J Chem Ecol (2006) 32: 2543–2558 DOI 10.1007/s10886-006-9163-3
Male-Produced Aggregation Pheromone Compounds from the Eggplant Flea Beetle (Epitrix fuscula): Identification, Synthesis, and Field Biossays
Bruce W. Zilkowski & Robert J. Bartelt & Allard A. Cossé & Richard J. Petroski
Received: 4 April 2006 /Revised: 26 June 2006 / Accepted: 6 July 2006 / Published online: 31 October 2006 # Springer Science + Business Media, Inc. 2006
Abstract Volatiles from the eggplant flea beetle, Epitrix fuscula Crotch (Coleoptera: Chrysomelidae), feeding on host foliage, were investigated. Six male-specific compounds were detected and were identified through the use of mass spectrometry, nuclear magnetic resonance (NMR) spectrometry, chiral and achiral gas chromatography, high-performance liquid chromatography, electrophysiology (gas chromatography-electroantennography, GC–EAD), and microchemical tests. The two most abundant of the six compounds were (2E,4E,6Z)-2,4,6-nonatrienal (1) and (2E,4E,6E)-2,4,6-nonatrienal (2). The other four compounds, present in minor amounts, were identified as himachalene sesquiterpenes; two of these, 3 and 4, were hydrocarbons and two, 5 and 6, were alcohols. All four sesquiterpenes were previously encountered from male flea beetles of Aphthona spp. and Phyllotreta cruciferae. Synthetic 1 and 2 matched the natural products by GC retention times, mass spectra, and NMR spectra. Sesquiterpenes 3–6 similarly matched synthetic standards and natural samples from the previously studied species in all ways, including chirality. Both natural and synthetic 1 and 2 gave positive GC–EAD responses, as did sesquiterpenes 3, 5, and 6. Field trials were conducted with a mixture of 1 and 2, and the baited traps were significantly more attractive than control traps to both male and female E. fuscula. The E. fuscula pheromone has potential for monitoring or controlling these pests in eggplants.
Key words Flea beetles . Epitrix fuscula . Coleoptera . Chrysomelidae . aggregation pheromone . (2E,4E,6Z)-2,4,6-nonatrienal . (2E,4E,6E)-2,4,6-nonatrienal . sesquiterpene . himachalene . field trials
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the Department of Agriculture. B. W. Zilkowski (*) : R. J. Bartelt : A. A. Cossé : R. J. Petroski USDA Agricultural Research Service, National Center for Agricultural Utilization Research, Crop Bioprotection Research Unit, 1815 North University Street, Peoria, IL 61604, USA e-mail: [email protected] 2544 J Chem Ecol (2006) 32: 2543–2558
Introduction
The eggplant flea beetle, Epitrix fuscula Crotch (Coleoptera: Chrysomelidae), is an important pest of eggplants. Found throughout most of the United States, E. fuscula is most common in the south. It is the most prominent insect pest of eggplant in Arkansas, requiring multiple insecticide applications after transplanting for beetle control (McLeod et al., 2002). Adult beetles can be especially damaging in the spring and are capable of defoliating young eggplant transplants in as little as 12 hr (Sorensen and Baker, 1994; Andersen, 2000). Flea beetle feeding, including from E. fuscula, is an important cause of crop failure in eggplants grown by organic methods (Patton et al., 2003). A number of cultural practices recommended for flea beetle control include trap crops, row covers, removal of crop residues, sticky traps, and careful timing of plantings (Sorensen and Baker, 1994; Delahaut, 2001; Kuepper, 2003). An effective detection and monitoring tool, such as a pheromone, could assist in the control of E. fuscula. The life history of E. fuscula was reported by Sorensen and Baker (1994). Briefly, adults overwinter in soil or ground debris and emerge and mate in early spring. Females lay eggs near the base of host plants. Larvae feed on roots or tubers and pupate in the soil when mature. Adult beetles emerge in about 7 to 10 days and begin to feed on foliage. In the southern U.S., the eggplant flea beetle is thought to have at least two generations per year (Sorensen and Baker, 1994; Capinera, 2001). This species has a limited host range, feeding primarily on solanaceous plants (Capinera, 2001). It causes damage to potatoes and sugar beets, and will feed on some weed species such as horse nettle and pokeweed (Sorensen and Baker, 1994). Adults cause the same type of characteristic, leaf-feeding damage associated with other flea beetles, consisting of numerous, small, irregular “shot holes” (Kuepper, 2003). Previously, pheromones have been reported from both male and female flea beetles. Males of Longitarsus jacobaeae Waterhouse were reported to be attracted to cues associated with conspecific females, although no chemical compounds were identified (Zhang and McEvoy, 1994). The first report of a male-produced pheromone in a flea beetle was for Phyllotreta cruciferae (Peng and Weiss, 1992; Peng et al., 1999), detected from feeding beetles by laboratory and field bioassays. Subsequently, six male-specific sesquiterpenes were found in this species and fully characterized, and four of the six were synthesized (Bartelt et al., 2001; Muto et al., 2004; Mori, 2005). Soroka et al. (2005) found that a blend of these compounds was attractive in the field to both male and female P. cruciferae and that even greater attraction was possible when the blend was combined with allyl isothiocyanate (AITC), a previously known, host-related attractant for P. cruciferae (Vincent and Stewart, 1984; Pivnick et al., 1992). Tóth et al. (2005) performed a similar field trial in Hungary by using pure enantiomers of P. cruciferae pheromone components in combination with AITC and achieved similar results, attracting P. cruciferae as well as some other, congeneric species. The same male-specific compounds emitted by P. cruciferae, plus two additional sesquiterpene alcohols, have also been found in the volatiles emitted from males of three Aphthona flea beetle species, A. flava, A. czwalinae, and A. cyparissiae (Bartelt et al., 2001), but field bioassays have not yet been performed. By using techniques that were employed with P. cruciferae and Aphthona spp., a male- produced pheromone was demonstrated in E. fuscula. The identification of six male- specific compounds and the synthesis and field activity of the two major components are reported. J Chem Ecol (2006) 32: 2543–2558 2545
Methods and Materials
Insects
In early May of 2004, overwintered E. fuscula adults were collected from eggplants on an organic farm located in Farmington, IL, USA. In 2005, beetles were again collected from the same site and from an eggplant research plot at National Center for Agricultural Utilization Research (NCAUR), in Peoria, Illinois. The sex was determined under a microscope, by using the abdominal characteristics described for Phyllotreta (Smith, 1983). The fifth abdominal sternum of females appears as a simple, smooth surface, but in males contains an apical median lobe. The beetles were used for collection of volatiles in the laboratory.
Collection of Volatiles
Volatiles were collected from groups of males, females, or mixed-sex beetles feeding on eggplant leaves, and from eggplant leaves alone, as described previously for another species and host (Bartelt et al., 2006). Briefly, beetles and foliage were placed in 45×3 cm (ID), horizontal glass tubes, equipped with Super-Q (Alltech, Deerfield, IL, USA) filters on both ends and through which air was drawn (300 ml/min) by vacuum. The inlet filter cleaned incoming air, and the second filter trapped the volatiles emitted within the tube. Typically, 10 beetles were used per tube, but numbers ranged from 3–20. A single eggplant leaf, about 9 cm in length and 5 cm at its greatest width, served as a food source and was replaced every time volatiles were collected. To keep the leaf fresh, the petiole was placed in a 5-ml glass vial containing water. A Teflon® seal, held in place with a ring-shaped screw cap, kept water from spilling. Collection duration was 1 to 4 d, and collected volatiles were recovered by rinsing the outlet Super Q filter with 400 μl of hexane into a vial. Collectors were kept in an incubator at 27°C with a relative humidity of about 50%. Light was provided by eight 40-W fluorescent tubes set about 0.5 m above and behind the collection tubes, and the daily light cycle was 16:8 hr light/dark.
Gas Chromatographic/Mass Spectrometric and Gas Chromatographic Analysis
All volatile collections were analyzed by coupled gas chromatography/mass spectrometry (GC–MS), and comparisons were made among collections from feeding males, females, and mixed sexes and from host plants only. The analyses were conducted on a Hewlett Packard 5973 mass selective detector, interfaced to a Hewlett Packard 6890 GC. For most analyses, a 30-m DB-5MS capillary column (0.25 mm ID, 1.0 μm film thickness, J&W Scientific, Folsom, CA, USA) was used. The temperature program was 50°C for 1 min, then rising to 280°C at 10°C per min and holding for 5 min at 280°C. The temperature of the splitless inlet was 200°C, and the transfer line temperature was 285°C. The Wiley MS library (Wiley, 1995) was installed on the data system. Chiral GC–MS analysis was conducted for some samples (see below) by using a 30-m Cyclodex-B column (0.25 mm ID, 0.25 μm film thickness, J&W Scientific). Temperature program was 50°C for 1 min, then rising at 30°C/min to a final temperature of either 120° C or 130°C. A Hewlett Packard 5890 GC was used for quantitation and was equipped with a DB-1 column (as above), splitless and cool-on-column inlets, and flame-ionization detector. Estimation of amounts of selected compounds in samples was by the external standard method, relative to nonadecane, by using the splitless inlet (200°C). Septum release rates 2546 J Chem Ecol (2006) 32: 2543–2558 were measured using the internal standard method (nonadecane, 4.09 μg per sample) and cool-on-column injections (with inlet temperature tracking the oven temperature).
Electrophysiology
Coupled GC–electroantennographic (GC–EAD) analyses were carried out on a Hewlett Packard 6890 GC, interfaced to antennal preparations. Amplified EAD and GC profiles were obtained simultaneously and analyzed by Syntech GC–EAD software. General methods and equipment have been previously described by Cossé and Bartelt (2000).
Liquid Chromatography
Fractionation of collected volatiles on open columns of silica gel (6×0.5 cm ID, in Pasteur pipettes), followed by GC–MS analysis of fractions, gave information on compound polarity and served as an initial purification step. Elution was with hexane, then 10% ether in hexane, and finally with 25% ether in hexane (3 column volumes per solvent). High-performance liquid chromatography (HPLC) and other techniques were applied to these silica gel fractions to gain further information about compounds 1–6 (Fig. 1). For HPLC, a Waters 515 pump (flow rate=1 ml/min) and a Waters R401 differential refractometer detector were employed. A Supelcosil LC-SI silica column (25 cm, 0.46 cm ID, 5 μm particle size, Supelco, Bellefonte, PA, USA) was used for purifying aldehydes 1 and 2 before hydrogenation and nuclear magnetic resonance (NMR), and the solvent was 10% ethyl ether (redistilled) in hexane. A silica column (Adsorbosphere Silica 5μ, a 25×4.6-mm ID silica column; Alltech) that had been treated with silver nitrate
O O 9 H 1 1 2 4 6 9 H 2 4 6 1 2
H H H H 1 9 1 9 1 9 1 9 9a 9a 9a 9a 3 4a 3 4a 3 4a 3 4a 5 5 HO 5 5 HO 3 456
5 4a 2 1 9a 9 7
Fig. 1 Compounds 1–6, detected during the analysis of volatiles from male E. fuscula feeding on eggplant leaf. Compound 7, ar-himachalene, derived from E. fuscula 3, to determine absolute configuration (see text). Chemical Abstracts index names: 1,(2E,4E,6Z)-2,4,6-nonatrienal; 2,(2E,4E,6E)-2,4,6-nonatrienal; 3, (9R,9aS)-5,6,7,8,9,9a-hexahydro-3,5,5,9-tetramethyl-1H-benzocycloheptene; 4,(9R,9aS)-2,3,5,6,7,8,9, 9a-octahydro-5,5,9-trimethyl-3-methylene-1H-benzocycloheptene; 5,(3R,9R,9aS)-2,3,5,6,7,8,9,9a-octahy- dro-3,5,5,9-tetramethyl-1H-benzocyclohepten-3-ol; 6,(3S,9R,9aS)-2,3,5,6,7,8,9,9a-octahydro-3,5,5,9-tetra- methyl-1H-benzocyclohepten-3-ol; 7,(5R)-6,7,8,9-tetrahydro-2,5,9,9-tetramethyl-5H-benzocycloheptene J Chem Ecol (2006) 32: 2543–2558 2547
(Heath and Sonnet, 1980) was used for separating hydrocarbons 3 and 4, with 0.5% 1- hexene in hexane as solvent. All effluent was collected in consecutive 1-ml fractions.
Hydrogenation
Microscale hydrogenation of HPLC-purified 1 and 2 (combined) was conducted over 10% palladium on carbon. A sample of 1 and 2 (ca. 100 ng) in a tapered vial was taken to dryness under a stream of argon and immediately redissolved in CH2Cl2 (100 μl). A small amount of catalyst (<1 mg, barely visible in the solution) was added. By using a fine needle, hydrogen was bubbled through the solution for 5 min at room temperature. Then, the solution was concentrated about 10-fold under argon and analyzed by GC–MS.
NMR Spectroscopy
NMR spectra were acquired on a Bruker Avance 500 instrument with a 5-mm inverse broadband probe with a Z-gradient. Proton and COSY spectra were obtained for beetle- derived 1 and 2 in CDCl3 and for synthetic 1 and 2 in both CDCl3 and deuterobenzene, as described below.
Chiral Analysis of Compounds 3, 4, 5, and 6
The enantiomers of 4 are known to separate on a Cyclodex-B capillary column (Bartelt et al., 2001), and the hydrocarbon silica gel fraction of the natural sample, which contained 4,was compared with racemic synthetic 4 on that column (final temperature 130°C). The enantiomers of 3 do not separate on Cyclodex-B (Bartelt et al., 2001), but an alternative analysis was possible: Compound 3 in the hydrocarbon silica fraction was separated from 4 by HPLC on the silver nitrate column (3 eluting 3–4 ml after injection, and 4,at6–7ml),and then 3 was converted to ar-himachalene (7; Fig. 1) by treatment with 2,3,5,6-tetrachlorohy- droquinone (Mehta and Singh, 1977; Bartelt et al., 2001), and the product compared with racemic 7 on Cyclodex-B (final temperature 120°C). The 25% ether hexane silica gel fraction from E. fuscula, which contained alcohols 5 and 6 but was free of 3 and 4,wastreatedwitha
O O
H O 8 a 9
9 HO 2 b 10 c
O
H 1 abc 11
Fig. 2 Synthesis of aldehydes 2 and 1 (see text). Reagents and details for steps: (a) triethyl 2- phosphonoacetate, either in tetrahydrofuran with BuLi as base or in hexane with Li t-OBu as base, 0°C, warming to room temperature (RT), 1 hr, 88% yield; (b) diisobutylaluminum hydride in hexane/ether (1:1), 0°C, 1 hr, 90% yield; (c) MnO2 in methylene chloride, RT, 16 hr, 85%. Aldehyde 1 was prepared in the same way except that (2E,4Z)-2,4-heptadienal was the starting material instead of 8 2548 J Chem Ecol (2006) 32: 2543–2558 strong cation exchanger (acidic form) to dehydrate 5 and 6 (Bartelt et al., 2001). The resulting compound 4 was analyzed on the Cyclodex-B column as above.
Chemicals
Aldehydes 1 and 2 were synthesized, and sesquiterpenes 3 to 7 were available from previous research (Bartelt et al., 2003; Muto et al., 2004). Synthesis of 2 (Fig. 2) used commercial (2E,4E)-2,4-heptadienal (8) (Sigma-Aldrich, St. Louis, MO, USA) as the starting material. The chain was extended by a Wittig–Horner reaction with triethyl 2-phosphonoacetate (Boutagy and Thomas, 1974) to form the triene ester (9); both lithium t-butoxide in hexane (Petroski and Weisleder, 2001)andn-butyllithium in tetrahydrofuran (Bartelt et al., 1990) were satisfactory base/solvent combinations. The ester was reduced to alcohol 10. In an initial trial, reduction was performed with LiAlH4 (Bartelt et al., 1990), but this reagent also gave some reduction of the olefin system. Subsequent reductions were performed with diisobutylaluminum hydride (Miller et al., 1959; Kreft, 1977), and the unwanted overreduction did not occur. In either case, the resulting alcohol was oxidized to aldehyde 2 with MnO2 (Petroski, 2003). Aldehyde 1 was prepared by the same route (Fig. 2), but the starting material, (2E,4Z)- 2,4-heptadienal (11), was not commercially available. Instead, this was prepared by the method of Petroski (2003). By GC–MS analysis, synthetic 1 contained 12% of 2 as an impurity immediately after synthesis. The product was diluted to a concentration of 27 μg/μl in CH2Cl2 and stored at −70°C until needed for field tests. Instability of 1 was a concern, and to evaluate its lability a solution was kept at ambient laboratory conditions and periodically analyzed. Synthetic 1 (100 μg in 1.8 ml of hexane) was added to a 2-ml glass autosampler vial with crimp-top septum cap, along with undecanol (22.7 μg) as internal standard. GC analysis was performed immediately after preparing the sample (1-μl injection through the cool-on-column inlet), and subsequent injections were made every 24 hr for 3 d (the Teflon® lined septum top was not replaced during the test).
Field Lures
Red rubber septa (Sigma-Aldrich) were cleaned by Soxhlet extraction for 6 hr with CH2Cl2 and allowed to air dry overnight. To prepare septa, a solution of synthetic 1 was applied (500 μgin20μl), followed by CH2Cl2 (300 μl). After the solvent soaked in, the septa were aired 1 hr in a fume hood and then kept in a tightly capped glass jar. Septa were always used in the field the same day they were prepared and discarded after 24 hr. The emissions of compounds 1 and 2 from freshly prepared septa were analyzed in the laboratory. Three septa were placed individually in volatile collectors, and collections of approximately 1-d duration were made for 3 consecutive days and quantitated by GC.
Field Tests
Field tests were conducted during three periods and in two areas. The three periods were late spring (7 d, May 26 to June 1, 2005), mid summer (5 d, July 11 to July 15, 2005), and late summer (10 d, August 2 to August 19, 2005). The first test area was a commercial organic vegetable farm in Farmington, IL, that contained both eggplant (var. Black Bell, Rosa Bianca, and Pingtung Long) and potatoes (var. Kennebec, Pontiac, Red Norland, All J Chem Ecol (2006) 32: 2543–2558 2549
Blue, Cranberry Red, and Onaway). The second area was a research plot located at the NCAUR that was planted only in eggplant (var. Black Beauty and Salangana hybrid). Traps used in the field trials were yellow sticky cards, coated with adhesive on both sides (“Sticky Strips,” Olson Products, Medina, OH); these were cut in half (final size, 15×15 cm). Holes were punched on opposite sides, and the traps were secured to bamboo stakes (1 m high) with twist ties. Traps were situated so that their bottom edges were about 5–10 cm above the tops of the plants. Lures were attached with wire to the tops of traps. A paired experimental design was used. Each pair consisted of a baited and unbaited trap, separated by about 5 m. Within the pairs, assignment of treatments to traps was by coin flip and, thereafter, was alternated each day. Traps were replaced with new ones each day, and the used ones were taken to the laboratory for examination. All flea beetles were removed from the traps (by using hexane to help dissolve the glue) and examined under a microscope to confirm identity, and the numbers of E. fuscula were recorded. Analysis of variance was conducted, as described with results. Sex ratios were determined from 5 subsamples of 10 beetles, each chosen randomly from both the treatment and control trap catches. Sex was confirmed by dissection (whether aedeagus was present).
Results
Volatile Collections
Comparisons of volatile collections from adult E. fuscula males and females revealed six male-specific peaks (1–6 in Fig. 3). The most abundant (1) was detected in 82 out of the 93 male volatile collections made in 2004 and 2005. In the 25 collections from 2005, the mean amount of 1 was 5.5 ± 4.2 (SD) ng per male per day, and the maximum was 16 ng per male per day. The minor compounds were also detected consistently whenever 1 was prominent. Typical ratios are summarized in Table 1. Compounds 1–6 were not found in the 50 volatile collections from females or in the 9 collections from eggplant leaves only. In GC traces for females or eggplant only, peaks sometimes occurred at the same, or similar, retention times as the numbered compounds from males (e.g., Fig. 3), but in these cases the mass spectra did not agree with the male compounds. Nine of 10 mixed-sex collections contained compounds 1–6 in amounts and proportions consistent with the collections from males.
Fig. 3 Comparisons of volatiles collected from male (top) and 1 female (bottom, mirror view) E. fuscula beetles feeding on eggplant leaf. Compounds 1–6 are detectable only from the male 2 volatiles. Some peaks in female collections had similar retention times to male-specific com- pounds (e.g., peaks 3 and 5) but 3 4 5 differed in mass spectra. Peaks Males 6 marked with asterisks are proba- * bly geometrical isomers of 1 and * 2 (see text) 0
Females
11 12 13 14 15 16 17 18 19 20 21 Time (min after injection) 2550 J Chem Ecol (2006) 32: 2543–2558
Table 1 Mean Percentage of each Male-specific Compound in Male-specific compound Mean (%) to 1±SD the Volatile Collections Relative a to Compound 1 based on GC– 1 100 MS Peak Area 226±13a 3 7.5±3.9b 4 8.2±4.2b 5 0.70±0.31b a N=23 6 2.2±1.1b b N=12
Electrophysiological (GC–EAD) Analysis
Gas chromatographic-electroantennographic analysis of collected volatiles of male beetles feeding on eggplant foliage showed that the natural 1 and 2 were readily detected by the antennae of males (N=3) and females (N=4; Fig. 4, male example shown). Similar results were obtained for the synthetic aldehydes. Male and female antennae did not respond to 3– 6 in volatile collections from males feeding on eggplant foliage, perhaps because of the relatively small amounts (<1 ng/compound). However, when synthetic 3–6 were injected at 10 ng/compound, compounds 3 and 5 did elicit antennal responses in five out of five tests. Compound 6 produced responses in two of the five tests, but no response was seen to compound 4. Male and female antennae also responded to some foliage-specific compounds, but these were not addressed further.
Identification of Compound 1
Compound 1 eluted from silica gel with 10% ether in hexane, a polarity consistent with an aldehyde, ketone, or ester. Silica HPLC of volatile collections rich in 1 (10 of these, combined) afforded a sample suitable for NMR spectroscopy and microhydrogenation; compound 1 (and also 2) eluted between 8 and 9 min after injection. The mass spectrum of 1 (Fig. 5, top) suggested a molecular weight of 136 but did not match any library spectra. An aldehyde or ketone with molecular weight 136 (C9H12O) would have four degrees of unsaturation, and an ester of the same weight (C8H8O2) would have five. Hydrogenation over palladium increased the molecular weight to 142 (Fig. 5, bottom), indicating the uptake of six hydrogen atoms and, therefore, the presence of three
Fig. 4 Positive GC–EAD re- 1 sponse (male antennae) to com- pounds 1 and 2 in a volatile collection from male beetles feeding on eggplants 2
GC
EAD
5 6 7 8 9 10
Time (min from injection) J Chem Ecol (2006) 32: 2543–2558 2551
Fig. 5 Positive EI mass spectrum 79 of compound 1 after HPLC iso- 136 lation (top). Nonanal the resulting Natural compound 1 product after hydrogenenation 77 (bottom) 91 107
81 39 121 53 65 103
35 45 55 65 75 85 95 105 115 125 135 145 m / z
57 41 After Hydrogenation
43
70 98 82 39 95
114 109 124 142 35 45 55 65 75 85 95 105 115 125 135 145 m / z
carbon–carbon double bonds in the original compound. The mass spectrum of the hydrogenation product gave a good library match to nonanal, and authentic nonanal had the same mass spectrum and GC retention time as the hydrogenation product. Because hydrogenation over palladium was not expected to affect an aldehyde group or the carbon skeleton, compound 1 was concluded to be a nonatrienal (the fourth degree of unsaturation required by the molecular weight being accounted for by the aldehyde carbonyl). The 1H and COSY NMR spectra of HPLC-purified 1 (Fig. 6; Table 2) defined the 1 locations and configurations of the double bonds. The H spectrum in CDCl3 indicated six olefinic protons (δ 5.79–7.18), an aldehydic proton (δ 9.57), and an ethyl group (terminal methyl group at δ 1.09, split into a triplet by the methylene group at δ 2.30). The olefinic protons had to be on six consecutive carbons in the middle of the nine-carbon chain because the one-carbon aldehyde and two-carbon ethyl groups occupied the ends. Thus, compound 1 was a 2,4,6-nonatrienal. As shown in Fig. 6, the coupling involving olefinic protons was readily followed in the COSY spectrum from the aldehyde end to the alkyl end of the structure, and the olefinic proton resonances were thus assigned to specific chain 1 positions. The key coupling constants J2,3, J4,5, and J6,7 were read from the assigned H spectrum as 15.2, 15.4, and 10.7 Hz, indicating configurations of E, E, and Z, respectively (Williams and Fleming, 1980). Thus, compound 1 was (2E,4E,6Z)-2,4,6-nonatrienal. Synthetic 1 matched the natural compound by mass and NMR spectra and by GC retention time. 2552 J Chem Ecol (2006) 32: 2543–2558
O 3 5 1 8 H 1 1 2 4 67 9
O 3' 5' 7' 9' Solvent CDCl3 X H 2 8 1' 2' 4' 6' 8' 3’ 4’
3 6 1’ 2 4 5 7 8’ 5’
(9 off scale)
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 Chemical Shift (ppm)
(2’) 3 2 6 54 6’ 7’ 7 3’ 5’ 4’ ppm
55.6.6
7 55.8.8
7’ 66.0.0 6 (2’) 2 6.2 6’ 7 also correlates to 8 (at 2.30 ppm) 4’ 4 66.4.4 7’ also correlates to 8’ (at 2.24 ppm) 66.6.6 5’
66.8.8
5 77.0.0
3’ 2’ also 3 correlates to 1’ 77.2.2 (at 9.56 ppm) 2 also correlates to 1 (at 9.57 ppm) 77.4.4
7.4 7.2 77.0.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 ppm J Chem Ecol (2006) 32: 2543–2558 2553
Table 2 Proton NMR Data for Aldehydes 1 and 2
NMR property Compound 1 Compound 2
CDCl3 C6D6 CDCl3 C6D6
Position number, description and shifts (ppm) 1 (1H, d) 9.57 9.55 9.56 9.51 2 (1H, dd) 6.17* 6.06 6.15 6.06 3 (1H, dd) 7.18 6.56§ 7.12 6.55 4 (1H, dd) 6.43 5.95* 6.36 5.89* 5 (1H, dd) 6.98 6.58§ 6.65 6.21 6 (1H, dd) 6.11* 5.91* 6.20 5.91* 7 (1H, dt) 5.79 5.56 6.10 5.74 8 (2H, p) 2.30 2.08 2.24 1.96 9 (3H, t) 1.09 0.93 1.08 0.91 Coupling constants (Hz)
J1,2 8.0 7.8 8.0 7.8 a a a a J2,3 15.2 15.1 15.2 15.2 J3,4 11.2 11.4 11.1 11.1 a a a a J4,5 15.4 15.1 14.9 14.9 J5,6 11.6 11.4 10.6 10.7 b b a a J6,7 10.7 11.0 15.2 15.2 J7,8 7.9 7.6 6.5 7.4 J8,9 7.5 7.5 7.4 7.5
Within a column, signals followed by the same symbol (* or §) partially overlap at 500 MHz. a Evidence for an E double bond (J > 12 Hz). b Evidence for a Z double bond (J < 12 Hz).
Identification of 2
Compound 2 had a mass spectrum that was nearly identical to 1 and was believed to be a geometrical isomer. The beetle-derived NMR sample (Fig. 6) contained a small amount of 2, and most of its resonances were visible, being somewhat offset from those of 1. Assignment of resonances was performed from the COSY spectrum as with 1. Coupling constants J2,3 and J4,5 were 15.2 and 14.6 Hz, indicating E double bonds, but resonances for the olefinic protons at positions 6 and 7 were obscured, so that J6,7 could not be observed. However, given that 2 is not identical to 1, it was concluded that the 6 double bond of 2 must be E and, therefore, that compound 2 was (2E,4E,6E)-2,4,6-nonatrienal. The mass spectrum and GC retention time of synthetic 2 were identical to the natural product, and so was the NMR spectrum, to the extent that resonances could be compared. When deuterobenzene was used as the NMR solvent instead of CDCl3, shifts of the olefinic protons changed, but overlap of signals in 1 and 2 was not completely avoided (Table 2). Minor amounts of compounds, believed to be geometrical isomers of 1 and 2 based on mass spectra, were sometimes found in the male-produced volatiles (peaks identified with asterisks, Fig. 3), but these did not elicit antennal responses.
R Fig. 6 The 1H (top) and COSY (bottom) NMR spectra of natural sample from E. fuscula that contained compounds 1 and 2. Assignment of resonances to compound position numbers are shown in upper panel; data for compound 2 are indicated with (′) 2554 J Chem Ecol (2006) 32: 2543–2558
Compounds 3–6
Compounds 3 and 4 eluted from silica gel with hexane (polarity consistent with hydrocarbons), whereas 5 and 6 eluted with 25% ether in hexane (polarity consistent with alcohols). Mass spectra of compounds 3, 4, 5, and 6 (Fig. 1) were recognized from previous flea beetle research (compounds A, C, F, and G, respectively, in Bartelt et al., 2001). The GC retention times of the E. fuscula compounds also matched those of authentic standards on the achiral column. Analysis of 4 and of derivatives of 3, 5, and 6 from E. fuscula on the Cyclodex-B GC column allowed assignment of the absolute configurations. Compound 4 from E. fuscula had a retention time of 20.50 min, the earlier of the enantiomers of 4 (racemic 4 gave peaks at 20.51 and 20.75 min). P. cruciferae and A. flava also have the earlier eluting enantiomer of 4 (Bartelt et al., 2001), which was shown by chiral synthesis (Muto et al., 2004) to have the (9R,9aS) configuration (see Fig. 1). ar-Himachalene (7) derived from E. fuscula 3 had a GC retention of 39.70 min, the latter of the enantiomers (racemic 7 gave GC peaks at 39.17 and 39.70 min). Mori (2005) established that the later-eluting enantiomer has the (5R) configuration (Fig. 1), and therefore the 3 from which it was derived had to have the (9R,9aS) configuration. Finally, alcohols 5 and 6 from E. fuscula dehydrated to give just one enantiomer of 4, which eluted at 20.50 min, corresponding to the (9R,9aS) configuration. Thus, 5 and 6 must have been (3R,9R,9aS) and (3S,9R,9aS), respectively.
Labile Compounds
Lability of compounds 1, 5, and 6 made it difficult to accurately characterize the chemical blend of 1–6 at the instant it was emitted from the beetles. Isomerization of 1 into thermodynamically more stable 2 occurs over time if 1 is not carefully protected (e.g., kept in solution in a dark freezer at −70°C). For example, immediately after the sample of synthetic 1 was placed in the autosampler vial, the amount of 2 was 18% of the amount of 1, by GC analysis. When the vial was left at ambient conditions in the laboratory, the amount of 2 as a percentage of 1 increased to 31%, 66%, and 93% after 24, 48, and 72 hr, respectively. However, the total amount of sample in the vial (1 plus 2) decreased by less than 3% after 72 hr (measured with internal standard), indicating most of the relative decrease in 1 must have been due to isomerization to 2. Isomerization of 1 was also likely in the Super Q collections from live beetles because the measured relative amounts of 2 increased as durations of volatile collections increased. For collections of 1-d duration, the amount of 2 as a percentage of 1 (mean±SD) was 26±
Table 3 Mean Number of E. fuscula Adults Caught on Baited and Unbaited Sticky Traps during Three Trial Periods in 2005
Time period Mean trap catch (E. fuscula/trap/d)
Baited Control N Ratio baited:control
May 26–June 10 26.9a 5.8 35 4.6:1.0 July 11–15 7.6a 2.3 25 3.3:1.0 August 2–18 10.6b 6.5 50 1.6:1.0 a Significant difference between treatment and control at the 0.001 level. b Significant difference between treatment and control at the 0.01 level. J Chem Ecol (2006) 32: 2543–2558 2555
13 (N=23). For collections of 2-, 3-, and 4-d duration, the corresponding amounts of 2 were 33±10 (N=23), 40±19 (N=29), and 43±12 (N=13), respectively. From linear regression analysis, the amount of 2 relative to 1 increased by 5.1±1.7 (SE) percentage points per day (t=4.38, P<0.001). The predicted fitted value of 2 for time=0 d (i.e., time of emission from beetles) was 21.4±3.0% (SE), a value that was significantly different from zero (t=7.12, P<0.001). Thus, the apparent rate of isomerization of 1 was not high enough to account totally for the presence of 2. It was concluded that 2 was, in fact, being emitted by the beetles. It has been reported that compounds 5 and 6 both decompose in a heated GC inlet into compounds 3 and 4 (Bartelt et al., 2001); 50% degradation was typical, and the resulting ratio of the 3 and 4 that were formed was about 1:1. Thus, it is likely that the amounts of 3 and 4 actually in beetle emissions are considerably lower than the measured values in Table 1, and correspondingly, the real amounts of 5 and 6 from the beetles are higher than the values in Table 1.
Field Lures
Aldehydes 1 and 2 were readily released from the rubber septa, but isomerization of 1 was a problem. In 17-hr collections of emitted volatiles in the laboratory, mean amounts of compounds 1 and 2 were as follows: 27 μg±8.0 (SD) and 28 μg±9.2 SD, respectively, (N=3). Although the solution applied to the septa contained only 18% of 2,relativeto1,this percentage increased to 104% in the 17-hr collection, and by 72-hr collection, the amount of 2 relative to 1 was 257%.
Field Experiments
Traps baited with synthetic 1 (also emitting 2 at a level that was at least 18% as high as 1) performed significantly better than control traps in all three trial periods (Table 3). In a three-way ANOVA (factors being treatment, seasonal period, and trap pair), the overall treatment effect was highly significant (F=98.3, df=1,107, P<0.001), but there was also a significant interaction between treatment and seasonal period (F=11.5, df=2,107, P<0.001). Thus, the relationship between the treatment and control catches changed with time of year. As shown in Table 3, the ratio between treatment and control (by using newly prepared septa each day) was 4.6:1 in late spring, but decreased to 3.3:1 in early summer and, further, to1.6:1 in late summer. The effect of 1 and 2 was strongest in late spring, although it was significant (P<0.01) in all three periods. In the subsamples from treatment and control traps, captured beetles were 64% and 48% females, respectively (N=50 for each treatment).
Discussion
Volatile collections of male E. fuscula feeding on eggplant foliage showed six compounds that were not present in volatile collection from feeding females or from foliage alone. The two major male-specific compounds, (2E,4E,6Z)-2,4,6-nonatrienal (1) and (2E,4E,6E)- 2,4,6-nonatrienal (2), were identified by GC–MS, NMR, and microscale hydrogenation. Both compounds were synthesized and matched natural 1 and 2 by all available criteria. The true amount of 2 emitted from the beetles, relative to 1, is not yet known because of the tendency for 1 to isomerize into 2, but it was concluded that at least some 2 is naturally 2556 J Chem Ecol (2006) 32: 2543–2558 present. The beetle antennae respond to both compounds in GC–EAD tests. Field tests of baits containing synthetic 1 and 2 were attractive to both male and female E. fuscula, compared with unbaited traps. Although the blend of 1 and 2 was attractive in all three time periods, it performed best early in the season, shortly after the emergence of overwintered adults. Aldehyde 1 has been reported previously in the volatiles from blended endive leaves (Götz-Schmidt and Schreier, 1986), and has recently been identified from oat flakes and Darjeeling black tea (Schuh and Schieberle, 2005, 2006). Aldehyde 1 has also been reported as being among the most odor-active compounds found in food, detectable to humans at an odor threshold of 0.0002 ng/l in air and with an oat-flake-like aroma (Schuh and Schieberle, 2005). In our research, it was not uncommon to detect an odor similar to bread baking when volatile collections from males were processed. Aldehyde 2 was first identified from the volatile oil of blended dry beans (Buttery, 1975), and has also been reported in the volatile constituents of endive leaves (Götz-Schmidt and Schreier, 1986) and cooked spinach (Näf and Velluz, 2000). Interestingly, the human nose is 20,000 times less sensitive to 2 than it is to 1 (Schuh and Schieberle, 2005). Schuh and Schieberle (2005) provided proton NMR data for both 1 and 2 that generally agree with ours. One difference for 1 is that they reported the resonance for the proton at position 7 to be within a multiplet at 6.05 to 6.25 ppm, whereas we found its shift to be 5.79 ppm. Another difference is that the aldehydic proton of 1 was reported to be slightly upfield of that for 2 (9.55 vs. 9.58 ppm, respectively), whereas the reverse was true in our spectra (9.57 and 9.56 ppm, respectively). The Schuh and Schieberle (2005) synthesis of 1 resulted in a mixture of isomers, in which 1 was not the major product. This situation, along with the absence of COSY data, may have made peak assignment difficult. Compounds 1 and 2 have not been previously reported from other flea beetle species or from any other insect species. However, it is interesting to note that males of another chrysomelid beetle, Diorhabda elongata Brullé, emit a homologous compound, (2E,4Z)- 2,4-heptadienal, as part of their aggregation pheromone (Cossé et al., 2005). Small amounts of other isomers of 1 and 2 were detected in the male-produced volatile collections, although their origin, exact configuration, and attractiveness are unknown. Considering the instability of compound 1, the presence of these other isomers is not unexpected. Schuh and Schieberle (2005) also encountered additional isomeric compounds of 2,4,6-nonatrienals during synthesis of (2E,4E,6Z)- and (2E,4Z,6Z)-2,4,6-nonatrienal. The additional four minor male-specific compounds (3, 4, 5,and6) matched synthetic and natural male-specific sesquiterpenes previously identified from Phyllotreta and Aphthona spp. flea beetles in GC retention times, mass spectra, and chirality (Bartelt et al., 2001;Tóthetal., 2005). The configurations of all four compounds are identical at ring positions 9 and 9a (Fig. 1), suggesting a common biosynthetic origin. The GC–EAD activity of 3, 5,and6 in E. fuscula is consistent with, but does not prove, a pheromonal function. Field tests were attempted with racemic 3–6 during the late summer period (when results for 1 and 2 were weakest) but were inconclusive. Pheromonal activity has been demonstrated at least for compound 3 in P. cruciferae (Soroka et al., 2005; Tóth et al., 2005); the males of this species emit both 3 and 4. Males of Aphthona flava, A. czwalinae, and A. cyparissiae also produce 3 and 4 as well as 5 and 6, with the proportions of each being species specific (Bartelt et al., 2001). Such a situation might be expected if the species share a common habitat and if the suites of compounds serves as pheromones, but field bioassays with synthetic compounds have not yet been conducted for the Aphthona species. J Chem Ecol (2006) 32: 2543–2558 2557
Further enhancement of field attractant lures may be possible once true emission rates of 1–6 from E. fuscula males are determined, taking into account distortions in ratios caused by chemical changes occurring before analysis (i.e., 1 isomerizing into 2) or during analysis (i.e., 5 and 6 dehydrating into 3 and 4). Additional compounds (i.e., other 2,4,6-nonatrienal isomers, or plant volatiles) may also increase attraction. Finally, finding a delivery system that will avoid or minimize the isomerization of 1 into 2 is also needed. In spite of these unresolved issues, a new pheromone attractant has been demonstrated to be effective in capturing E. fuscula, and adds to the growing list of pheromone systems identified in the flea beetle family.
Acknowledgments We are grateful to Anne Patterson of Living Earth Farm for assistance and to Richard Stessman and Nathan Deppe for growing and tending eggplants at the NCAUR garden plots. David Weisleder and Karl Vermillion of NCAUR acquired the NMR spectra. Dr. Alexander S. Konstantinov (Chrysomelidae), Systematic Entomology Laboratory, Agriculture Research Service, US Department of Agriculture, provided insect identification.
References
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