This application claims the benefit of U.S. Provisional patent application 61/513,054 filed Jul. 29, 2011.
BACKGROUND OF THE INVENTION
The emerald ash borer, Agrilus planipennis Fairmaire, (Coleoptera: Buprestidae) is an invasive Palearctic species that has killed millions of ash trees (Fraxinus spp. L.) (Oleaceae) in the USA and Canada (Cappaert et al. 2005; Poland and McCullough 2006). Although initially detected near Detroit, Mich. in 2002, there is evidence that populations of this invasive species had been present in Michigan, USA and Ontario, Canada since the mid-1990s (Seigert et al. 2007). Since then, it has spread rapidly and has been detected in 15 states and two provinces, Ontario and Quebec, in Canada (EAB 2010). Movement of infested firewood and nursery stock has exacerbated its spread and large scale devastation of ash trees is predicted (Marchant 2006). Early detection of A. planipennis infestations has proven difficult because visual signs and symptoms, such as D-shape exit holes, epicormic shoots, bark deformities, and thinning crowns, usually appear only on heavily infested trees a year or more after populations have been established (Cappaert et al. 2005; de Groot et al. 2006, 2008; Poland and McCullough 2006). Development of a monitoring system is critical for early detection of A. planipennis populations, which would aid in management and control decisions. In order to maximize detection efficacy, a better understanding of the behavior and chemical ecology of adult A. planipennis is needed.
Adult A. planipennis are typically active between 0600-1700 h, particularly when the weather is warm and sunny (Yu 1992; Rodriguez-Saona et al. 2007), with mating occurring from 0900-1500 h and lasting for 20-90 min. Yu (1992) observed that adults preferred trees in open areas with direct sunlight and that during rainy or cloudy weather they tended to rest in cracks in the bark or on the foliage. Adult beetles, particularly males, spend much of their time in the canopy feeding and flying short distances (Lance et al. 2007; Lelito et al. 2007; Rodriguez-Saona et al. 2007). Indeed, traps in the mid-upper ash canopy capture more adults than traps hung below the canopy (Lance et al. 2007; Francese et al. 2007, 2008; Crook et al. 2008, 2009) and traps in locations exposed to direct sunlight (i.e. on the edge or near a gap) generally catch more adults than those in shaded locations (Poland et al. 2005; McCullough et al. 2006, 2009; Francese et al. 2008; Lyons et al. 2009).
Crook and Mastro (2010) reviewed the considerable progress made towards developing a trap that is effective at capturing A. planipennis (Francese et al. 2005, 2007, 2008, 2010; Crook et al. 2008, 2009; Lelito et al. 2007, 2008; McCullough et al. 2008). Color has been identified as an important factor affecting trap captures, with purple shown to be highly attractive (Francese et al. 2005, 2008; Crook et al. 2008). Purple traps typically catch more females than males (Francese et al. 2008; Crook et al. 2009), due to A. planipennis response to light in both the blue and red range of the visible spectrum (Crook et al. 2009). Currently, a sticky purple prism trap is utilized in surveys for A. planipennis in the United States (Francese et al. 2008; Crook and Mastro 2010). Adult A. planipennis also respond to green light in the 540-560 nm range of wave length 540-560 nm (Crook et al. 2009), with green traps capturing two to three times as many adults as purple traps. Crook et al. 2009 also found that dark green (24% reflectance) and light green (64% reflectance) caught more beetles than purple. Also, Francese et al (2010b) Can. Entomol. 142: 596-600 tested purple vs light green (540 nm, 64% reflectance) traps and reported that green caught more EAB, particularly males. Also, Francese et al. (2010a) J Econ Ent 103: 1235-1241 studied different green wavelengths and different reflectances, and concluded that the best trap would be a green trap with a wave length of 540 nm and 49% reflectance. Green traps typically have a bias towards males in trap captures (Lance et al. 2007; Rodriguez-Saona et al. 2007; Lelito et al. 2008; Crook et al. 2009). However, green traps typically catch more adults only when deployed high in the tree canopy. Thus, trap deployment, as well as color and lure combination, must be considered when evaluating traps for a monitoring program, as trap captures are likely influenced by adult preferences and behavioral activity patterns.
Numerous studies have described the chemical ecology of A. planipennis (Crook and Mastro 2010) and two types of host volatiles have been demonstrated to be attractive: bark sesquiterpenes (Poland and McCullough 2006; Crook et al. 2008) and green leaf volatiles (Poland et al. 2004, 2005, 2006, 2007; Rodriguez-Saona et al. 2006; de Groot et al. 2008; Grant et al. 2010). Girdled or stressed ash (Poland and McCullough 2006; Crook et al. 2008) are attractive to both sexes, as are Manuka and Phoebe oils which contain, in part, the sesquiterpenes emitted by stressed Fraxinus spp. (Crook et al. 2008; Crook and Mastro 2010; Grant et al. 2010). Of the green leaf volatiles, one compound in particular, (3Z)-hexenol, is highly antennally active and attractive to males (de Groot et al. 2008; Grant et al 2010). These results indicate that specific host volatiles act as kairomones in the chemical ecology of A. planipennis and these compounds may provide useful detection tools.
Much of the literature on the mating behavior of buprestids (e.g. Rodriguez-Saona et al. 2006; Lelito et al. 2007; Akers and Nielsen 1992; Gwynne and Rentz 1983; Carlson and Knight 1969) has described the use of visual and tactile cues for mate location. For buprestids, including those in the genus Agrilus, host location has been described as occurring first by olfactory processes and then mate location by visual, or by vibratory and/or tactile cues (Carlson and Knight 1969). However, Dunn and Potter (1988) showed attraction of A. bilineatus (Weber) males to cages containing females compared to host-logs only, suggesting the use of a female-produced pheromone.
Limited progress has been made into the pheromone chemistry of A. planipennis. Previous work suggested the presence of a contact pheromone (Lelito et al. 2007), subsequently identified by our research group as 9-methylpentacosane, which appears only on the cuticle of female A. planipennis at sexual maturity (7-10 d old) and stimulates full copulatory activity in males upon antennal contact (Silk et al. 2009), although 3-methyltricosane may also be involved as an additional component (Lelito et al. 2009). Bartelt et al. (2007) identified a volatile, antennally-active predominantly female-produced macrocyclic lactone, (3Z)-dodecen-12-olide [(3Z)-lactone], which was the first putative volatile pheromone described for A. planipennis, but no behavioral activity was reported.
Pureswaran and Poland (2009) reported that males were able to locate and identify females at close range using olfaction and an unidentified volatile cue. Here, we use GC-EAD in combination with field trapping and olfactometry to test whether (3Z)-lactone elicits behavioral responses in A. planipennis either alone or in combination with host kairomones (bark sesquiterpenes or green leaf volatiles). We tested various lure combinations on both purple and green traps, as both colors have been shown to be attractive. We also tested the lactone stereoisomer, (3E)-lactone, for its effect on A. planipennis behavior because preliminary studies suggested that exposure to UV-light catalyzes the isomerization of (3Z) to the (3E)-lactone and A. planipennis adults are known to favor sunny locations.
SUMMARY OF THE INVENTION
This invention provides the first behavioral evidence for a volatile pheromone of A. planipennis in combination with host foliar volatiles in association with a trap of a color in the green range of the visible light spectrum, contributing to the knowledge of the chemical ecology and the development of improved tools for the detection of A. planipennis infestations.
According to one aspect of the present invention, a composition for the attraction of A. planipennis is provided, comprising
(a) (3Z)-dodecen-12-olide and
(b) ash foliar volatiles, associated with a trap of a color in the green range of the visible light spectrum.
According to one embodiment of the composition aspect of the invention, the amount of (3Z)-dodecen-12-olide is a source dosage which emits 2.4-160 μg of (3Z)-dodecen-12-olide per day at about 25° C.
According to another embodiment of the composition aspect of the invention, the ash foliar volatiles comprise (3Z)-hexenol.
According to yet another embodiment of the composition aspect of the invention, the ash foliar volatiles comprise (3Z)-hexenol of a source dosage which emits 50-100 mg per day at about 25° C.
According to yet another embodiment of the composition aspect of the invention, the trap is a sticky prism tap of a green color defined by a wave length of 540-560 nm and a reflectance of 24-64% e.g. 540 nm and 49% reflectance.
According to yet another embodiment of the invention, the amount of (3Z)-dodecen-12-olide is a source dosage which emits 2.4-22 μg per day of (3Z)-dodecen-12-olide and the amount of (3Z)-hexenol is a source dosage which emits 40-60 mg per day of (3Z)-hexenol.
According to another aspect of the present invention, a method for the attraction of A. planipennis is provided, comprising applying to an insect habitat an insect attracting amount of (3Z)-dodecen-12-olide and ash foliar volatiles, associated with a trap of a color in the green range of the visible light spectrum.
According to one embodiment of the method aspect of the invention, the amount of (3Z)-dodecen-12-olide is a source dosage which emits 2.4-160 μg of (3Z)-dodecen-12-olide per day at about 25° C.
According to another embodiment of the method aspect of the invention, the ash foliar volatiles comprise (3Z)-hexenol.
According to yet another embodiment of the method aspect of the invention, the ash foliar volatiles comprise (3Z)-hexenol of a source dosage which emits 50-100 mg per day at about 25° C.
According to yet another embodiment of the method aspect of the invention, the trap is a sticky prism tap of a green color defined by a wave length of 540-560 nm and a reflectance of 24-64% e.g. wavelength 540 nm and 49% reflectance.
According to yet another aspect of the invention, a kit for the attraction of A. planipennis is provided, wherein the components (a) and (b) of the composition are maintained separately until use, and associated for use with a trap of a color in the green range of the visible light spectrum. The kit comprises a septum containing an insect attracting amount of (3Z)-dodecen-12-olide dissolved in a volatile solvent e.g. hexane which evaporates before use, leaving the (3Z)-dodecen-12-olide to emit therefrom, and a bubble cap containing an effective amount of neat ash volatiles e.g. (3Z)-hexenol, associated with the trap and maintained separately until use.
According to one embodiment of the kit aspect of the invention, the amount of (3Z)-dodecen-12-olide is a source dosage which emits 2.4-160 μg of (3Z)-dodecen-12-olide per day, and the ash foliar volatiles comprise (3Z)-hexenol of a source dosage which emits 50-100 mg per day, at about 25° C., and wherein the trap is a prism sticky trap of a green color defined by a wave length of about 540-560 nm and a reflectance of 24-64%.
When a sticky trap is used, no insecticide is required. However, if a non-sticky trap is used, an appropriate insecticide is included.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a flow diagram illustrating a process according to the invention for the synthesis of (3Z)-dodecen-12-olide.
FIG. 2 is a flow diagram illustrating a process according to the invention for the synthesis of (3E)-dodecen-12-olide.
FIG. 3 is a graph illustrating electroantennographic (EAG) dose-response curves of male and female A. planipennis antennae to (3Z)-dodece-12-olide (3ZLac) and (3E)-dodece-12-olide (3ELac) according to dosages applied to stimuli cartridges. EAG dose-responses (mean±SEM) are presented relative to a positive control standard (geranyl acetone, 1 μg applied dose).
FIG. 4 illustrates GC-FID/EAD responses of male and female A. planipennis antennae. The FID trace is a synthetic mixture of (3E)- and (3Z)-lactones.
FIGS. 5a-d are graphs illustrating proportions of male A. planipennis crawling up the test vs. control arms of a Y-tube olfactometer in 12 independent trials in response to: (a) (3E)-lactone, (3Z)-lactone or a 60:40 combination; (b) Phoebe oil (25 μl) alone or combined with either (3E)-lactone or (3Z)-lactone); (c) Phoebe oil (2.5 μl) alone or combined with (3E)-lactone or (3Z)-lactone; and (d) (3Z)-hexenol alone or combined with either (3E)-lactone or (3Z)-lactone. For each stimulus, the test treatment was compared with the control using a chi-square goodness of fit test.
FIG. 6 are bar graphs illustrating mean (±SE) catches of male and female A. planipennis on purple sticky prism traps baited with various combinations of (3Z)- and/or (3E)-lactone and host volatiles in field experiments carried out at two sites in (a) 2008 and two sites (b, c) in 2009. Sites were analyzed separately in 2009 due to the differences in sex ratio. Note differences in scale of X-axis. Prior to analyses, data were transformed using a natural log (n+1), however untransformed data are presented. Error bars reflect + or − one standard error of the least squares means. In 2008 (FIG. 2a), (3E)-lactone was not tested except that it was present in the synthetic (3Z)-lactone at 2%.
FIG. 7 are bar graphs illustrating mean (±SE) catches of male and female A. planipennis on green sticky prism traps baited with the different attractant combinations at (a) Anika and McKellar sites combined and (b) sites in Michigan, USA. Plotted values reflect the least squares means of 12 replicate blocks in total (untransformed data). Statistics (P>F) apply to natural log (n+1)-transformed data following ANOVA. Error bars reflect + or − one standard error of the least squares means.
FIG. 8. Dose-response curve for male and female A. planipennis to increasing dose of (3Z)-lactone loaded on rubber septa and deployed on dark green sticky traps in combination with (3Z)-hexenol. Plotted values reflect the means of 10 replicate blocks in total (untransformed data). Statistics (p>F) apply to natural log (n+1)-transformed data following ANOVA. Error bars reflect + or − one standard error of the mean. Letters above bars indicate significant differences among treatments compared against the control within each sex.
FIG. 9 (a and b). Influence of (3Z)-lactone with and without (3Z)-hexenol on attraction of male and female A. planipennis to dark green sticky traps placed at different heights with respect to the ash canopy: (a) low and (b) high. Plotted values reflect the least squares means of 10 replicate blocks in total (untransformed data). Statistics (p>F) apply to natural log (n+1)-transformed data following ANOVA. Error bars reflect + or − one standard error of the least squares means.
DETAILED DESCRIPTION OF THE INVENTION
Methods and Materials
Source of Insects.
Trees with larval A. planipennis were felled near Windsor and Sarnia, Ontario; infested logs were transported to the Great Lakes Forestry Centre in Sault Step Marie, Ontario. Storage and rearing protocols have been previously reported (Silk et al. 2009). Emerged adults were sexed and virgin males and females were kept on a 16:8 h L:D cycle and supplied with water and foliage of evergreen ash, Fraxinus uhdei (Wenzig) Linglesh.
Volatiles were collected from two groups of virgin adult males (n=18 and n=8) and two groups of virgin adult females (n=17 and n=18) feeding on ash leaves in separate 250 ml glass chambers (16:8 L:D at 22° C.). Adults were 10 d old when placed in the chambers in groups of 6-8 at one time; and were replaced as they died over the volatile collection period. Filtered air was drawn from the chambers at ˜0.1 L/min onto a pre-conditioned Super-Q® filter (˜200 mg) for 10-11 d. Volatiles were eluted using methylene chloride (3×2 mL) and concentrated to 10-20 μl under dry nitrogen.
Analytical Techniques and Purification.
Synthetic samples and extracts were analyzed by GC/MS on a Hewlett-Packard 5890 GC and a 5971 mass selective detector in the electron impact (EI, 70 eV) mode (Silk et al. 2007). The column used for analysis was a Supelco SPB-5 capillary (30 m×0.32 mm×0.25 μm film) in the splitless mode with helium as carrier gas. The injection port was at 220° C. and the oven temperature was programmed from 70° C., held for 1 min and then increased at 10° C./min to 240° C. and held for 30 min. Compounds were purified by flash chromatography on silica gel and, when required, by Kugelrohr distillation.
NMR (1H and 13C) was carried out on a Varian Innova 300 MHz spectrometer in CDCl3 with TMS as internal standard. IR spectra were recorded as thin liquid films on KBr discs with a Perkin Elmer 727B IR-spectrometer.
The macrocyclic lactone, (3Z)-dodecen-12-olide (1) (FIG. 1), was synthesized according to the procedure described by Boden et al. (1993) and used by Bartell et al. (2007) with the addition of a tert-butyldimethylsilyl (TBS) protecting group (which doubled the yield of the Wittig step). This involved ozonolysis of a TBS-protected alkenol (5) into a protected hydroxyaldehyde (6), Wittig reaction with a Wittig salt containing a protected aldehyde (3), removal of the TBS group to give 8, then hydrolysis of the acetal to give a (3Z)-unsaturated aldehyde 9, Lindgren oxidation (Lindgren and Nilsson 1973) to a carboxylic acid (10) and finally a Mitsunobu esterification (Kurihara et al. 1976) to effect the macrolactonisation. The synthesis of (3Z)-dodecen-12-olide was, therefore, successfully accomplished with the IR spectra, EI (70 eV) mass spectra and 1H and 13C NMR spectra closely matching those reported (Boden et al. 1993). Formation of (2E)-dodecen-12-olide and (3E)-dodecen-12-olide were found to be intrinsic to the synthesis at approximately 3% each. The (2E)-product, characterized by 1H NMR, was readily separated from the desired (3Z)-lactone by column chromatography. The (3E)-lactone, however, could not be separated from the (3Z)-lactone. 1H NMR supported the presence of ca. 3% of (3E)-lactone in the product.
The 3E-lactone [(3E)-dodecen-12-olide] (11) (FIG. 2) synthesis was successfully accomplished by a Julia-Kocienski olefination according to the methodology described by Blakemore et al. (1998). The Julia-Kocienski olefination of aldehyde 17 proceeded with 34% yield and ca 97% E stereochemistry (FIG. 2) to give olefin 18a. Thus, protection of alkenol 4 with ethyl vinyl ether (EVE) proceeded smoothly to give 16, and ozonolysis with reductive workup gave aldehyde 17. The phenyltetrazole (PT) sulfone 15 was synthesized by deprotonating 1-phenyl-1H-tetrazole-5-thiol 13 with sodium hydride and coupling it with commercially available 12 to give thioether 14. mCPBA oxidation of 14 furnished the PT sulfone 15. After the Julia-Kocienski olefination, double hydrolysis of the two acetals of 18a gave 19 and Lindgren oxidation of 19 gave the hydroxyacid 20. Finally, as reported by Boden et al. (1993), activation of the hydroxyl group using the Mitsunobu method modified according to Steglich (Justus and Steglich 1991) gave (3E)-dodecen-12-olide 11 in an overall yield of 14% from alkenol 4. Spectral data for (3E)-lactone [(3E)-dodecen-12-olide)] 11:
1H NMR (CDCl3, 400 MHz): δ 5.47-5.62 (10 line symmetrical multiplet, 2H), 4.12 (AA′XX′, 2H), 2.98 (d, 2H, J=7.0 Hz), 2.05 (m, 2H), 1.57 (m, 2H), 1.29-1.42 (m, 10H); 13C NMR (CDCl3, 100 MHz): δ 172.0, 135.4, 123.2, 64.5, 39.0, 31.4, 27.2, 26.34, 26.28, 25.7, 24.9, 23.6. IR (neat): cm−1 3027 (w), 2928 (s), 2855 (s), 1733 (s), 1666 (w), 1457 (w), 1375 (w), 1348 (w), 1246 (s), 1143 (m), 1111 (m), 1039 (m); MS (EI) Major peaks: 41 (base peak), 54, 67, 81, 95, 109, 121, 136, 150, 168, 178, 196.
Schlosser modification of the Wittig reaction (Schlosser and Christmann 1966) was initially employed in an attempt to make 18b starting from Wittig salt 3 and aldehyde 17, however, the E-selectivity of the reaction was very capricious, with 80% stereochemical purity being the best result out of a dozen attempts at the reaction. This was deemed to be unacceptable, and the much better ˜97% stereochemical purity obtained with the Julia-Kocienski olefination which gave 18a was much more satisfactory. Reagents and conditions of the syntheses of (3Z)-lactone and (3E)-lactone are as follows.
Synthesis of (3Z)-Dodecen-12-olide 1 ((3Z)-lactone) (after Boden et al. 1993).
a) 2-Propanol, HBr, CH2Cl2. PPh3, −10° C.−RT b) HC(OiPr)3, one pot c) TBSCl, imidazole, DMF, RT d) O3. Sudan III, CH2Cl2, −78° C. e) PPh3, −78° C.—RT f) 3+NaHMDS, PhCH3/THF (4:1), 0° C.−RT, then 6, −99° C.−RT g) TBAF, THF, RT h) TsOH, wet THF, reflux i) NaClO2, H2NSO3H, 1-methylcyclohexene, CH2Cl2/H2O (1:3), 0° C.−RT j) DEAD, PPh3, PhCH3, RT. HBr=Hydrobromic acid, CH2Cl2=Dichloromethane, PPh3=Triphenylphosphine, HC(OiPr)3=Triisopropylorthoformate, TBSCl=tert-butyldimethylsilyl chloride, DMF=Dimethylformamide, O3=Ozone, NaHMDS=Sodium Hexamethyldisilylamide, PhCH3=Toluene, THF=Tetrahydrofuran, TBAF=tetrabutylammonium fluoride, TsOH=para-Toluenesulfonic acid, NaClO2=Sodium chlorite, H2NSO3H=Sulfamic acid, DEAD=Diethyl azodicarboxylate
Synthesis of (3E)-Dodecen-12-olide 11 ((3E)-lactone); modified Julia-Kocienski olefination. k) 13+NaH, DMF, 0° C.-60° C., then 12, NaI, 60° C. l) mCPBA, NaHCO3, CH2Cl2, RT m) EVE, PPTS, CH2Cl2, RT n) 15+KHMDS, DME, −55° C., then 17, −55° C.−RT. NaH=Sodium hydride, NaI=Sodium iodide, mCPBA=meta-Chloroperoxybenzoic acid, NaHCO3=Sodium bicarbonate, EVE=Ethyl vinyl ether, PPTS=Pyridinium para-toluenesulfonate, KHMDS=Potassium hexamethyldisilylamide, DME=1,2-Dimethoxyethane.
GC-EAD Analysis and EAG Dose-Response Study.
EAG analyses were made by methods and equipment generally described by Cossé and Bartelt (2000). EAG connections were made by inserting a glass-pipette silver-grounding electrode into the back of an excised beetle head. A second glass-pipette silver-recording probe was placed in contact with the distal end of one antenna. Both pipettes were filled with Beadle-Ephrussi (Ephrussi and Beadle 1936) saline.
For the EAG-dose-response study, (3Z)- and (3E)-lactones were purified (99.9% purity by GC/MS) by high performance liquid chromatography (HPLC) using a Waters 515 pump, a Waters R401 refractive index detector, and a 25 cm by 0.46 cm i.d. silica column (Adsorbosphere Silica 5 μm, Alltech, Deerfield, Ill.), treated with silver nitrate as described by Heath and Sonnett (1980). Solvent was 8% ether in hexane. Ten micro liters of serially diluted solutions (methylene chloride) of synthetic (3Z)-lactone and (3E)-lactone were applied to filter paper strips (0.5 cm×5 cm, Whatman no. 1). The filter paper strips were placed in 14-cm-long Pasteur pipettes, hereafter referred to as stimulus cartridges, after 5 min at room temperature. Stimulus doses tested were 0.01, 0.1, 1, 10, and 100 μg. Male and female antennae were exposed to single 0.2s puffs of odor-bearing air at 5 ml/s by placing the tip of an stimulus cartridge into a hole of a glass tube (0.7 cm ID×20 cm), 10 cm from the outlet and 11 cm away from the antennal preparation. Airflow through the glass tube was humidified and set at 10 ml/s. Puff duration and airflow speeds were maintained by a stimulus flow controller (SFC-2, Syntech, Hilversum, The Netherlands). Stimuli cartridges were selected in random order, beginning with the lowest dosages and working upward to the highest dosages. Each puffed dosage was preceded and followed by a puff from a solvent blank cartridge (filter paper plus solvent). To compensate for possible deterioration of the antennal preparation, a standard control compound, geranyl acetone (1 μg dose) preceded dosages of stimuli compound. EAG amplitudes were normalized according to the responses to geranyl acetone by dividing the amplitude of the EAG generated by the test compounds by that of geranyl acetone. Dose-response series were replicated, using different antennal preparation for each replication, and the EAG responses were expressed as a percentage of the EAG responses to geranyl acetone. Each antennal preparation was tested with freshly prepared sets of stimuli cartridges. Male and female EAG responses were submitted to analysis of variance (ANOVA) using Statistica for Windows software (StatSoft Inc. Tulsa, Okla.)).
A Varian CP-3380 gas chromatograph with FID detector was modified for use with a GC-EAD signal recording device (IDAC-232). EAG data were analyzed using Syntech GC-EAD software v.2.6 (SYNTECH, The Netherlands). The column used for analysis was a Supelco SPB-5 capillary (30 m×0.32 mm×0.25 μm film) in the splitless mode with helium as carrier gas. The injection port was at 220° C. and the oven temperature was programmed from 70° C., held for 1 min and then increased at 10° C./min to 240° C. and held for 30 min. A number of GC-EAD runs on male and female volatiles were carried out. Both the (3Z)-lactone and (3E)-lactone were diluted to 10 μg/ml in hexane; 1 μl of diluted pheromone was injected for each GC-EAD run.
Ten nanograms was injected for the GC-EAD analysis consisting of 90% (3Z)-lactone and 10% (3E)-lactone using a DB-1 (15m×0.25 mm ID, 1 μm film) capillary column (J&W Scientific, Folsom, Calif.). The GC oven temperature program was 50° C. for 1 min, then increased at 20° C./min and held at 280° C. for 2 min. The GC-EAD responses of five male and five female EAB antennae were analyzed.
Effect of Light on (3Z)-Lactone.
To determine whether light would promote the isomerization from (3Z) to (3E)-lactone, 20 mg of (3Z)-lactone was placed neat on a glass slide 10 mm below a UV light (UVG-54 handheld UV-lamp, 254 nm, 6w; UVP Upland Calif., USA) for three d. Subsamples (taken as ˜1 mg in a pipette) were analyzed by GC/MS at regular intervals and the ratio of (3E):(3Z)-lactone was recorded. In addition, 6 mg of each of (3Z)- and (3E)-lactones (neat) were coated on the quartz surface of a cuvette and exposed outdoors to sunlight at 11° C. mean temperature for 9 d for an average of 5 h a day. Finally, (3Z)-lactone was coated (4 mg) on the dorsal surface of abdomen and elytra of 3 female EAB cadavers that were exposed to sunlight for 6 h per day for 1, 2 or 3 d at 10° C. mean temperature; cadavers were stored at 4° C. between sunny days. The lactones were removed from cuvettes and cadavers with hexane washing and analysed by GC/MS to determine the E:Z ratio.
Two-Choice Olfactometer Assays.
A Y-tube olfactometer (Analytical Research Systems Inc, Gainsville, Fla.) was used to test for attraction of A. planipennis to lactone isomers and host volatiles. The glass olfactometer (1.5 cm i.d.) had an 11 cm main stem that branched into two 9-cm arms. Each arm was connected to a cylinder that contained the stimulus. Charcoal filtered air was passed into each arm at a flow rate of 1.2 L/min. Treatments included the pheromone alone: (3Z)-lactone (10 μg); (3E)-lactone (10 μg); and 60:40 (3E):(3Z)-lactone (10 μg). Next, we tested bark sesquiterpenes and a green leaf volatile alone: Phoebe oil (25 μg and 2.5 μg) and (3Z)-hexenol (5 μg). We then tested the pheromone combined with bark sesquiterpenes: (3Z)-lactone (10 μg)+Phoebe oil (at both 25 μg and 2.5 μg); (3E)-lactone (10 μg)+Phoebe oil (at both 25 μg and 2.5 μg). Finally, we tested the pheromone combined with the green leaf volatile: (3Z)-lactone (10 μg)+(3Z)-hexenol (5 μg); and (3E)-lactone (10 μg)+(3Z)-hexenol (5 μg). Each stimulus (1 μl for single compound treatments and a total of 2 μl for two-compound treatments) was diluted in hexane, placed on a strip of filter paper and given one minute for the solvent to evaporate before being placed in the olfactometer. A second filter paper, treated with the equivalent volume of solvent was placed in the other arm of the olfactometer to serve as the control. The apparatus was rinsed with acetone after each treatment, and the arm attached to the test stimulus was randomized between replicates.
For each treatment, we tested increasing numbers of adults until we obtained a minimum of 12 beetles responding to the stimuli (either positively or negatively). To obtain this minimum, we tested 15-54 beetles per treatment. For each trial, a single A. planipennis (mature virgin male or female, >10 days old) was given ten minutes to choose between the two stimuli; adults were used only once in the bioassay. A choice was recorded when the beetle passed a “finish line”, 7 cm beyond the branching point of each arm. ‘No choice’ was recorded if the beetle failed to pass either finish line after the ten minutes. Beetles that did not select either the stimulus or the control (i.e., no choice) were excluded from a subsequent chi-square goodness of fit test used to test whether the ratio of beetles choosing the stimulus vs. the hexane control differed significantly from 1:1. A chi-square test was conducted for each independent trial.
Three trapping experiments were carried out in green ash plantations (F. pennsylvanica Marsch) with low-to-moderate A. planipennis populations about 40 km southeast of Sarnia, Ontario (42° 58′ 0 N, 82° 24′ 0 W) in 2008, 2009 and 2010. Trees at these sites were generally healthy in appearance with low or no signs of decline, and only a small number of trees had obvious signs/symptoms of infestation by A. planipennis. In Ontario sites, trees were 20-25 years old, 4-6 m tall, 10-15 cm in diameter, and spaced about 2 m apart within a row and 2.5 m between rows. In 2010, the trapping experiment was replicated at four sites in Michigan, USA, in addition to the sites in Ontario. Sites in Michigan were 10-100 years old, 10-30 m tall, 15-70 cm in diameter, and located in a mixed woodlot. Corrugated plastic “prism” traps (0.30 cm×35.00 cm×58.75 cm) were coated with stickem (Crook et al. 2008) (Synergy Semiochemicals Corp., Burnaby, BC) and hung using rig spreaders (Zing Products, Westport Mass., USA). Purple traps were suspended from metal stands at a height of 1.5 m (2008-2009), whereas green traps were hung in the mid-canopy from ropes tied between two trees at 2.5 m in Ontario and at 6 m height in Michigan (2010). In Michigan, traps were hung from a single line thrown over the lowest canopy branch. Light green traps (approx 540 nm wavelength) were the same as used by Francese et al. (2010). Traps were set within 1.5-2 m of trees, spaced 20-30 m apart, in a randomized complete block design. Traps were checked every 2 weeks and A. planipennis were collected, counted and sexed.
Experiment 1, conducted in Ontario in 2008, was designed to test for attractiveness of (3Z)-lactone (Bartell et al. 2007), alone and in combination with two types of host volatiles: bark sesquiterpenes (Crook et al. 2008) and a binary blend of green leaf volatiles ((3Z)-hexenol and (2E)-hexenol) (Poland et al. 2005, de Groot et al. 2008). We used purple prism traps, which at the time of this experiment were shown to be more attractive than traps of other colors (Francese et al. 2005), and which had been used successfully in other recent trapping experiments for A. planipennis (Crook et al. 2008, de Groot et al. 2008). Traps were baited with one of the following treatments: (3Z)-lactone; Phoebe oil (Synergy Semiochemicals Corp., Burnaby, BC); (3Z)-lactone+Phoebe oil; green leaf volatiles (GLVs) consisting of two bubblecaps, one containing (3Z)-hexenol and the other containing (2E)-hexenol (ConTech, BC); (3Z)-Lactone+GLVs; and unbaited controls. We selected Phoebe oil because it contained two additional sesquiterpenes that had been detected in ash trees and appeared to be more attractive than Manuka oil (Crook et al. 2008) and the (3Z)-hexenol and (2E)-hexenol combination based on results from de Groot et al. (2008). Release rates at 20° C. were estimated by weight loss as ca. 50 mg/d, 17 mg/d and 16 mg/d for Phoebe oil, (3Z)-hexenol, and (2E)-hexenol, respectively. (3Z)-lactone was emitted at ca. 80 μg/d at 20° C. from red rubber septa (Wheaton) impregnated with 5.0 mg per lure. Traps were out 10-24 Jun. 2008, replicated with 3 blocks at one site (Site A: Conservation area) and 7 blocks at the second site (Site B: Union Gas site). Lures were not changed during the experiment.
Experiment 2, conducted in Ontario in 2009, was designed to test the attractiveness of (3E)- vs. (3Z)-lactone, alone and in combination with Phoebe oil, based on results from 2008. Purple prism traps were baited with the following lure treatments: (3Z)-lactone; (3E)-lactone; Phoebe oil; (3Z)-lactone+Phoebe oil; (3E)-lactone+Phoebe oil; and unbaited controls. As in 2008, release rate of phoebe oil was ca. 50 mg/d at 20° C. The lactone lure consisted of a 1.5 ml PCR tube containing 50 mg of either (3E)- or (3Z)-lactone; a pipe cleaner wick was placed into the vial through a 1.0 mm hole with 2.0 mm of the wick protruding through the top of the tube (release rate=˜0.5 mg/d at 20° C. Traps were in the field from 2 Jun.-4 Aug. 2009, with 7 blocks at one site (Site B: Union Gas site) and 8 blocks at the other (Site C: Anika Mills site). Lures were not changed during the experiment.
Experiment 3, conducted in 2010, was designed to test the effect of the single green leaf volatile, (3Z)-hexenol (de Groot et al. 2008, Grant et al. 2010), as a potential kairomone in combination with either (3Z)- or (3E)-lactone. We used green sticky prism traps deployed in the ash canopy, which had recently been demonstrated to capture more A. planipennis than purple traps (Francese et al. 2008; Crook et al. 2009) particularly when baited with (3Z)-hexenol (Grant et al. 2010). Treatments tested were: (3Z)-lactone; (3E)-lactone; (3Z)-hexenol; (3Z)-lactone+(3Z)-hexenol; (3E)-lactone+(3Z)-hexenol; and unbaited controls. (3Z)- and (3E)-lactone were loaded at a source dosage of 1.0 mg each and emitted ˜22 μg/d at 25° C. from red rubber septa (Wheaton). The source dosage of 1.0 mg is taken from a solution of the lactone in hexane, which is absorbed into the red rubber septum. The solvent evaporates from the septum, which then emits the lactone at an effective rate of ˜22 ng/d at 23° C. This experiment was replicated in Ontario and the Michigan. In Ontario, traps were out 1 Jun.-14 Jul. 2010 with 7 blocks at one site (Anika Mills site) and 5 blocks at another site (McKellar conservation area). Traps were hung at 2.5 m above the ground in the bottom edge of the canopy. In Michigan, traps were out from 25 May-7 July at four different sites. All traps in Michigan in 2010 were deployed below the canopy; the trees were 10-30 m in height. The lactone lures were replaced every two weeks; the other lures were unchanged.
The effect of each attractant on mean catch of female and male A. planipennis was analyzed independently using ANOVA and a randomized complete block design. Sites were analyzed separately in 2009 due to differences in sex ratios. In 2010, sites in Ontario were analyzed separately from those in Michigan due to the considerable differences in stand conditions and height of traps with respect to the ash canopy. In all three experiments, a priori hypotheses about the treatments were tested with contrasts; tests were conducted as one-sided tests for increases in trap captures. The first contrasts tested whether a single-component lure ((3E)- or (3Z)-lactone, Phoebe oil or GLV) caught more beetles than the unbaited control; a second set of contrasts compared captures of two-component lures vs. single component lures to test for the effect of adding the second component. Residuals were tested for homogeneity of variance and normality, and a ln(y+1) transformation was used where necessary. We present the untransformed least squares treatment means and their standard errors, along with statistics (P>F) from ANOVA of transformed data. For the two component traps, the foliar volatiles, in this case comprising (3Z)-hexenol, is provided in a separate emitter ie. a ‘bubble cap” emitter from Contech of Vancouver, Canada, associated with the trap and containing a source concentration of 2-3 g of neat material, which emits 40-60 mg per day.
GC/MS of Collected Volatiles.
GC/MS analysis of extracts from female volatiles confirmed the presence of the (3Z)-lactone with retention time and EI-mass spectra identical with the synthetic material. The (3E)-lactone, if present, was below the detection limit (ca. <200 picograms injected) and could not be confirmed as being emitted by females in the laboratory. Neither lactone was detected in volatiles collected from male A. planipennis.
EAG Dose-Response Study and GC-EAD Analysis.
The EAG dose-response curves of male and female A. planipennis antennae for the two isomers of synthetic lactone are presented in FIG. 3. Female antennae did not respond differently to the (3Z)- and (3E)-lactone (F1,149=0.01, P=0.91). Similar results were obtained with the male antennae (F1,149=2.3, P=0.14). However, female antennae were more responsive to both (3Z)-lactone (F1,149=45.3, P<0.0001) and (3E)-lactone (F1,149=39.8, P<0.0001) than male antennae, particularly at higher doses. The mean responses of A. planipennis antennae to the geranyl acetone standard (1 μg applied dose) was −0.06±0.03 mV (±SD, n=80, 15 antennal preparations), while those to the solvent/air controls measured −0.03±0.03 mV (±SD, n=45, 15 antennal preparations).
GC/EAD analysis showed responses at the retention time of (3Z)-lactone (not (3E)-lactone) produced by females only confirming previously published results (Bartelt et al. 2007). This was confirmed by GC/MS analysis. GC-FID/EAD responses of male and female A. planipennis antennae is shown in FIG. 4 to a synthetic mixture of (3E)- and (3Z)-lactones; note the significant responses to both stereoisomers.
Effect of Light on (3Z)-lactone.
Exposure to UV-light had a considerable impact on the ratio of (3E):(3Z)-lactone The initial lactone sample had a (3E):(3Z) ratio of 0.028 which increased with time of exposure to UV light, reaching a ratio of 0.60 after three d. GC/MS confirmed that exposure to UV-light resulted in isomerization without producing any other secondary products except a small amount (<1%) of the conjugated isomer. Preliminary studies found that under our normal laboratory fluorescent lighting conditions, (3Z)-lactone is very stable and did not readily isomerize to the (3E)-lactone. In addition, storing (3Z)-lactone in a pyrex container filtered out the UV-light, also preventing photoisomerisation. Exposure of either lactone isomer in a quartz cuvette or on the surface of female A. planipennis cadavers in direct sunlight resulted in very slow isomerization even after 2-3 days.
Y-Tube Olfactometer Assays.
In the Y-tube olfactometer assay, males were significantly attracted to the (3E)-lactone (χ2=6.76, n=25, P=0.009), but not the (3Z)-lactone (χ2=2.88, n=17, P=0.09) or the 60:40 ratio (χ2=0.17, n=24, P=0.68) (FIG. 5a). Low doses of Phoebe oil were attractive to males (χ2=5.54, n=26, P=0.018) (FIG. 5c), whereas higher doses were significantly repellant (χ2=7.12, n=17, P=0.008) (FIG. 5b). Combining either lactone isomer with a low dose of Phoebe oil was not attractive to males (χ2=0.0, n=38, P=1.0 and χ2=0.08, n=48, P=0.773, for (3E) and (3Z)-lactone, respectively). Similarly, combining (3E)-lactone with the high dose of Phoebe oil was not attractive (χ2=0.11, n=9, P=0.74) and (3Z)-lactone combined with high dose of Phoebe oil was significantly repellant (χ2=8.33, n=12, P=0.004). Finally, males were highly attracted to (3Z)-hexenol (χ2=9.0, n=25, P=0.003) (FIG. 5d), the (3Z)-lactone+(3Z)-hexenol combination (χ2=5.4, n=15, P=0.02) (FIG. 5d), but not the (3E)-lactone+(3Z)-hexenol combination (χ2=0.059, n=17, P=0.88). Females were slightly attracted to a low dose of Phoebe oil (70% responded) (χ2=3.52, n=23, P=0.061) and to the (3Z)-hexenol (75% responded) (χ2=6.00, n=24, P=0.014), but did not respond in sufficient numbers for analysis in any other treatment.