but rather colonizes diseased and dying limbs and small trees (Lekander
et al., 1977). The avoidance of monoterpenes from resin exuding from Scotch
pines would enable flying P. bidentatus to save time and energy while
locating parts of the tree suitable for colonization. Some of the monoterpenes,
e.g., alpha-pinene, are also found in Norway spruce, Picea abies, which is
avoided in nature (Byers, unpublished). The bark beetle also avoids volatiles
from Scotch pine needles or bark, Norway spruce needles or bark, and birch
(Betula pendula) leaves or bark (Byers, unpublished).
beetles are known to avoid host monoterpenes. A plastic tube releasing GC
effluents with monoterpenes from frass of male I. paraconfusus feeding in
ponderosa pine caused walking females of this species to turn away from the
effluents (Pitman et al., 1966). An attractive component (probably ipsenol),
when eluting, caused a positive taxis toward the tube. The specific monoterpenes
were not identified precisely but included one or more of the following:
alpha-pinene, myrcene, B-pinene, 3-carene, and limonene (Pitman et al., 1966).
Bordasch and Berryman (1977) reported that the fir engraver,
Scolytus ventralis LeC., was repelled by resin vapors, monoterpene
fractions, and alpha-pinene from grand fir, Abies grandis (Dougl.) Lindl.
On the other hand, Rudinsky et al. (1971) reported that the European spruce bark
beetle, Ips typographus (L.), is attracted to alpha-pinene, B-pinene, and
limonene (rates unknown) as compared to camphene (since there was no control).
Later studies implied that the attraction by host monoterpenes must be rather
weak since freshly cut Norway spruce did not attract I. typographus
initially, but after storage some beetles were attracted (Lindelow et al.,
1992). In other field studies, traps with freshly cut logs or bark chips did not
catch I. typographus (Byers, unpublished). In addition, volatiles
(monoterpenes) from freshly cut host logs did not synergize attraction to
pheromone components of I. typographus (Schlyter et al., 1987). In
contrast, Reddemann and Schopf (1996) found that the attraction of I.
typographus to aggregation pheromone is enhanced by large amounts (2
ml/dispenser) of (-)-alpha-pinene and (+)-limonene but decreased by
(+)-alpha-pinene and (-)-B-pinene. (+)-alpha-pinene reduced trap catch to only
6%. However, these results can be questioned if alpha-pinene oxidized to
verbenone (an inhibitor) (Bakke, 1981 ) or to cis-verbenol (an
aggregation pheromone component) (Bakke et al., 1977).
In the case of
North American spruce beetles, Dedroctonus rufipennis (Kirby), Werner
(1995) found that limonene, 4-allylanisole, myrcene, and B-phellandrene
inhibited the response to frontalin, an aggregation pheromone component, while
in the eastern larch beetle, Dendroctonus simplex LeC., myrcene and
limonene inhibited response to seudenol, an aggregation pheromone component. As
mentioned earlier, many bark beetles are known to be attracted directly by
monoterpenes (Byers et al., 1985; Byers, 1989, 1992; Phillips et al., 1988) or
they synergire response to aggregation pheromones (Bedard et al., 1969; Werner,
1972; Rudinsky et al., 1972; Byers et al., 1988; Reddemann and Schopf, 1996).
These diverse behaviors indicate a need for further research into the roles of
host volatiles, especially monoterpenes, in the chemical ecology of bark
The piezoelectric sprayer should prove useful in many kinds of
studies where precise quantities of semiochemicals need to be released in the
lab or field. The piezoelectric sprayer can be constructed from a kit, including
electronic components and the pump, obtainable from the first author.
Acknowledgements The study was supported in part by grants
from the Swedish Council for Forestry and Agricultural Research (SJFR) and a
postdoctoral grant to A. El-Sayed from the Schweizer Nationalfonds zur Forderunt
der wissenschaftlichen Forschung (SNF). Ch. Pfaffenhichler provided helpful
comment on the manuscript. J. Jönsson provided technical assistance and advice
on construction of the trap rotor.
|A. M. El-Sayed1 and J. A.
Department of Crop Science, Swedish University of
Agricultural Sciences, SE-230 53 Alnarp, Sweden
1Present address: Agricultural and Agri-Food Canada,
P.O. Box 6000, Vineland Station, Ontario, Canada L0R 2E0
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