Pheromone of the Banana-Spotting Bug , Amblypelta lutescens lutescens Distant ( Heteroptera : Coreidae ) : Identification , Synthesis , and Field Bioassay

1 Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Building 007, Room 326, BARC-West, 10300 Baltimore Avenue, Beltsville, MD 20705, USA 2 Department of Employment, Economic Development and Innovation, Horticulture and Forestry Science, 28 Peters Street, P.O. Box 1054, Mareeba, QLD 4880, Australia 3 School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia


Introduction
The fruit-spotting bug, Amblypelta lutescens lutescens Distant (Heteroptera: Coreidae), commonly known as the bananaspotting bug, is one of the most important insect pests of fruit and nut crops in tropical and subtropical Australia [1][2][3][4].Nymphs and adults feed on shoots and developing fruits of a wide range of commercial tree crops, causing significant production losses if pesticides are not regularly applied.Monitoring banana-spotting bug activity in crops is extremely difficult and currently relies on scouts assessing damage at weekly or fortnightly intervals throughout the season [5].This approach is reactive and not always effective, as well as being time consuming and expensive since a large proportion of a crop must be monitored because damage can be unevenly distributed throughout an orchard.Alternative monitoring tools based on semiochemicals, such as host plant volatiles and/or pheromones, are highly desirable because they could potentially be more reliable and easier to standardize.Studying male-specific compounds from Nearctic and Australasian true bugs, Aldrich et al. identified (E,E)-α-farnesene and (R,E)-nerolidol from A. lutescens lutescens, whereas a third compound with molecular weight 220 remained unknown [6].Blends of these chemicals could serve as an attractant pheromone for A. lutescens lutescens but their biological importance has not been demonstrated, nor has the structure of the unknown compound been elucidated.The aims of this paper were to address both of these issues.

Material and Methods
2.1.Insects.Amblypelta lutescens lutescens males were handcollected from a row of Murraya paniculata (orange jessamine) bushes on the northwest outskirts of Mareeba, Australia (16 • 59 S, 145 • 25 E), at the end of March 2009.Males were separated from females by the squarer shape of the tip of the abdomen when viewed laterally.The collected males were held in a 30 × 30 × 30 cm gauze-covered cage with M. paniculata fruit, green beans, and water at approximately 26 • C and 60-70% RH under natural light until aeration experiments.

Collection of Airborne
Volatiles from Males.Two samples of airborne extracts were collected in Mareeba from 14 male A. lutescens lutescens over a 4-and then a 3-day period in early April 2009.During aerations, the males were held in a 500 mL triple-neck vacuum flask with 3 green beans as food.The flask inlet was connected by silicone tubing (8 mm i.d.) to a modified Pasteur pipette (5 mm i.d.) containing 50 mm of acid-cleansed activated charcoal to filter the incoming air.The flask outlet was also connected by silicone tubing to an 18 cm long glass tube (5 mm i.d.) containing 50 mm of Super Q absorbent (Alltech, USA) held in place on each end by glass wool.Silicone tubing connected the glass tube to an Airchek 224-PCXR8 Sampler pump (SKC Inc., Eight-Four PA), which operated at 250-500 mL/min during the aerations.All connections in the inlet and outlet lines were sealed with Teflon tape.Sampling was conducted at approximately 26 • C and 60-70% RH.No bugs died during either sampling period.Once collected, the aeration samples in their glass tubes were sealed in individual airtight containers and sent to USDA-ARS, Beltsville, where they were eluted with 600 μL of hexane.The extracts were concentrated to 50 μL under a gentle stream of N 2 and analyzed by gas chromatographymass spectrometry (GC-MS) on an Rtx-5MS and by GC on Chiraldex B-DM columns (see below).

Analytical Methods.
Electron-impact (EI) mass spectra were obtained at 70 eV with an Agilent Technologies 5973 mass selective detector interfaced with a 6890 N GC equipped with a 30 m × 0.25 mm i.d.× 0.25 μm film Rtx-5MS (Restek Corporation, Bellefonte, PA, USA) column.Column temperature was maintained at 50 • C for 5 min and then raised to 260 • C at 10 • C/min.Helium was used as a carrier gas at 1 mL/min.Enantioselective GC analyses of nerolidol and racemic and optically active (E,E)-α-farnesene oxide were performed on an Agilent Technologies 6890 N GC equipped with an FID detector and 30 m × 0.25 mm i.d.× 0.12 μm film Chiraldex B-DM column (Astec, Whippany, NJ, USA).Column temperature was maintained at 100 • C for 2 min and then raised to 180 • C at 2 • C/min.Hydrogen was used as a carrier gas at 1 mL/min. 1 H and 13 C NMR spectra of compounds 6 and 8 were obtained on a Bruker AVIII-600 MHz spectrometer. 1 H NMR spectra of compounds 4 and 10 and 1 H-1 H COSY of 6 were obtained on a Bruker AV-400 MHz spectrometer.The 1 H NMR spectrum of compound 5 was obtained on an Anasazi 90 MHz spectrometer (Indianapolis, IN).Chemical shifts are reported in δ units and referenced to the residual CDCl 3 solvent signal.Optical rotations were obtained on a Perkin-Elmer 241 polarimeter with a 1.0 mL cell.Thin layer chromatography analyses were conducted on Whatman AL SIL G/UV plates using 20% ethanol solution of phosphomolybdic acid and/or UV for visualization of spots.Flash chromatography was carried out with 230-400 mesh silica gel (Fisher Scientific, Fair Lawn, NJ).

(10R,3E,6E)-3,7,11-Trimethyl-10,11-epoxy-1,3,6-dodecatriene ((R,E,E)-α-farnesene
[α] D 25 = +0.2E,E)-α-farnesene (2), 1.2 mg (R,E)-nerolidol (3), and 0.6 mg (R,E,E)-α-farnesene oxide (4); no. 2, 0.2 mg (E,E)-α-farnesene, 1.2 mg (R,E)-nerolidol, 0.6 mg (R,E,E)-αfarnesene oxide, and 0.6 mg hexyl hexanoate (1).Relative proportions of components in these blends were based on relative areas of peaks in the aeration extract.The pheromone components plus 5% BHT (butylated hydroxytoluene, or 2,6-di-tert-butyl-4-methylphenol) stabilizer were applied to rubber septa in hexane solutions [16] at Beltsville and shipped to Australia.The test site was located at 16 • 58 S, 145 • 20 E at an elevation 450 m, and has a mean annual rainfall of 1,110 mm.The station has several thousand mango trees, including an extensive gene pool block, mixed varietal block, a large number of hybrids, and pure blocks containing Kensington Pride, R2E2, and Keitt varieties.The traps were set out in two rows of mature trees (16-20 years old) of mixed varieties at one trap per tree with several trees without traps between them.All trees contained maturing fruit at the time of trap deployment in October.Traps consisted of panels of green-colored twin wall polypropylene sheeting (Corflute BFS Plastic Pty. Ltd., Salisbury, QLD, Australia, 0.3 × 25 × 30 cm), placed 2-2.5 m above ground and fixed to branches with wire so that they remained rigid even in windy conditions.Traps were covered on each side with double-sided cloth tape coated with emulsion acrylic adhesive (Henkel Aust.Pty. Ltd., Kilsyth, VIC, Australia).Each trap had a 3 cm diameter hole in the center in which the rubber septum containing the pheromone was fastened.There were five replicates of each pheromone treatment and five untreated control traps.Traps were assigned sequentially for treatments, repeated for each subsequent replicate down the rows.Pheromone lures were stored, sealed, and refrigerated until use, and traps were baited the morning of deployment.Traps were inspected 4 days after their initial deployment, when adult males, females, and nymphs were removed and recorded.

Analysis of Trapping Data.
The data are presented as number of A. lutescens lutescens adult males, adult females, and nymphs per trap per day.Data were (x + 1)-transformed to compare treatments by analysis of variance using GenStat 11th Edition [17].Significantly different means were compared by the least significant difference method at P = 0.05.

Results and Discussion
3.1.Identification and Synthesis.The total ion chromatogram (Figure 1) revealed several compounds, among which were the previously identified (E,E)-α-farnesene ( 2) and (E)nerolidol (3) [6], plus compound 4, the mass spectrum of which matched that of the unknown male-specific compound showing a molecular weight 220, previously found by Aldrich et al. [6].In addition to these compounds, in the aeration sample from males we found a relatively large amount of hexyl hexanoate (1), a trace of which was previously found in female but not male A. lutescens lutescens [6].Because the unidentified compound 4 conceivably could have been an epoxide of (E,E)-α-farnesene, we decided to start with a random epoxidation of (E,E)-α-farnesene that had already been reported [8,15].We repeated the mchloroperbenzoic acid epoxidation of (E,E)-α-farnesene in methylene chloride and found that the monoepoxidation product at the 10,11 double bond matched compound 4 by GC retention time and mass spectrum.The Chiraldex B-DM GC column resolved (Figure 2) synthetic racemic farnesene oxide 4 (lower chromatogram) and racemic (E)nerolidol to baseline (middle chromatogram).Analysis of the A. lutescens lutescens aeration sample of males on the Chiraldex B-DM column (upper chromatogram) revealed that only one enantiomer of (E)-nerolidol and farnesene oxide were produced by the bug.Aldrich et al. [6] had already determined that (E)-nerolidol produced by A. lutescens lutescens had the (R)-configuration, and we confirmed this assignment by showing that synthetic (R,E)-nerolidol had the same GC retention time as the natural compound, or the first eluting enantiomer of racemic (E)-nerolidol.Determination of the enantiomeric composition of (E,E)-α-farnesene-10,11-epoxide 4 produced by A. lutescens lutescens necessitated synthesis of both enantiomers.Thus, we considered a route developed by Moore et al. [18] for synthesis of ocimene epoxides, which was based on a Sharpless asymmetric dihydroxylation and further stereoselective cyclization of intermediate chiral diols to epoxides through secondary mesylates.In fact, asymmetric dihydroxylation of (E,E)-α-farnesene 2 had been reported in the literature [14] but only in very low yields (11 and 15% for (−) and (+) diols, resp.) for a 10,11-dihydroxylation product.The low yield of the desired diol was a reflection of the multiple sites of unsaturation present in (E,E)-α-farnesene that were susceptible to asymmetric dihydroxylation.To overcome this shortcoming, we developed a new route presented in Scheme 1, which was essentially an enantioselective version of the synthesis of racemic (E,E)-α-farnesene-10,11-epoxide developed by Fielder and Rowan [15].The synthesis started with alkylation of 3-methyl-3-sulfolene with geranyl bromide, as described in several reports (see Section 2).The key step, asymmetric dihydroxylation of the intermediate 5 with ADmix α, was highly regioselective (as was MCPBA epoxidation of 5 [15]) and offered sulfolenediol 6 in 88% yield. 1 H NMR spectrum of diol 6 recorded at 600 MHz clearly shows the presence of two diastereomers with separation of several signals, including (CH 3 ) 2 COH, even though the two chiral centers are separated by five carbon atoms (see Section 2).The observed regioselectivity of asymmetric dihydroxylation of 5 was in stark contrast with direct asymmetric dihydroxylation of (E,E)-α-farnesene [14] and was similar to the analogous dihydroxylation of ocimene [18] and squalene [19], whereby the reactions occurred primarily at the terminal trisubstituted double bonds.
Thermal elimination of sulfur dioxide in 6 was accomplished in pyridine [10] to minimize side reactions and polymerization due to the acidity of SO 2 .For the same reason, mesylation of diol 8 was also conducted in pyridine, and in the last step, the crude mesylate was cyclized to epoxide 4 with potassium hydroxide in methanol [20].Analogously, sulfolene 5 was dihydroxylated with AD-mix β to provide diol 7, which was then thermally transformed to diol 9. Cyclization of the latter as described above furnished enantiomeric farnesene epoxide 10.Both 4 and 10 were prepared in ≥94% ee as judged by GC analysis on the Chiraldex B-DM column, which indicated that asymmetric dihydroxylation proceeded with high enantioselectivity, as anticipated from the original report by Sharpless et al. [13] and the subsequent results of Moore et al. [18].High optical purities of epoxides 4 and 10 were also consistent with previous reports [18,20] and indicative of highly stereoselective, base-catalyzed S N i substitution in the cyclization step.Overall, the route capitalizes on the much higher regioselectivity of dihydroxylation of sulfolene 5 compared to direct dihydroxylation of (E,E)-α-farnesene and, in fact, does not add additional steps to the synthesis of diols 8 and 9 (and epoxides 4 and 10) because one of the best known synthetic routes to (E,E)α-farnesene includes intermediate 5 [11].With both enantiomers of farnesene epoxide 4 and 10 available, we identified the later eluting peak in the gas chromatogram of the racemic epoxide on Chiraldex B-DM (Figure 2, lower chromatogram) as the (R)-enantiomer and, hence, determined that the natural farnesene epoxide (Figure 2, upper chromatogram) collected from A. lutescens lutescens had the (R)-configuration.

Field Bioassay.
In preliminary trapping experiments pheromone lures were deployed on painted plywood panels covered with Tangle-Trap insect barrier gel (The Tanglefoot Co., Grand Rapids, MI, USA).When deployed in avocado and mango orchards, these traps baited with either the 3-or 4-component pheromone lures (see Section 2) only caught 0.01-0.02 A. lutescens lutescens/trap/day.It was observed that many bugs attracted to the pheromone lures were able to escape from the traps unless caught on their backs or sides.
The Corflute panels covered by adhesive tape were much more effective at trapping A. lutescens lutescens and, when deployed with the 3-and 4-component pheromone lures in mangoes, caught up to ± 0.23 fruit-spotting bugs/trap/day (mean ± SE) (Table 1).When run in the same trial, the plywood panels with a tangle-trap gel caught a maximum of 0.10 ± 0.06 fruit-spotting bugs/trap/day.
There was no statistical difference in the total numbers of A. lutescens lutescens attracted to either the 3-or 4component lures, and traps baited with both caught significantly more bugs than the untreated control traps (Table 1).Thus, hexyl hexanoate does not seem to be a component of the attractant pheromone for A. lutescens lutescens.Both pheromone treatments attracted adult males, adult females, and nymphs, without a clear dominance of one sex or stage over another.Hence, our results indicate that the blend of male-specific compounds 2-4 found in the male A. lutescens  lutescens aerations contained the aggregation pheromone for this species.Whereas (R,E)-nerolidol had been identified previously from at least two bug species [6] and (E,E)α-farnesene was recently identified as a termite alarm pheromone [21], to our knowledge, (R,E,E)-α-farnesene-10,11-oxide 4 has not been reported as a component for any other insect species.More research is needed to determine whether the actual pheromone consists of one, two, or all three of these compounds, and for practical purposes, the optimal dose(s) and importance of chirality of the farnesene oxide must be determined.Preliminary experiments have indicated that doubling or tripling the emission rate of the 3-component lure as used above did not increase bug catches.The recent deregistration of endosulfan in Australia has significantly reduced the insecticidal options for the control of A. lutescens lutescens, and an effective monitoring tool based on a pheromone lure may become an essential tool in the management of the banana-spotting bug.
followed by the same letter are not significantly different at P = 0.05.

Table 1 :
Mean numbers of A mblypelta lutescens lutescens trapped in a mango crop in north Queensland by sticky panels containing 3-and 4-component pheromone lures over a 4-day period in October 2011.