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
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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]
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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 maleproduced pheromone was demonstrated in E. fuscula. The identification of six malespecific compounds and the synthesis and field activity of the two major components are reported.
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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
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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
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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-octahydro-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-tetramethyl-1H-benzocyclohepten-3-ol; 7, (5R)-6,7,8,9-tetrahydro-2,5,9,9-tetramethyl-5H-benzocycloheptene
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(Heath and Sonnet, 1980) was used for separating hydrocarbons 3 and 4, with 0.5% 1hexene 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 (
