A male-produced aggregation pheromone was identified for the Colorado potato beetle Leptinotarsa decemlineata (Say) (Coleoptera:Chrysomelidae). While male beetles produced only minor amounts of the pheromone, its production could be enhanced by topical application of juvenile hormone III (JH III) (eightfold), by antennectomy (40-fold) or by the combined treatment of JH III and antennectomy (almost 200-fold); this enhancement enabled the identification of the compound as(S)-3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol [(S)-CPB I], a unique structure for an insect pheromone. Antennal receptors of both sexes responded selectively to the (S)-enantiomer. Both male and female Colorado potato beetles were attracted to serial source loads of(S)-CPB I in laboratory bioassays; (R)-CPB I was inactive or inhibitory, as demonstrated by the inactivity of the racemate. This is the first identification of a pheromone for the Colorado potato beetle and differs from the paradigm of a female-produced pheromone for this insect. The attractant is also the first male-produced pheromone identified for the Chrysomelidae. The discovery that both JH III and antennectomy increase levels of the pheromone (S)-CPB I indicates the existence of a feedback system involving antennal input, and this system may be under hormonal control.

Insect behavior is governed by complex interactions among chemical and physical signals in the environment. For example, host plant volatiles may (i)facilitate orientation of insects to potential feeding sources(Bernays and Chapman, 1994),(ii) provide a mechanism for insects to avoid non-hosts(Dickens et al., 1992) or(iii) either enhance (Dickens,1989; Dickens et al.,1990) or disrupt behavioral responses to pheromones(Dickens et al., 1992). Insect-produced volatiles or pheromones attract conspecifics for mating(Cardé and Baker, 1984)or disrupt behavioral responses of closely related species(Hansen, 1984; Borden, 1997).

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.

Animals

Adult Colorado potato beetles, Leptinotarsa decemlineata Say, were obtained from a colony infused every year with field-collected insects; all life stages were reared on Solanum tuberosum var. Kennebeck. Emerging adults were collected daily, identified to gender and isolated in Petri dishes with moistened filter paper and fresh potato foliage, which was replenished daily. Insects were kept in an environmental chamber (16 h:8 h L:D) at 25°C until use. For volatile collections, insects more than 11 days old were used because both sexes are sexually mature at this time(Dickens, 2000a).

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 grandisBoh. (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 m×0.53 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 (0.00001-0.1μg 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-1by 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).

Neural detection of volatiles released by the host plant and Colorado potato beetle feeding

Searching for sex-specific compounds emitted from Colorado potato beetles,we investigated volatiles released by undamaged plants, by mechanically damaged plants and by females or males feeding on the plant. Coupled gas chromatography/electroantennogram detection with both male and female antennal preparations revealed similar selectivity for both sexes for volatiles released from these sources (Fig. 1).

Fig. 1.

Gas chromatographs (FID) with coupled electroantennograms (EAD) from Colorado potato beetles in response to volatiles emitted over a 3 h test period by an undamaged potato plant (A), a mechanically damaged potato plant(B), 10 female beetles feeding on a potato plant (C) and 10 male beetles feeding on a potato plant (D). EADs in A, C and D were recorded from female antennae; the EAD in B was recorded from a male antenna. A, nonanal; B, 2-phenyl ethanol; CPB I, male-specific compound; IS, internal standard (10 ng of decane); Sesquiterpene region, retention times of various sesquiterpenes. No differences were observed between EADs of male and female antennae.

Fig. 1.

Gas chromatographs (FID) with coupled electroantennograms (EAD) from Colorado potato beetles in response to volatiles emitted over a 3 h test period by an undamaged potato plant (A), a mechanically damaged potato plant(B), 10 female beetles feeding on a potato plant (C) and 10 male beetles feeding on a potato plant (D). EADs in A, C and D were recorded from female antennae; the EAD in B was recorded from a male antenna. A, nonanal; B, 2-phenyl ethanol; CPB I, male-specific compound; IS, internal standard (10 ng of decane); Sesquiterpene region, retention times of various sesquiterpenes. No differences were observed between EADs of male and female antennae.

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.

Fig. 2.

Production of the male-specific compound (24h collection) CPB I by 20 untreated male Colorado potato beetles (N=4) (A) and enhancement of CPB I production by experimental treatments (1 μl injection into the gas chromatograph of 50 μl of rinse): (B) topical treatment with 10 μg of juvenile hormone III (JH III) (N=4); (C) antennectomy (N=3);and (D) combined treatment of topical application of JH III and antennectomy(N=3). CPB I, male-specific compound; P, 6-methyl-5-hepten-2-one;FID, response of flame ionization detector. The electroantennogram (EAD)response is included for A—C. EADs in A and B were recorded from female antennae; the EAD in C was recorded from a male antenna. No differences were observed in EADs of male and female antennae. SR, sesquiterpene region.

Fig. 2.

Production of the male-specific compound (24h collection) CPB I by 20 untreated male Colorado potato beetles (N=4) (A) and enhancement of CPB I production by experimental treatments (1 μl injection into the gas chromatograph of 50 μl of rinse): (B) topical treatment with 10 μg of juvenile hormone III (JH III) (N=4); (C) antennectomy (N=3);and (D) combined treatment of topical application of JH III and antennectomy(N=3). CPB I, male-specific compound; P, 6-methyl-5-hepten-2-one;FID, response of flame ionization detector. The electroantennogram (EAD)response is included for A—C. EADs in A and B were recorded from female antennae; the EAD in C was recorded from a male antenna. No differences were observed in EADs of male and female antennae. SR, sesquiterpene region.

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.

Fig. 3.

Structure of the Colorado potato beetle male-specific compound(S)3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol (A) (compound 1, CPB I) and the related compound 6-methyl-5-heptene-2-one (B) (compound 2, P).

Fig. 3.

Structure of the Colorado potato beetle male-specific compound(S)3,7-dimethyl-2-oxo-oct-6-ene-1,3-diol (A) (compound 1, CPB I) and the related compound 6-methyl-5-heptene-2-one (B) (compound 2, P).

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). [α]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.

Fig. 4.

Mean electroantennogram responses (in mV) from Colorado potato beetles elicited by a 10ng injection of (S)-enantiomer,(R)-enantiomer and racemic CPB I. Responses of males and females were not significantly different and were therefore combined. Values are means± S.E.M. (N=6, three males and three females). Columns with different letters are significantly different, P<0.01, Duncan's multiple-range test.

Fig. 4.

Mean electroantennogram responses (in mV) from Colorado potato beetles elicited by a 10ng injection of (S)-enantiomer,(R)-enantiomer and racemic CPB I. Responses of males and females were not significantly different and were therefore combined. Values are means± S.E.M. (N=6, three males and three females). Columns with different letters are significantly different, P<0.01, Duncan's multiple-range test.

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.

Fig. 5.

Behavioral responses (percentage of choices) in an open Y-track olfactometer of male (A) and female (B) Colorado potato beetles to volatiles emanating from serial source loads of (S)-CPB I=(S)-enantiomer, (R)-CPB I=(R)-enantiomer and racemic CPB I=Racemate versus solvent control. Each individual was tested only once. Shaded columns represent responses to experimental treatment; open columns represent responses to the solvent control. Asterisks indicate that the response to an experimental treatment differs significantly from the control (**P<0.01) by testing the hypothesis that the binomial proportion is significantly different from P=0.05 using the standard normal approximation(Brase and Brase, 1983). Values are percentages of 20 beetles responding to each treatment ±S.E.M..

Fig. 5.

Behavioral responses (percentage of choices) in an open Y-track olfactometer of male (A) and female (B) Colorado potato beetles to volatiles emanating from serial source loads of (S)-CPB I=(S)-enantiomer, (R)-CPB I=(R)-enantiomer and racemic CPB I=Racemate versus solvent control. Each individual was tested only once. Shaded columns represent responses to experimental treatment; open columns represent responses to the solvent control. Asterisks indicate that the response to an experimental treatment differs significantly from the control (**P<0.01) by testing the hypothesis that the binomial proportion is significantly different from P=0.05 using the standard normal approximation(Brase and Brase, 1983). Values are percentages of 20 beetles responding to each treatment ±S.E.M..

We report here a volatile attractant pheromone (CPB I) for the Colorado potato beetle that is produced only by male beetles. Previous studies on pheromonal communication in Colorado potato beetles indicated the presence of a volatile female-produced attractant that increased positive anemotaxis in males (DeWilde et al., 1969) or movement of males towards a potato plant housing females(Edwards and Seabrook,1997).

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.

J.C.D. thanks Dr A. Mattoo, USDA, ARS, BARC and The Vegetable Laboratory for support. J.C.D. was supported by grants from the National Potato Council. Critical reviews of the manuscript were provided by Professor K.-E. Kaissling,Max Planck Institute for Behavioral Physiology, Seewiesen, Germany, Professor F. E. Hanson, Department of Biological Sciences, University of Maryland,Baltimore, MD, USA, Dr Ulla Klein, Department of Zoology, University of Munich, Germany, and Dr Ashok Raina, USDA, ARS, SRRC, Formosan Termite Research Unit, New Orleans, LA, USA.

Bernays, E. A. and Chapman, R. F. (
1994
).
Host Plant Selection by Phytophagous Insects
. New York: Chapman & Hall.
312
pp.
Boiteau, G. (
1988
). Sperm utilization and post-copulatory female-guarding in the Colorado potato beetle, Leptinotarsa decemlineata.
Entomol. Exp. Appl.
47
,
183
-187.
Bolter, C. J., Dicke, M., Van Loon, J. J. A., Visser, J. H. and Posthumus, M. A. (
1997
). Attraction of Colorado potato beetle to herbivore-damaged plants during herbivory and after its termination.
J. Chem. Ecol.
23
,
1003
-1023.
Borden, J. H. (
1997
). Disruption of semiochemical-mediated aggregation in bark beetles. In
Insect Pheromone Research New Directions
(ed. R. T. Cardé and A. K. Minks), pp.
421
-438. New York: Chapman &Hall.
Borden, J. H., Nair, K. K. and Slater, C. E.(
1969
). Synthetic juvenile hormone: Induction of sex pheromone production in Ips confusus.
Science
166
,
1626
-1627.
Brase, C. H. and Brase, C. P. (
1983
).
Understanding Statistics
. Lexington, MA: D. C. Heath.
Cardé, R. T. and Baker, T. C. (
1984
). Sexual communication with pheromones. In
Chemical Ecology of Insects
(ed. W. J. Bell and R. T. Cardé), pp.
355
-383. New York: Chapman & Hall.
Devi, J. R. and Bhattacharyya, P. K. (
1977
). Microbiological transformations of terpenes. XXIII. Fermentation of geraniol,nerol and limonene by a soil pseudomonad, Pseudomonas incognita(linalool strain).
Indian J. Biochem. Biophys.
14
,
359
-363.
DeWilde, J., Hille Ris Lambers-Suverkropp, K. and Van Tol,A. (
1969
). Responses to airflow and airborne plant odour in the Colorado beetle.
Neth. J. Plant Pathol.
75
,
53
-57.
Dickens, J. C. (
1986
). Specificity in perception of pheromones and host odours in Coleoptera. In
Mechanisms in Insect Olfaction
(ed. T. L. Payne, M. C. Birch and C. Kennedy), pp.
253
-261. Oxford: Oxford University Press.
Dickens, J. C. (
1989
). Green leaf volatiles enhance aggregation pheromone of boll weevil, Anthonomus grandis Boh.(Coleoptera: Curculionidae).
Entomol. Exp. Appl.
52
,
191
-203.
Dickens, J. C. (
1999
). Predator—prey interactions: olfactory adaptations of generalist and specialist predators.
Agric. Forest Entomol.
1
,
47
-54.
Dickens, J. C. (
2000a
). Sexual maturation and temporal variation of neural responses in adult Colorado potato beetles to volatiles emitted by potato plants.
J. Chem. Ecol.
26
,
1265
-1279.
Dickens, J. C. (
2000b
). Orientation of Colorado potato beetle to natural and synthetic blends of volatiles emitted by potato plants.
Agric. Forest Entomol.
2
,
167
-172.
Dickens, J. C., Billings, R. F. and Payne, T. L.(
1992
). Green leaf volatiles interrupt aggregation pheromone response in bark beetles infesting southern pines.
Experientia
48
,
523
-524.
Dickens, J. C., Jang, E. B., Light, D. M. and Alford, A. R.(
1990
). Enhancement of insect pheromone responses by green leaf volatiles.
Naturwissenschaften
77
,
29
-31.
Dickens, J. C., McGovern, W. L. and Wiygul, G.(
1988
). Effects of antennectomy and a juvenile hormone analog on pheromone production in the boll weevil, Anthonomus grandis Boh.(Coleoptera: Curculionidae).
J. Entomol. Sci.
23
,
52
-58.
Dubis, E. E., Malinski, A., Dubis, J., Szafranek, J., Nawrot,J., Poplawski, J. and Wróbel, J. T. (
1987
). Sex-dependent composition of cuticular hydrocarbons of the Colorado potato beetle, Leptinotarsa decemlineata Say.
Comp. Biochem. Physiol.
87A
,
839
-843.
Duncan, D. B. (
1955
). Multiple range and multiple F tests.
Biometrics
11
,
1
-42.
Edwards, M. A. and Seabrook, W. D. (
1997
). Evidence for an airborne sex pheromone in the Colorado potato beetle, Leptinotarsa decemlineata.
Can. Entomol.
129
,
667
-672.
Hansen, K. (
1984
). Discrimination and production of disparlure enantiomers by the gypsy moth and the nun moth.
Physiol. Entomol.
9
,
9
-18.
Heath, R. R. and Manukian, A. (
1994
). An automated system for use in collecting volatile chemicals released from plants.
J. Chem. Ecol.
20
,
593
-608.
Hughes, P. R. and Renwick, J. A. A. (
1977a
). Neural and hormonal control of pheromone biosynthesis in the bark beetle, Ips paraconfusus.
Physiol. Entomol.
2
,
117
-123.
Hughes, P. R. and Renwick, J. A. A. (
1977b
). Hormonal and host factors stimulating pheromone biosynthesis in female western pine beetles, Dendroctonus brevicomis.
Physiol. Entomol.
2
,
289
-292.
Jermy, T. and Butt, B. A. (
1991
). Method for screening female sex pheromone extracts of the Colorado potato beetle.
Entomol. Exp. Appl.
59
,
75
-78.
Levinson, H. Z., Levinson, A. R. and Jen, T.-L.(
1979
). Sex recognition by a pheromone in the Colorado beetle.
Naturwissenschaften
66
,
472
-473.
Mayer, M. S. and McLaughlin, J. R. (
1991
).
Handbook of Insect Pheromones and Sex Attractants
. Boca Raton, FL: CRC Press.
1083
pp.
McIndoo, N. E. (
1926
). An insect olfactometer.
J. Econ. Entomol.
19
,
545
-571.
Oliver, J. E. (
2001
). (S)(+)-Linalool from oil of coriander.
J. Essent. Oil Res.
(in press).
Otto, D. (
1996
). Further evidence for the presence of a female sex pheromone in the Colorado potato beetle Leptinotarsa decemlineata Say. and its biological characterization. In
Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones IV
(ed. H. Kleeberg and V. Micheletti), pp.
135
-147. Lahnau, Germany: Trifolio-M GmbH.
Palaniswamy, P., Sivasubramanian, P. and Seabrook, W. D.(
1979
). Modulation of sex pheromone perception in female moths of the eastern spruce budworm, Choristoneura fumiferana by altosid.
J. Insect Physiol.
25
,
571
-574.
Peng, C., Bartelt, R. J. and Weiss, M. J.(
1999
). Male crucifer flea beetles produce an aggregation pheromone.
Physiol. Entomol.
24
,
98
-99.
Renwick, J. A. A. and Dickens, J. C. (
1979
). Control of pheromone production in the bark beetle, Ips cembrae.
Physiol. Entomol.
4
,
377
-381.
Schanz, M. (
1953
). Der Geruchssinn des Kartoffelkäfers (Leptinotarsa decemlineata Say).
Z. Vergl. Physiol.
35
,
353
-379.
Schütz, S., Weißbecker, B., Klein, A. and Hummel, H. E. (
1997
). Host plant selection of the Colorado potato beetle as influenced by damage induced volatiles of the potato plant.
Naturwissenschaften
84
,
212
-217.
Visser, J. H. (
1976
). The design of a low-speed wind tunnel as an instrument for the study of olfactory orientation in the Colorado potato beetle (Leptinotarsa decemlineata).
Entomol. Exp. Appl.
20
,
275
-288.
Visser, J. H. and Piron, P. G. M. (
1998
). An open Y-track olfactometer for recording aphid behavioural responses to plant odours.
Proceedings of the Section Experimental and Applied Entomology of the Netherlands Entomological Society Amsterdam
9
,
41
-46.