Breaking a paradigm: male-produced aggregation pheromone for the Colorado potato beetle
1 United States Department of Agriculture, Agricultural Research Service,
Plant Sciences Institute, Vegetable Laboratory, Beltsville, MD 20705,
USA
2 United States Department of Agriculture, Agricultural Research Service,
Plant Sciences Institute, Chemicals Affecting Insect Behavior Laboratory,
Beltsville, MD 20705, USA
* Present address: United States Department of Agriculture, Agricultural
Research Service, Plant Sciences Institute, Chemicals Affecting Insect
Behavior Laboratory, Beltsville, MD 20705, USA
(e-mail: dickensj{at}ba.ars.usda.gov )
Accepted 9 April 2002
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Summary |
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Key words: pheromone, insect, neural regulation, juvenile hormone, feedback loop, host plant, Colorado potato beetle, Leptinotarsa decemlineata
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Introduction |
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The existence of a sex attractant pheromone for the Colorado potato beetle
Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) has been
a subject of dispute. Boiteau
(1988) considered that plant
odors attracted both sexes to the crop, where sexual encounters were random.
The existence of a short-range or contact sex pheromone on the elytra of
female Colorado potato beetles that elicited copulatory behavior in males was
first demonstrated by Levinson et al.
(1979
) and later verified by
others (Jermy and Butt, 1991
;
Otto, 1996
).
Prior to the work of Boiteau
(1988) cited above, DeWilde et
al. (1969
) observed that female
emissions `enhanced the anemotactic response of males' in a laboratory
behavioral bioassay. Experiments by Levinson et al.
(1979
) showed that males
responded differentially to male and female extracts from a distance of 8 mm.
These observations could not be verified in a different behavioral assay in
which male Colorado potato beetles `did not show any sign of percepting the
presence of females kept in small cages'
(Jermy and Butt, 1991
). Later,
Edwards and Seabrook (1997
)
demonstrated that males move upwind towards females from a distance of at
least 50 cm. Their results were based on greenhouse studies in which all
possible sex combinations placed on potted potato plants were tested; however,
only 22 % (11 of 49) of the male beetles moved towards the female-containing
plant.
On the basis of laboratory behavioral studies in which antennal segments
were extirpated, olfactory receptors for a sex attractant pheromone in male
Colorado potato beetle were localized to the terminal and penultimate antennal
segments (DeWilde et al.,
1969). Electroantennograms elicited by pentane extracts of female
beetles were nearly twice as large as those elicited by extracts of males or
potato foliage (Levinson et al.,
1979
). Dubis et al.
(1987
) demonstrated chemical
differences in the cuticular hydrocarbons of male and female beetles; such
differences could function in the recognition of females by males and as a
releaser of copulatory behavior.
In contrast to previous studies, we report the discovery of a male-specific
compound (S)-3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol [(S)-CPB
I] released by male Colorado potato beetles feeding on potato plants; this
compound is absent from collections of volatiles from females feeding on
potato plants. Both male and female beetles are attracted to CPB I in
laboratory behavioral bioassays. Since the accepted paradigm for chrysomelid
beetles (Mayer and McLaughlin,
1991), in general, and the Colorado potato beetle
(DeWilde et al., 1969
;
Edwards and Seabrook, 1997
),
in particular, was a female-produced attractant pheromone, our discovery of a
male-produced pheromone in Colorado potato beetles breaks this previous
paradigm and provides a new model for chemical communication in these
insects.
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Materials and methods |
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Collection of plant and insect volatiles
Potato plants, Solanum tuberosum var. Kennebeck, were grown in a
greenhouse under a 16 h:8 h L:D photoperiod at 25 °C in potting
soil/vermiculite mix (Jiffy mix). Undisturbed, single-stemmed potato plants
were used for volatile collections. All plants were 5-7 weeks old and
approximately 35 cm in height with a canopy of 30-35 cm.
An automated volatile collection system (Analytical Research Systems, Inc.,
Gainesville, FL, USA) modified from one described by Heath and Manukian
(1994) was used for collection
of plant- and insect-produced volatiles. The system consists of a humidified
air delivery system with mass flow controllers to regulate airflow into a
volatile collection chamber. An inlet took laboratory air, regulated to 420
kPa (60 p.s.i.), which was then filtered and further regulated to 125 kPa
(18.5 p.s.i.). The air was split between `dry' and `wet' air lines, controlled
to 1-31 min-1 flow rate. The `dry' line passed directly into the
volatile collection chamber; the `wet' air line was bubbled through distilled
and deionized water prior to entering the chamber.
Two glass volatile collection chambers were used: a 45 l carboy for collection of volatiles from individual plants or insects feeding on a plant, and a 3 l jar for collection of volatiles from insects alone. The 45 l carboy sat atop a guillotine base assembly with two Teflon plates coming together in a tongue-and-groove joint; the main stem of the plant passed through a 2.5 cm diameter hole in the center, which was then sealed with cotton. A manifold lid with eight ports to hold volatile collection traps was attached to the top of the chambers with an O-ring and C-clamp. Volatile collection traps consisted of glass tubes 8 cm in length, 0.5 cm in outside diameter (0.4 mm internal diameter) and filled with 30 mg of 80/100 mesh Super-Q as the adsorbent. Air was pulled through individual volatile collection traps with a vacuum (-80 kPa) regulated to -34 kPa and controlled to 1-21 min-1 with a mass flow controller. Solenoid switches, controlled with a GE Fanuc PLC programmed with Timed Event Sequencing Software (Analytical Research Systems, Inc., Gainesville, FL, USA), sampled air through eight valves attached distally to the volatile collection traps with Tygon tubing. The contents of the traps were extracted with 100 µl of hexane; 50 µl of this wash was collected in 300 µl cone vials for gas chromatography/electroantennogram analysis. n-Decane (10 ng µl-1) was added to each sample as an internal standard.
Volatiles were collected from undamaged or mechanically damaged potato plants and from plants being fed upon by Colorado potato beetle males or females. Volatiles from individual plants placed into the 45 l carboy were collected continuously for a 24 h period in eight volatile collection traps programmed to sample for 3 h per trap. A light shield covered the 45 l volatile collection chamber to simulate lighting conditions in the greenhouse (16 h:8 h L:D). Undamaged plants were placed undisturbed into the volatile collection chamber. Mechanically damaged plants were placed into the chamber after cutting five 1 cm long incisions around the perimeter of six leaves with dissection scissors washed in methanol. Volatiles from plants infested with 10 males or 10 females were also collected.
Subsequently, volatiles were collected from 20 male or 20 female beetles feeding on 4.8 g of potato foliage in the 3 l volatile collection chamber. Collections were made continuously for a 24 h period with a single volatile collection trap.
Gas chromatography/electroantennogram analysis of volatile
collections
Samples (1 µl) of volatile collections were injected into a Hewlett
Packard (model 5890A) gas chromatograph (GC) equipped with an HP-5 capillary
column (crosslinked 5 % PH ME Siloxane; film thickness 0.25 µm; length 30
m; internal diameter 0.25 mm) and flame ionization detector (FID). The
effluent from the column was split using a Gerstel GraphPack-3D/2 splitter
with a ratio of approximately 1 part to the GC (FID):4parts to an
electroantennogram (EAD) preparation. The EAD preparation was an adult
Colorado potato beetle antenna removed and mounted between two glass
capillaries filled with 0.1 mol l-1 NaCl. Ag/AgCl wires were
inserted into the ends of the glass capillaries, which then served as the
recording and ground electrodes. An effluent conditioning assembly to carry GC
effluent over a Colorado potato beetle antennal preparation and the hardware
and software for data collection and analyses using a computer were obtained
from Syntech (Hilversum, the Netherlands). After an initial temperature of
50°C, which was held for 2 min following injection, the temperature of the
GC oven was increased at 15°C min-1 to 235°C, which was
held for 8 min.
Isolation and identification of the male-specific compound
In an attempt to increase production of the male-specific volatile, 20
males were subjected to the following treatments: juvenile hormone III (JH
III) in acetone, extirpation of both antennae, and extirpation of both
antennae plus JH III in acetone. JH III [synthetic
(±)-10,11-epoxy-3,7,11-trimethyl-trans-trans-2,6-dodecadienoic
acid methyl ester], as obtained from Sigma-Aldrich, St Louis, MO, USA, was 75%
pure. Treatment with a JH analog and antennectomy increased pheromone
production in another coleopteran, the boll weevil Anthonomus grandis
Boh. (Curculionidae) (Dickens et al.,
1988). All treatments were repeated at least three times. For the
JH III treatment, 2µl of a 5µgµl-1 solution of JH III in
acetone was applied to the prothoracic sternum between the coxae. For the
antennectomy treatment, antennae were removed at the third segment from the
proximal end. Treatment with 2 µl of acetone and extirpation of a
mesothoracic leg served as controls for the JH III and antennectomy
treatments, respectively. All treatments were performed 1 h before placing
beetles into the volatile collection chamber.
The major GC/EAD-active component (CPB I) in volatiles collected by aeration of feeding Colorado potato beetle males was isolated in pure form for nuclear magnetic resonance (NMR) spectroscopy by using a Gerstel (Baltimore, MD, USA) automated preparative fraction collector connected to an HP 6890 gas chromatograph with hydrogen as carrier gas at 50 cm s-1. Six Gerstel 100 µl U-shaped glass traps that had been baked overnight at 220°C were plumbed into the preparative fraction collector and were cooled to 0°C in an ethanol bath. The preparative fraction collector switching valve and transfer line were held at a constant 200°C. The HP 6890 injector, fitted with a Tenax-packed insert, was operated in the solvent venting mode: 59°C at manual injection with hexane as solvent, solvent venting at 100 ml min-1 for 0.45 min followed by heating to 250°C at 600°C min-1. The chromatographic column (HP-1, 60 mx0.53 mm internal diameter, 5 µm film thickness) was held at 46°C for 1.6 min after injection and then heated to 220°C at 30°C min-1. The column effluent was split approximately 95 parts to the preparative fraction collector and 5 parts to a flame ionization detector. These operating conditions were developed using 2-dodecanone as standard because it had chromatographic retention indices like that of the active Colorado potato beetle compound (CPB I) and they afforded approximately 70-80% recovery of the chromatographed standard. Five sequential 3-4µl injections of combined and concentrated hexane aeration extracts with collection in one trap over 16-17.25 min of each chromatographic run yielded approximately 1 mg of pure compound. The ends of the trap were sealed with small rubber septa, and the compound was subsequently eluted into an NMR tube with deutero-solvent for analysis and structure determination.
NMR spectra were obtained with a JEOL spectrometer (model Eclipse+ 500) with deuterobenzene as solvent. Proton spectra were recorded at 500 MHz and 13C-spectra at 125 MHz. Mass spectra were recorded with a Shimadzu GCMSQP5050A spectrometer or with a Hewlett Packard (model 5973) mass-selective detector. Electron ionization spectra were collected at 70eV, and ammonia and deuteroammonia were employed as reagent gases for chemical ionization spectra. Optical rotations were measured on chloroform solutions using a Perkin-Elmer (model 241) automatic polarimeter operated at the sodium-D (589 nm) wavelength. Mention of a proprietary product or company does not imply endorsement.
Assays of the biological activity of the male-specific compound
The sensitivity of antennal olfactory receptors for the racemate and
optical isomers of the male-specific compound was tested using coupled GC/EAD
(Dickens, 1999). GC/EAD tests
involved injection of 1 µl of a 10 ng µl-1 hexane dilution of
(S)-, (R)- and racemic 3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol
(CPB I) into the GC/EAD system described above.
Serial dilutions of CPB I were tested for behavioral activity in an open
Y-track olfactometer modified after Visser and Piron
(1998) and described in detail
by Dickens (1999
). In brief,
volatiles emanating from 10 µl samples of the serial dilutions (0.00001-0.1
µg per µl of solvent) eluted onto filter paper discs (2.5 cm diameter;
Whatman no. 1 filter paper) in Erlenmeyer flasks were delivered to one side of
the device; volatiles emanating from 10 µl of hexane solvent were delivered
to the other side of the device as the control. Hydrocarbon-free air that was
humidified by passing through distilled water carried the odor molecules to
either arm of the bioassay apparatus. Treatments were replenished after 30 min
of use in the bioassay apparatus. Airflow was regulated to 11 min-1
by flowmeters. Experiments were conducted at 22°C in a darkened room in
which the only source of light was that associated with the bioassay device.
Orientation was scored as soon as the test insect had moved completely from
the horizontal to one of the 4° extension arms of the bioassay device.
Following each test, the bioassay device was cleaned with acetone to remove
contamination left by the insect. For any given series of tests, approximately
half were performed with the treatment and associated test apparatus on one
side and half with them on the other side. For all bioassays, at least 20
males and 20 females were tested.
Prior to testing at 7-14 days, unmated insects were held individually in 162.6 ml cups, provided with fresh potato foliage on a continuous basis and maintained under incubator conditions of a photoperiod of 16 h:8 h L:D, at 80-90 % relative humidity and at `day' and `night' temperatures of 25 and 23°C, respectively. On the day of testing, insects were transferred to smaller 29.6 ml cups and held for 1-3 h with moist filter paper but no foliage; they were then held in darkness for an additional 1-2 h.
Laboratory bioassays were assessed for significant differences by the
hypothesis on binomial proportions based on the standard normal approximation
(Brase and Brase, 1983). EAD
responses to enantiomers of male-specific compound (CPB I) were compared by
analysis of variance and Duncan's multiple-range test
(Duncan, 1955
).
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Results |
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Nonanal was the most often detected compound released from an undamaged plant (Fig. 1A). For mechanically damaged plants, EAD responses were present for both nonanal and 2-phenyl ethanol (Fig. 1B). Small quantities of sesquiterpenes and other compounds were released by undamaged and mechanically damaged plants, but seldom were significant antennal responses noted for these compounds.
Similar to the mechanically damaged plants, EADs in response to female and male feeding on plants were recorded most often in response to nonanal and 2-phenyl ethanol (Fig. 1C). The quantities of sesquiterpenes emitted by female feeding were generally greater than the quantities emitted by either undamaged or mechanically damaged plants.
EAD responses to volatile collections during male feeding on potato plants differed from responses to undamaged, mechanically damaged plants and female feeding on plants: there was a large EAD consistently present in an area just prior to the sesquiterpenes (Fig. 1D). Responses at this retention time were observed only for volatile collections from males; thus, this EAD response represented a sex-specific, male-produced volatile (CPB I). However, under these conditions, no observable peak was recorded in the flame ionization detector.
Enhancement of sex-specific, male-produced volatile
Aerations of 10 Colorado potato beetle males feeding on a potato plant in
our initial experiments did not yield adequate amounts of CPB I for
visualization of a peak on the flame ionization detector. Therefore, volatiles
were collected from 20 males feeding on potato foliage in a collection chamber
with a smaller volume (31) (Fig.
2A). Collections performed in this manner generally presented a
visible peak representing only a few nanograms (mean 53 ng) for the 24h
collection period, which was still an inadequate amount for chemical
identification.
|
To enhance the production of CPB I, three techniques were tested
(Dickens et al., 1988): (i)
topical treatment with juvenile hormone III (JH III), (ii) antennectomy and
(iii) topical treatment with JH III together with antennectomy. Treatment with
JH III enhanced production of the male compound eightfold to 396.2 ng
(Fig. 2B). Antennectomy
resulted in a 40-fold increase in the production of CPB I
(Fig. 2C) relative to untreated
males, with little effect on quantities of sesquiterpenes collected. The
combined treatment of JH III and antennectomy enhanced collections of CPB I by
nearly 200-fold (to 8834 ng); these levels of CPB I enabled collection of
quantities adequate for identification. Concurrent with the increase in CPB I
collected from antennectomized males and males subjected to the combined
treatment was a notable increase in the amount of 6-methyl-5-hepten-2-one
(labeled `P' in Fig. 2C,D).
Neither control treatment (acetone solvent treatment or extirpation of a
mesothoracic leg) resulted in a significant increase in CPB I production
compared with untreated, intact insects.
Identification of the male-specific compound
The EAG-active compound was identified as
(S)-3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol (CPB I)
(Fig. 3A). Compound 1 has been
reported (Devi and Bhattacharyya,
1977) as a metabolite of geraniol; however, characterization was
incomplete, the absolute configuration was not determined and synthesis was
not attempted. A more detailed description of our identification and synthesis
will be reported elsewhere; briefly, the initial assignment was made from the
compound's electron ionization and chemical ionization mass spectra, and an
apparent relationship to 6-methyl-5-heptene-2-one (compound 2=P)
(Fig. 3B) (a peak for compound
2 always appeared in gas chromatograms of samples containing compound 1, and
the mass spectra of the two compounds suggested features in common).
1H- and 13C-NMR spectra from material isolated by
preparative gas chromatography supported the assignment, and the general
structure was finally confirmed by synthesis of racemic compound 1 from
geraniol via its 2,3-monoepoxoide. Chiral gas chromatographic
comparison with racemic compound 1 demonstrated that the insect-derived
material consisted of a single enantiomer.
|
Both enantiomers of compound 1 were then synthesized individually. The
terpene linalool was chosen as the starting material because both enantiomers
have been fully characterized. (R)()-Linalool is commercially
available, and the (S)(+)-enantiomer was isolated and purified from
oil of coriander (Oliver,
2001). Since the absolute configuration of C-3 of linalool does
not change during the synthetic transformations, the configurations of both
enantiomers of compound 1 were thereby established. The absolute configuration
of C-3 of insect-derived compound 1 was found to be (S). Synthetic
(S)-CPB I had more than 99 % optical purity; synthetic
(R)-CPB I was 96 % optically pure.
Compound (S)(+)-1 is a clear liquid, 1H-NMR 0.91 (s,
3H), 1.14 (s, 3H), 1.42 (s, 3H), 1.42-1.43 (m), 1.72-1.81 (complex multiplet),
2.02 (1H, dd, J=3.0 and 10.0), 2.62 (1H, br. s), 3.61 (1H, d, J=10.4), 3.82
(1H, d, J=10.5). 13C-NMR 93.63, 83.53, 79.97, 73.72, 48.48, 38.03,
28.03, 24.54, 22.57, 21.54. Mass spectrum (m/z, %) 127 (6), 109 (37),
104 (10), 86 (7), 83 (7), 71 (11), 70 (5), 69 (88), 67 (9), 58 (5), 55 (10),
53 (6), 43 (100), 41 (72). []D25=+0.73.
Antennal receptors for CPB I respond selectively to the
(S)-enantiomer
Mean EAG values were significantly greater (approximately 10-fold) for
(S)-CPB I than for the (R)-enantiomer (P<0.01)
(Fig. 4). An intermediate
response was elicited by an equal amount of the racemate. There were no sexual
differences in EAGs in response to either enantiomer or to the racemate at
this dose.
|
Behavioral activity of optical isomers
Both male and female Colorado potato beetles oriented preferentially to the
(S)-enantiomer of CPB I (Fig.
5A,B) (P<0.01). Responses of males had a threshold of
only 0.001 µg source load; female Colorado potato beetles had a slightly
higher behavioral threshold of 0.01 µg source load. Once the threshold had
been reached for both sexes, 80-90 % of all individuals were attracted to the
(S)-enantiomer up to the highest source load tested (1 µg). There
was no significant preference for serial source loads of either the
(R)-enantiomer or the racemate for either sex.
|
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Discussion |
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The conclusion of DeWilde et al.
(1969) of a female attractant
was based on the movement of males in a 123 cm long chamber in which air was
passed over 40 females. The number of males that moved upwind, remained
indifferent or responded negatively was noted; a mean excess was calculated as
the measure of response. Of 20 males placed in the chamber, a mean excess of
8.3 males moved upwind compared with 2.7 males for a control; the number of
males that responded either negatively or indifferently was not reported.
Responses of female beetles to air passed over males were not reported.
The approach of Edwards and Seabrook
(1997) to demonstrating a
Colorado potato beetle pheromone differed from that of DeWilde et al.
(1969
). They carried out
greenhouse experiments in which seven Colorado potato beetles of a specified
sex were placed on upwind and downwind potato plants; the number of insects
that moved upwind after a 10 h test period was then noted. Although
significant attraction was noted only for males moving upwind to females, only
11 of 47 males moved to the plant containing females. Levinson et al.
(1979
) demonstrated the arrest
of male Colorado potato beetles 8 mm above female extracts; Jermy and Butt
(1991
) could not verify the
volatile nature of this attractant. It would be difficult to eliminate the
importance of plant volatiles on the observed attraction of males in the
experiments of Edwards and Seabrook
(1997
) because all the insects
were in contact with and presumably feeding on the potato plants. While the
experimental conditions of DeWilde et al.
(1969
) are unclear, plant
volatiles may also have been involved in the observed responses. The
attraction of Colorado potato beetles to volatiles emanating from potato
plants is wellknown (McIndoo,
1926
; Schanz,
1953
; DeWilde et al.,
1969
; Visser,
1976
; Bolter et al.,
1997
; Schütz et al.,
1997
), and specific blends of volatiles emitted by potato plants
that attract Colorado potato beetles have recently been identified (Dickens,
1999
,
2000b
).
Emission of CPB I by male Colorado potato beetle was at extremely low
levels, hence the need to increase production levels for identification.
Although JH III clearly increased the amount of CPB I emitted by male Colorado
potato beetles, the effect of antennectomy was even greater
(Fig. 2B,C). The combination of
JH III treatment and antennectomy specifically increased quantities of CPB I
released by nearly 200-fold compared with control insects under similar
conditions. Previous research on other coleopterous insects showed that
topical application of juvenile hormone (JH) or a juvenile hormone analog
substantially increased pheromone production in bark beetles (Scolytidae)
(Borden et al., 1969; Hughes
and Renwick, 1977a
,
b
;
Renwick and Dickens, 1979
).
While application of a juvenile hormone analog (methoprene) increased
pheromone production in the boll weevil Anthonomus grandis Boh.
(Curculionidae) over that of control insects, antennectomy increased pheromone
production significantly more within 48 h
(Dickens et al., 1988
). Since
the juvenile hormone analog decreased the sensitivity of antennal receptors
for pheromones (Palaniswamy et al.,
1979
) and for plant odors in the boll weevil
(Dickens, 1986
), it was
proposed that decreased antennal input may be responsible for observed
increases in pheromone production. In other words, the low levels of CPB I
observed for male Colorado potato beetle might be monitored directly by the
beetle and subsequently regulated by antennal input. Thus, extirpation of the
antennae and subsequent deprivation of antennal input deprives Colorado potato
beetles of information on levels of CPB I necessary to regulate its release or
production levels. Extirpation of other appendages does not have this effect
in either the Colorado potato beetle (J. C. Dickens, unpublished observations)
or the boll weevil (Dickens et al.,
1988
).
The male-produced pheromone for Colorado potato beetles is the first to be
identified for a chrysomelid beetle; previous pheromones identified for
chrysomelids have been female-produced sex attractants
(Mayer and McLaughlin, 1991).
Although the structure of CPB I has been reported
(Devi and Bhattacharyya, 1977
)
as a bacterial metabolite of geraniol, it is unique for an insect pheromone.
Recently, field-trapping experiments indicated that male crucifer flea
beetles, Phyllotreta cruciferae (Goeze), may produce an aggregation
pheromone, but the nature of the attractant was not elucidated
(Peng et al., 1999
).
In conclusion, we have identified a male-produced aggregation pheromone,
(S)-CPB I, for Colorado potato beetles. Production of
(S)-CPB I was enhanced by topical application of JH III and
antennectomy and, thus, levels may be regulated by a feedback system using
antennal input which, in turn, may be under hormonal control. Only
(S)-CPB I is released by males; (S)-CPB I is attractive in
laboratory behavioral bioassays for both male and female Colorado potato
beetle, while (R)-CPB I is inactive and its presence in the racemate
seems to abolish the response to the (S)-enantiomer. This finding
suggests that there are receptor cells tuned to (R)-CPB I and that
the behavioral inhibition is due to central processing. The male-produced
aggregation pheromone will provide an additional tool for use in conjunction
with previously identified plant attractants for Colorado potato beetles
(Dickens, 1999,
2000b
) already being tested in
the field (A. R. Alford, J. Martel and J. C. Dickens, unpublished
observations) for manipulation of chemically mediated behavior for
environmentally sound pest management.
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Acknowledgments |
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References |
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