Identification of a Pheromone Component and a Critical Synergist for the Invasive Beetle Callidiellum rufipenne (Coleoptera: Cerambycidae)

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Chemical Ecology Environmental Entomology, 45(1), 2016, doi: /ee/nvv165 Advance Access Publication Date: 27 October 2015 Research article Identification of a Pheromone Component and a Critical
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Chemical Ecology Environmental Entomology, 45(1), 2016, doi: /ee/nvv165 Advance Access Publication Date: 27 October 2015 Research article Identification of a Pheromone Component and a Critical Synergist for the Invasive Beetle Callidiellum rufipenne (Coleoptera: Cerambycidae) Yunfan Zou, 1 Claire E. Rutledge, 2 Kiyoshi Nakamuta, 3 Chris T. Maier, 2 Lawrence M. Hanks, 4,5 Austin B. Richards, 6 Emerson S. Lacey, 1 and Jocelyn G. Millar 1 1 Department of Entomology, University of California, Riverside, CA jocelyn. 2 Department of Entomology, The Connecticut Agricultural Experiment Station, New Haven, CT Graduate School of Horticulture, Chiba University, Matsudo, Chiba , Japan 4 Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL Corresponding author, and 6 Aquatic Bioassessment Laboratory, California State University, Chico, CA Received 20 August 2015; Accepted 7 October 2015 Abstract The invasive Asian cerambycid beetle Callidiellum rufipenne (Motschulsky), informally known as the Japanese cedar longhorned beetle, was first detected in North America in North Carolina in The beetle has since been detected in neighboring states and is expected to further expand its range. However, delineating the current distribution of C. rufipenne has been hindered by the lack of efficient sampling methods. Here, we present the results of research on the chemistry of volatile pheromones of C. rufipenne. Analyses of headspace odors revealed that males produce (R)-3-hydroxyhexan-2-one, with lesser amounts of (S)-3-hydroxyhexan-2-one, and (R)- and (S)-2-hydroxyhexan-3-one. In field bioassays conducted over several years in Connecticut, where populations of the beetle were well established, no reconstructed blend of these compounds was significantly attractive to beetles of either sex. However, during field trials in Japan that targeted another species, we discovered that adult male and female C. rufipenne were attracted to a blend of racemic 3-hydroxyhexan-2-one and a novel natural product, 1-(1H-pyrrol-2-yl)-1,2-propanedione. Attraction to (R)-3-hydroxyhexan-2-one and the pyrrole subsequently was confirmed in field trials in Connecticut. Although it is unclear why the pyrrole acts as a synergist for a species that apparently does not produce it, the serendipitous discovery that adult C. rufipenne are attracted by the blend of ketone and pyrrole provides a badly needed method for monitoring its ongoing range expansion within North America, and for detecting new introductions in other parts of the world. Key words: longhorned beetle, semiochemical, synergism, monitoring, invasive species Insect pests of woody plants and the pathogens they vector are commonly spread to new regions of the world by global trade (Chornesky et al. 2005). In their new habitats, exotic and invasive species may multiply rapidly and become a threat to forest sustainability and productivity, and/or to tree crops (Gandhi and Herms 2010, Boyd et al. 2013). Wood-boring insects are among the most important of these invasive species, because the long-lived immature stages are easily transported while concealed and protected within wooden products and packing materials (e.g., Brockerhoff et al. 2006, Haack 2006, Cocquempot and Lindelöw 2010). A number of exotic wood-boring pests have established in North America in recent years and become important pests, including cerambycid beetles such as eucalyptus longhorned borers (Phoracantha species; Paine et al. 2010) and the Asian longhorned beetle (Anoplophora glabripennis (Motschulsky); Haack et al. 2010), buprestid beetles such as the emerald ash borer (Agrilus planipennis Fairmaire; Poland and McCullough 2006), and scolytine beetles such as the redbay ambrosia beetle (Xyleborus glabratus Eichhoff; Hanula et al. 2008) and the Euwallacea species complex of shothole borers (Eskalen et al. 2013). The Asian cerambycid Callidiellum rufipenne (Motschulsky), informally known as the Japanese cedar longhorned beetle, was detected in North Carolina in 1997 (Hoebeke 1999), and later in several northeastern states (Maier 2007, Maier and Graney 2012). In Connecticut, adults of this univoltine species emerge from their pupal cells in wood between late March and mid-may (Maier 2008). Based on observations in Japan (Shibata 1994) and Connecticut (Maier 2001, 2008), females mate on the host and begin to lay eggs in cracks and crevices in the bark of hosts within a few days of emergence, apparently not feeding on solid food. VC The Authors Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please 216 Environmental Entomology, 2016, Vol. 45, No In Japan, C. rufipenne is considered a secondary pest of cedars, cypress, and arborvitae (Shibata 1994, Iwata et al. 2007). In the northeastern United States, the larvae develop within cupressaceous plants that are stressed, dying, or dead (Maier and Lemmon 2000; Maier 2007, 2009). Study of the beetle s biology, and especially its distribution in North America, has been hindered by the lack of efficient sampling methods (Maier 2008). Research over the past decade has revealed that cerambycids of many species produce pheromones, and can be effectively monitored with pheromone-baited traps (e.g., Sweeney et al. 2010, Barbour et al. 2011, Hanks and Millar 2013). Adult male C. rufipenne have pores in the pronotum which in other species in the same subfamily are the source of volatile pheromones which attract both sexes (Ray et al. 2006). Males of C. rufipenne on larval hosts also have been observed to perform a stereotyped calling behavior (standing still for as long as 20 min, with front legs extended; C.E.R., personal observation) similar to the push-up stance calling behavior of other cerambycids in the same subfamily (Lacey et al. 2007a,b, 2009). Both of these characteristics suggested that male C. rufipenne probably produce attractant pheromones. As part of an ongoing project aimed at identifying pheromones and patterns of pheromone use within the family Cerambycidae, particularly for potentially invasive species, we began to investigate the semiochemistry of C. rufipenne in Analyses of headspace volatiles from both sexes revealed that only males produced (R)-3- hydroxyhexan-2-one, with lesser amounts of (S)-3-hydroxyhexan-2- one, and (R)- and (S)-2-hydroxyhexan-3-one. These structures are typical pheromone components of many cerambycid beetles in the subfamily Cerambycinae, to which C. rufipenne belongs (e.g., Hanks and Millar 2013). To our frustration, in field bioassays conducted over several years in southern Connecticut, where populations of the beetle were well established, no reconstructed blend of these compounds was significantly attractive to either sex (J.G.M. and C.E.R., unpublished data). However, during field trials in Japan which targeted another cerambycid species, we discovered that C. rufipenne of both sexes were attracted to a blend of racemic 3-hydroxyhexan-2-one and a novel natural product, 1-(1H-pyrrol-2-yl)-1,2-propanedione (henceforth pyrrole), which we recently identified from several other cerambycid species (J.G.M., unpublished data). We report here the details of these experiments, the synthesis of the key pyrrole compound, and the results of confirmatory field trials conducted in Connecticut with the blend of compounds. Materials and Methods Identification of Pheromone Components Adults of C. rufipenne for pheromone identification were collected by girdling or felling living Juniperus virginiana L. in spring 2004, to encourage females to oviposit. The trees were standing near Dooley Pond, Middletown, Middlesex County, CT (41.511, ). On 23 March 2005, the trees were cut into bolts that were placed in emergence barrels (19-liter plastic buckets with an inverted funnel in the lid that lead to a container to trap insects). Newly emerged adults were housed individually in 9-cm plastic petri dishes with water provided (8-ml vial with a cotton roll), and were held in a dark incubator at 15 C for 7 d prior to aeration. Volatile compounds produced by adults were collected by placing individual beetles in glass vacuum traps (0.3 liter) that were lined with aluminum screen to provide perches. Volatiles were trapped with a glass tube (6 cm by 4 mm i.d.) containing 100 mg of 80/100 mesh SuperQ (Alltech Associates, Deerfield, IL) that was attached to the outlet of each chamber with Teflon tubing. Charcoal-purified air was pulled through the apparatus with a water aspirator (1 l/min). A male and female were aerated simultaneously on a laboratory windowsill from 9:30 15:30 h EST on 25 April 2005, and a second set of adults was aerated from 12:00 to 16:00 h on 26 April Collectors were eluted into silanized glass vials (Cat. no , Supelco, Bellefonte, PA) with three 0.5-ml aliquots of methylene chloride, and the resulting extracts were analyzed with an HP-6890 gas chromatograph (GC; Hewlett-Packard, Avondale, PA) fitted with a DB5-MS column (30 m by 0.25 mm, 25 mm film thickness; J&W Scientific, Folsom, CA), and programed from 40 C/2 min, 10 C/min to 250 C, hold 15 min. The absolute configurations of the insect-produced compounds were determined by analysis of aliquots of extracts on a Cyclodex-B GC column (30 m by 0.25 mm, 0.25 micron film thickness, J&W Scientific) with the GC programmed from 50 C/1 min, 5 C/min to 200 C, detector 200 C. The injector temperature was set to 100 C to minimize the thermal isomerization of the hydroxyketones. Compounds were conclusively identified by coinjections of extracts with authentic standards to ensure that retention times matched exactly. Synthetic Pheromones Racemic 3-hydroxyhexan-2-one was purchased from Bedoukian Research (Danbury, CT), and (R)-3-hydroxyhexan-2-one was synthesized as described in Lacey et al. (2007a). General Conditions for the Synthesis of the Pyrrole Tetrahydrofuran (THF) was distilled from sodium/benzophenone under argon. NMR spectra of 1 H- and 13 C (400 and MHz, respectively), in CDCl 3 solution, were taken on a Varian INOVA-400 spectrometer (Palo Alto, CA). Chemical shifts are expressed in ppm relative to residual CHCl 3 ( 1 H-NMR, 7.27 ppm, 13 C-NMR, ppm). Unless otherwise stated, solvent extracts of reaction mixtures were concentrated by rotary evaporation under reduced pressure. Crude products were purified by flash chromatography on silica gel ( mesh; Fisher Scientific, Fair Lawn, NJ). Mass spectra were obtained with a HP-6890 GC interfaced to an HP mass selective detector in EI mode (70 ev) with helium carrier gas. The GC was equipped with a DB17-MS column (25 m by 0.20 mm i.d., 0.33 mm film; J&W Scientific). High resolution mass spectra were taken on a Waters GCT GC-MS instrument (Waters Corp., Milford, MA). Reactions with air- or water-sensitive reagents were carried out in oven-dried glassware under argon. 2 -(2-Methyl-acryloyl)-pyrrole-1-carboxylic acid tertbutyl ester (2) (Fig. 1) n-buli (2.3 M, 9.6 ml, 22 mmol) was added to a cold ( 78 C) solution of 2,2,6,6-tetramethylpiperidine (3.7 ml, 22 mmol) in THF (40 ml). The mixture was stirred 10 min at 78 C and 10 min at 0 C and then cooled again to 78 C. To this mixture was added N- Boc-pyrrole 1 (3.3 ml, 20 mmol; Aldrich Chemical Co., Milwaukee, WI) in THF (5 ml), and the mixture was stirred 50 min at 78 C. This preformed anion then was transferred to a cold ( 78 C) solution of methacrylic anhydride (4.5 ml, 30 mmol) in THF (20 ml) via a short, dry ice-cooled, double-ended needle. The mixture was stirred 2 h at 78 C, then poured into saturated NaHCO 3 solution and extracted with ether. The combined organic layers were washed with H 2 O and brine, then dried over anhydrous Na 2 SO 4, and concentrated. The crude product was first Kugelrohr distilled (0.2 torr, 60 C) to remove 2,2,6,6-tetramethylpiperidine, unreacted 218 Environmental Entomology, 2016, Vol. 45, No. 1 Fig. 1 Synthesis of 1-(1H-pyrrol-2-yl)-1,2-propanedione. N-Boc-pyrrole 1, and excess methacrylic anhydride, and the residue was purified by flash chromatography (hexanes/etoac ¼ 30/1) to give 2 as a light yellow oil (2.68 g, 57%). 1 H NMR (CDCl 3, 400 MHz): d 7.33 (dd, J ¼ 3.2, 1.6 Hz, 1H), 6.55 (dd, J ¼ 3.6, 1.6 Hz, 1H), 6.16 (t, J ¼ 3.4 Hz, 1H), 5.85 (m, 1H), 5.80 (m, 1H), 2.02 (s, 3H), 1.53 (s, 9H); 13 C NMR (CDCl 3, MHz): d , , , , , , , , 84.79, 27.73, 17.79; MS (m/z, relative abundance): 41 (31), 57 (100), 94 (58), 107 (32), 120 (14), 134 (23), 135 (93), 162 (25), 235 (M þ, 14). 2-(2-Oxo-propionyl)-pyrrole-1-carboxylic acid tert-butyl ester (3) Ruthenium (III) chloride hydrate (31 mg, 0.15 mmol) was added to a cold (0 C) mixture of 2 (1.41 g, 6 mmol) and NaIO 4 (7.70 g, 36 mmol) in CCl 4 (12 ml), CH 3 CN (12 ml), and H 2 O (18 ml), and the mixture was stirred vigorously for 1 h. Then CH 2 Cl 2 and H 2 O were added, the phases were separated, and the aqueous phase was extracted three times with CH 2 Cl 2. The combined organic extracts were washed with H 2 O and brine, dried over anhydrous Na 2 SO 4, and concentrated. The residue was diluted with ether, filtered through a pad of Celite (Fisher Scientific), and concentrated. The crude product was purified by flash chromatography (hexanes/ EtOAc ¼ 95/5) to give 3 as a yellow solid (0.58 g, 41%). 1 H NMR (CDCl 3, 400 MHz): d 7.31 (dd, J ¼ 3.2, 2.0 Hz, 1H), 7.00 (dd, J ¼ 3.2, 2.0 Hz, 1H), 6.27 (t, J ¼ 3.2 Hz, 1H), 2.47 (s, 3H), 1.56 (s, 9H); 13 C NMR (CDCl 3, MHz): d , , , , , , , 85.90, 27.88, 24.77; MS (m/z, relative abundance) 41 (19), 43 (15), 57 (100), 66 (12), 94 (50), 138 (63), 164 (24), 194 (35, M þ -43). 1-(1H-Pyrrol-2-yl)-propane-1,2-dione (4) Diketone 3 (0.81 g, 3.4 mmol) was heated under argon in an oil bath ( C) for 20 min. After cooling to room temperature, the residue was diluted with ether, filtered through a pad of Celite, and concentrated. The crude product was purified by flash chromatography (hexanes/etoac ¼ 5/1) to give 4 as a yellow solid, mp 55 C (0.41 g, 87%). 1 H NMR (CDCl 3, 400 MHz) d 9.96 (broad s, 1H), 7.35 (m, 1H), 7.17 (m, 1H), 6.36 (m, 1H), 2.49 (s, 3H); 13 C NMR (CDCl 3, 150 M Hz) d , , , , , , The 13 C spectrum was taken at 50 C on a 600 MHz spectrometer because the signals from the quaternary carbons at , , and ppm were not detected due to slow 13 C relaxation when the sample was run at room temperature. MS (m/z, relative abundance) 66 (45), 94 (100), 137 (M þ, 33). HRMS calculated for C 7 H 7 NO , found Field Bioassays The initial field experiment was conducted in Japan to test the attraction of cerambycid beetles to racemic 3-hydroxyhexan-2-one, the pyrrole, and a blend of the two. Insects were captured with black cross-vane flight intercept traps (0.4 m tall, 0.3 m in diameter; Sankei Chemical Co. Ltd., Tokyo, Japan; see Shibata et al. 1996) with basins filled with water to which was added a few drops of detergent and sorbic acid as a preservative. Traps were positioned within the tree canopy with a pulley and rope system, with trap bottoms 2 m above the ground. Pheromone lures consisted of polyethylene sachets (Bagette model 14770, 5.1 by 7.6 cm, Cousin Corp., Largo, FL) containing a cotton dental wick, that were loaded with 25 mg of the racemic hydroxyketone, 12.5 mg of the pyrrole, or the same amounts of each chemical for blends, in 0.5 ml isopropanol. The experiment consisted of four traps per linear transect (traps at least 15 m apart) that were baited with the ketone, the pyrrole, the blend of ketone and pyrrole, or a control lure (sachet with wick containing 0.5 ml neat isopropanol). The experiment was set up in an experimental planting of Japanese cedar, Cryptomeria japonica (L. f.) D. Don (trees 21 y old), at the Chiba Prefectural Forest Research Center, Chiba, Japan (35.347, , 22 m elevation), and was run from 2 April to 1 May 2015, within the flight period of the species (see Shibata 1994). The discovery that adults of C. rufipenne were attracted to the ketone pyrrole blend in Japan (see Results) inspired follow-up research in the northeastern United States to confirm that beetles of the introduced population also would be attracted. Field tests in Connecticut were started in mid-may 2015, even though the typical peak activity period had already passed (most adults are active for a short period in late April-early May; Maier 2008). However, the cool spring in 2015 in Connecticut may have delayed the flight period (C.T.M., personal observation). Experiments in Connecticut were of the same design as those in Japan and used black cross-vane flight intercept traps (Panel Trap model, AlphaScents, Portland, OR) coated with Fluon PTFE (AGC Chemicals Americas Inc., Exton, PA) with the catch basins filled part way with propylene glycol as a trapping agent and preservative. Traps were baited with lures of the same type as those used in Japan (see above), and loaded with 25 mg of (R)-3-hydroxyhexan-2-one, 25 mg of pyrrole, or the same amounts of each chemical in a blend, in 1 ml isopropanol. The experiment consisted of four traps in a linear transect (traps at least 10 m apart) that were baited with the ketone, the pyrrole, the blend of ketone and pyrrole, or a control lure (sachet containing 1 ml neat Environmental Entomology, 2016, Vol. 45, No isopropanol), with the order of treatments in each transect being rerandomized after each inspection. Field trials in Connecticut were conducted in areas known to harbor populations of C. rufipenne based on previous research (C.T.M., C.E.R., unpublished data) that were forested primarily with hardwood trees (especially Quercus species), but that also contained naturally growing or planted red cedar (Juniperus virginiana L.) and/or arborvitae (Thuja occidentalis L.), which are common hosts of C. rufipenne. Locations of transects were as follows; two transects in the town of Woodbridge (New Haven County; , ); two trap transects that bordered Lake Gaillard in the town of North Branford (New Haven County; , ; , ); two trap transects at Rocky Neck State Park in the town of East Lyme (New London County; , ); and one trap transect in the town of Old Saybrook (Middlesex County, , ). Traps at the Woodbridge and Rocky Neck State Park sites were suspended from L-shaped frames of polyvinyl chloride pipe mounted on steel rods (for details, see Graham et al. 2010), and were deployed from 20 May to 10 June Traps at the remaining sites were hung from branches of trees with the catch basins no more than 1 m above the ground, and were deployed from 22 May to 18 June Beetles were collected from traps at intervals of 3 to 7 d at which time treatments were rotated one position within transects. Beetles were sexed by color of the elytra (mostly black in males, reddish or testaceous in females; Hoebeke 1999), and confirmed by antennal length relative to body length (longer in males; Maier 2008). Differences between treatments in mean numbers of beetles captured were tested with the nonparametric Friedman s test (PROC FREQ with CMH option; SAS Institute 2011) because assumptions of analysis of variance were violated by heteroscedasticity (Sokal and Rohlf 1995). Pairs of treatment means were compared with the nonparametric Nemenyi multiple comparison test (Elliot and Hynan 2011, Zar 2010). Replicates that captured no beetles were dropped from analyses. The sex ratio of captured beetles was compared to a nominal 1:1 ratio with the v 2 test. Taxonomy of captured beetles follows Lingafelter (2007). Voucher specimens of C. rufipenne have been placed in the insect collection in the Department of Entomology at the Connecticut Agricultural Experiment Station, New Haven. Results Comparisons of extracts of headspace volatiles collected from males and females of C. rufipenne revealed three male-specific compounds that were tentatively identified (in order of elution) as 2,3-hexanedione, 3-hydroxyhexan-2-one, and 2-hydroxyhexan-3-one by matches of their mass spectra with spectra from our library of known cerambyc
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