Environmental Entomology, 45(5), 2016, 1271–1275 doi: 10.1093/ee/nvw101 Advance Access Publication Date: 14 August 2016 Chemical Ecology Research

Stereochemistry of Fuscumol and Fuscumol Acetate Influences Attraction of Longhorned (Coleoptera: Cerambycidae) of the Subfamily

G. P. Hughes,1 L. R. Meier,2 Y. Zou,3 J. G. Millar,3 L. M. Hanks,2 and M. D. Ginzel1,4

1Department of Entomology, Purdue University, 901 W. State St., West Lafayette, IN 47907 ([email protected]; mginzel@pur due.edu), 2Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 ([email protected]; [email protected]), 3Department of Entomology, University of California, Riverside, Riverside, CA 92521 ([email protected]; [email protected]), and 4Corresponding author, e-mail: [email protected] Downloaded from

Received 9 June 2016; Accepted 19 July 2016

Abstract http://ee.oxfordjournals.org/ The chemical structures of aggregation-sex pheromones of longhorned beetles (Coleoptera: Cerambycidae) are often conserved among closely related taxa. In the subfamily Lamiinae, adult males and females of several spe- cies are attracted by racemic blends of (E)-6,10-dimethyl-5,9-undecadien-2-ol (termed fuscumol) and the struc- turally related (E)-6,10-dimethyl-5,9-undecadien-2-yl acetate (fuscumol acetate). Both compounds have a chiral center, so each can exist in two enantiomeric forms. Males of many species of longhorned beetles only produce one stereoisomer of each pheromone component, and attraction may be reduced by the presence of stereoiso- mers that are not produced by a particular species. In a previous publication, analysis of headspace volatiles of adult beetles of the lamiine species Astyleiopus variegatus (Haldeman) revealed that males sex-specifically pro- at ESA Society Member on November 3, 2016 duced (S)-fuscumol and (S)-fuscumol acetate. Here, we describe field trials which tested attraction of this species to single enantiomers of fuscumol and fuscumol acetate, or to blends of enantiomers. We confirmed at- traction of A. variegatus to its species-specific blend, but during the course of the trials, found that several other species also were attracted. These included modestus (Gyllenhall), attracted to (S)-fuscumol acetate; Astylidius parvus (LeConte), attracted to (R)-fuscumol; macula (Say), attracted to (S)-fuscumol; and Graphisurus fasciatus (DeGeer), attracted to a blend of (R)-fuscumol and (R)-fuscumol acet- ate. These results suggest that chirality may be important in the pheromone chemistry of lamiines, and that spe- cific stereoisomers or mixtures of stereoisomers are likely produced by each species.

Key words: Cerambycidae, semiochemical, pheromone, chirality, enantiomer

The longhorned beetles (Coleoptera: Cerambycidae) are a large family the genus Monochamus (e.g., Pajares et al. 2010, Teale et al. 2011, of that exhibit considerable diversity in their chemical commu- Allison et al. 2012, Fierke et al. 2012). Attraction to monochamol in nication systems (reviewed by Millar and Hanks 2016). Males or fe- other species in the Lamiini (Wickham et al. 2014)providesfurther males of many species produce volatile aggregation-sex pheromones evidence that this compound is a conserved pheromone component (sensu Carde´2014), which serve to bring the sexes together on host within the subfamily. plants (Millar et al. 2009). Among species in the subfamily In contrast, males of the South American lamiine Cerambycinae, males commonly produce volatile pheromones which betulinus (Klug) (tribe ) produce the sesquiterpene are typically 6, 8, or 10 carbons in length with hydroxyl or carbonyl degradation product (E)-6,10-dimethyl-5,9-undecadien-2-one (gera- groups on carbons 2 and 3 (Millar and Hanks 2016). Less is known nylacetone), the corresponding alcohol (fuscumol), and the acetate about the pheromone chemistry of longhorned beetles in the subfam- ester of fuscumol (Fonseca et al. 2010). Blends of these compounds ily Lamiinae, but the evidence to date suggests that pheromone struc- were attractive to beetles in laboratory experiments, although at- tures vary across tribes. For example, males of species in the Lamiini traction in the field has yet to be confirmed. Fuscumol was first iden- and Monochamini produce hydroxyethers and related compounds, tified as the pheromone of the European longhorned including 4-(heptyloxy)butanol and the corresponding aldehyde from Tetropium fuscum (F.), in the small subfamily Spondylidinae (Silk species of Anoplophora (Zhang et al. 2002, Hansen et al. 2015), and et al. 2007, Sweeney et al. 2010). Field screening experiments in 2-(undecyloxy)ethanol (termed monochamol) from many species in North America revealed that several native lamiine species in the

VC The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: [email protected] 1271 1272 Environmental Entomology, 2016, Vol. 45, No. 5 tribes Acanthoderini and were attracted by racemic Table 1. Study sites for field experiments conducted in northwest- fuscumol and fuscumol acetate (Mitchell et al. 2011, Hanks and ern Indiana and east-central Illinois during 2012–2014 Millar 2013), indicating that this motif crosses subfamily State/County Name GPS coordinates Area (ha) boundaries. (long., lat.) Attraction of cerambycids to their aggregation-sex pheromones may be inhibited by compounds produced by related species, includ- IN/Warren Black Rock Barrens 40.358, 87.114 40 a ing stereoisomers of their pheromone components (Millar and Nature Preserve b Hanks 2016). For example, males of the cerambycine Neoclytus IN/Tippecanoe Ross Hills Park 40.404, 87.072 68 IL/Champaign Brownfield Woodsc 40.145, 88.165 26 acuminatus acuminatus (F.) produce a single-component pheromone IL/Vermillion Forest Glen Preserved 40.015, 87.567 3 comprising (2S,3S)-2,3-hexanediol, and attraction of both sexes IL/Champaign Nettie Hart 40.229, 88.358 16 to synthesized pheromone is unaffected by the nonnatural (2R,3R)- Memorial Woodsc enantiomer, but reduced by the (2S,3R)- and/or (2R,3S)- IL/Piatt Robert Allerton Parkc 39.996, 88.651 600 diastereomers (Lacey et al. 2004). Because fuscumol and fuscumol acetate have chiral centers, chirality could play an important role in a NICHES Land Trust (http://nicheslandtrust.org/, accessed 3 August 2016). b imparting species specificity to pheromones of species that utilize Tippecanoe County Parks & Recreation Department (http://www.tippeca these compounds. For example, male H. betulinus produce a blend noe.in.gov/187/, accessed 3 August 2016). c Property of the University of Illinois (http://research.illinois.edu/cna/, of geranylacetone, (R)-fuscumol acetate, and a mixture of (R)- and Downloaded from accessed 3 August 2016). (S)-fuscumol in a ratio of 82% and 18%, respectively (Vidal et al. d Vermilion County Conservation District (http://www.vccd.org/, accessed 2010). However, the activity of nonracemic blends of the enantio- 3 August 2016). mers of the latter two compounds have not been reported. These compounds have an additional stereochemical element in the C5 double bond, but the (Z)-isomer has not yet been identified from Table 2. Treatments for two field experiments which tested attrac- http://ee.oxfordjournals.org/ any longhorned beetle species, to our knowledge. tion of longhorned beetles to individual enantiomers of fuscumol Headspace collections from a male of the North American lam- and fuscumol acetate, or blends of the enantiomers of each iine Astyleiopus variegatus (Haldeman) (Acanthocinini) contained a component blend of (S)-fuscumol and (S)-fuscumol acetate (1:2 ratio; Hughes Treatment et al. 2013). This species already was known to be attracted by a blend of racemic fuscumol þ fuscumol acetate from field experi- Expt. 1 (R)-fuscumol ments (Mitchell et al. 2011). Here, we describe field experiments (R)-fuscumol acetate which tested the attraction of cerambycids to enantiomers of fuscu- (R)-fuscumol þ (R)-fuscumol acetate at ESA Society Member on November 3, 2016 mol and fuscumol acetate in Indiana and Illinois. These experiments (S)-fuscumol confirmed attraction of A. variegatus to its species-specific blend, (S)-fuscumol acetate (S)-fuscumol þ (S)-fuscumol acetate and also assessed attraction of sympatric lamiine species to these Solvent control compounds. Our results provide evidence that the pheromones of at Expt. 2 (R)-fuscumol least some lamiines may be delineated by chirality, and that the chir- (S)-fuscumol ality of fuscumol and fuscumol acetate allows for a variety of dis- (R)-fuscumol acetate tinct blends that could serve as species-specific pheromones among (S)-fuscumol acetate sympatric species. Solvent control

Materials and Methods efficiency (Graham et al. 2010, Allison et al. 2016). The supplied Sources of Chemicals trap basins were replaced with 2-liter plastic jars (General Bottle (R)-fuscumol (96.6% enantiomeric excess, ee), (S)-fuscumol (98% Supply Company, Los Angeles, CA) which had their threaded lids ee), (R)-fuscumol acetate (96.6% ee), and (S)-fuscumol acetate modified to accommodate a plastic funnel that received captured (98% ee) were synthesized by enzymatic kinetic resolution of the ra- beetles, allowing for quick removal of the jar when collecting speci- cemic compounds as described in Sweeney et al. (2010) and Hughes mens. Traps were suspended from L-shaped stands constructed of et al. (2013). polyvinylchloride irrigation pipe. Collection jars were filled with 0.1 liter of saturated aqueous NaCl solution to kill and preserve Study Sites specimens. Trap lures consisted of resealable polyethylene sachets Experiments to assess attraction of beetles to synthesized candidate (2 mil, 7 5 cm; Fisher Scientific, Waltham, MA) that were loaded pheromones were conducted at two study sites in northwestern with 25 mg per enantiomer of synthesized pheromone, dissolved in Indiana and four study sites in east-central Illinois (Table 1), all of 1 ml isopropanol, which was chosen as a solvent carrier because it which were wooded with mature second-growth or successional has not been shown to attract longhorned beetles. hardwoods and dominated by oak (Quercus species), hickory Two field experiments were conducted to assess attraction of (Carya species), maple (Acer species), and ash (Fraxinus species). adult beetles to enantiomers of fuscumol and fuscumol acetate. Experiment 1 tested attraction of beetles to individual (R)- and (S)- Field Testing of Candidate Pheromone Components enantiomers and blends of the two enantiomers of fuscumol and fus- Attraction of beetles to synthesized chemicals was tested using cumol acetate, respectively (Table 2). Seven traps were set up in a cross-vane panel traps (black corrugated plastic; AlphaScents, West linear transect (10 m apart), with individual treatments assigned Linn, OR) that were coated with undiluted Fluon (a Teflon emul- randomly to traps. The experiment was conducted during 24 June–4 sion; Northern Products Inc., Woonsocket, RI) to improve trap July 2012 at Black Rock Barrens Nature Preserve and Ross Hills Environmental Entomology, 2016, Vol. 45, No. 5 1273

Park in Indiana, during 28 June–3 October 2012 at Robert Allerton Table 3. and number of cerambycid beetles that were Park in Illinois, and repeated during 20 June–25 July 2014 in Illinois captured during field experiments in Indiana and Illinois in at the same site and at Nettie Hart Memorial Woods (Table 1). Experiments 1 and 2 Traps were serviced three times per week, at which time traps were Taxonomy Expt. 1 Expt. 2 Total rotated one position along transects to control for positional bias. Lures were replaced when they had little liquid remaining in the sa- Cerambycinae chets, which was approximately every 2–3 wk but varied with the Anaglyptini Cyrtophorus verrucosus (Olivier) 1 29 30 weather. Bothriospilini Experiment 2 tested attraction of beetles to individual enantio- Knulliana cincta cincta (Drury) 5 5 mers of fuscumol and fuscumol acetate (Table 2), with five traps set Clytini up in linear transects, and treatments randomly assigned to traps. Clytoleptus albofasciatus (Laporte & Gory) 1 1 During 2013, the experiment was conducted in Indiana at Black Clytus ruricola (Olivier) 1 1 Rock Barrens Nature Preserve and Ross Hills Park during 31 May– Neoclytus acuminatus acuminatus (F.) 17 10 27 20 September, and in Illinois at Robert Allerton Park during 8 Neoclytus mucronatus mucronatus (F.) 14 7 21 May–26 August and Forest Glen Preserve during 9 July–27 August Neoclytus scutellaris (Olivier) 10 1 11 (Table 1). The experiment was repeated at Black Rock Barrens dur- Sarosesthes fulminans (F.) 1 1

Xylotrechus colonus (F.) 49 68 117 Downloaded from ing 19 June–26 September 2014. Eburiini We tested differences between treatments, separately for each Eburia quadrigeminata (Say) 9 2 11 beetle species, using the nonparametric Friedman’s Test (PROC Elaphidiini FREQ, option CMH; SAS Institute Inc., 2015) because data violated Anelaphus parallelus (Newman) 2 15 17 the homoscedasticity assumption of ANOVA. Data were analyzed Anelaphus villosus (F.) 4 78 82

across six sites between 2012 and 2014 and, because the model re- Elaphidion mucronatum (Say) 14 9 23 http://ee.oxfordjournals.org/ vealed no significant year or site effect, data were pooled such that Parelaphidion aspersum (Haldeman) 13 13 experimental replicates for each species were defined solely by col- Parelaphidion incertum (Newman) 3 3 Neoibidionini lection date. Differences between pairs of treatment means were Heterachthes quadrimaculatus Haldeman 2 2 tested with the REGWQ means separation test, which controls max- Obriini imum experiment-wise error rates (PROC GLM; SAS Institute Obrium maculatum (Olivier) 9 16 25 2015). For each beetle species, only blocks of replicates in which at Tillomorphini least one beetle was caught were included in the analyses, i.e., elimi- Euderces picipes (F.) 1 1

nating periods when there was no beetle activity at all due to bad Lamiinae at ESA Society Member on November 3, 2016 weather or the termination of seasonal flight periods. Acanthocinini Taxonomy of captured beetles follows Monne´ and Hovore Astyleiopus variegatus (Haldeman) 127 6 133 (2005). Representative specimens from Illinois and Indiana are Astylidius parvus (LeConte) 2 7 9 Astylopsis collaris (Haldeman) 1 1 2 available from the laboratory collections of L.M.H. and M.D.G., re- Astylopsis macula (Say) 4 5 9 spectively. Voucher specimens collected in Illinois have been de- Graphisurus despectus (LeConte) 13 36 49 posited with the collection of the Illinois Natural History Survey, Graphisurus fasciatus (DeGeer) 108 69 177 Champaign, IL. Leptostylus transversus (Gyllenhal) 1 6 7 Lepturges angulatus (LeConte) 6 6 Lepturges confluens (Haldeman) 12 16 28 Results Lepturges symmetricus (Haldeman) 1 1 2 A total of 970 longhorned beetles representing 48 species were cap- Sternidius alpha (Say) 10 8 18 Urgleptes querci (Fitch) 2 1 3 tured during the two experiments (Table 3), including species in the Acanthoderini subfamilies Cerambycinae, Lamiinae, Lepturinae, Parandrinae, Aegomorphus modestus (Gyllenhal) 10 49 59 Prioninae, and one species in the closely related family Disteniidae. Desmiphornini The lamiine species that were captured in greatest numbers across Eupogonius pauper LeConte 1 1 experiments were Graphisurus fasciatus (DeGeer) and the target Dorcaschematini species A. variegatus (Table 3). Although >100 adults of the ceram- Dorcaschema cinereum (Olivier) 5 5 bycine species Xylotrechus colonus (F.), and relatively high numbers Onciderini of cerambycines such as N. a. acuminatus and its congener Oncideres cingulata (Say) 1 1 Neoclytus m. mucronatus (F.) were captured during our study, none dasycerus (Say) 2 6 8 of these species showed significant treatment effects, suggesting that Saperdini the catches were due to random encounters with traps, or possibly Saperda discoidea F. 5 5 to attraction to the visual silhouette of the traps. Saperda imitans Felt & Joutel 1 1 2 During Experiment 1, adults of A. variegatus were significantly Saperda tridentata Olivier 2 2 attracted only to traps baited with the blend of (S)-fuscumol and (S)- Lepturinae fuscumol acetate (Fig. 1; Friedman’s Q31,112 ¼5.76; P < 0.0001; Lepturini Fig. 1), consistent with the composition of the blend produced by Brachyleptura rubrica (Say) 3 3 males (Hughes et al. 2013). Additionally, adults of G. fasciatus were Strangalia bicolor (Swederus) 4 4 Strangalia luteicornis (F.) 1 1 more attracted to the blend of (R)-fuscumol and (R)-fuscumol acet- Typocerus deceptus Knull 1 1 ate than to the individual R-components (Fig. 1; Q34,112 ¼3.96, P < 0.0001). (continued) 1274 Environmental Entomology, 2016, Vol. 45, No. 5

Table 3. continued Taxonomy Expt. 1 Expt. 2 Total

Typocerus lugubris (Say) 1 2 3 Typocerus velutinus velutinus (Olivier) 3 3 Rhagiini Gaurotes cyanipennis (Say) 1 1 Parandrinae Parandrini Neandra brunnea (F.) 1 4 5 Prioninae Prionini Orthosoma brunneum (Forster) 15 4 19 Disteniidae Disteniini Elytrimitatrix undata (F.) 5 8 13 Total number of specimens: 476 494 970 Number of unique species: 39 37 48 Fig. 2. Mean (6 1 SE) number of beetles captured per trap and count period Downloaded from during Experiment 2. Means with different letters within species are signifi- cantly different (REGWQ means-separation test; P < 0.05).

(Mitchell et al. 2011), but was significantly attracted only by the blend of (R)-fuscumol and (R)-fuscumol acetate in this study. These http://ee.oxfordjournals.org/ findings suggest that weak attraction to incomplete simulations of pheromones may result in statistically significant treatment effects for some species of lamiines. On the other hand, the results of Experiment 2, when compared to earlier studies in which racemic compounds were tested, suggest that some species are attracted by blends even though they include components that a given species does not produce. For example, A. modestus was known to be at-

tracted by racemic fuscumol acetate, A. parvus to racemic fuscumol, at ESA Society Member on November 3, 2016 and A. macula to the blend of racemic fuscumol and racemic fuscu- mol acetate (Mitchell et al. 2011, Hanks et al. 2012, Hanks and Millar 2013). However, the present study suggests that the actual at- tractive components for these species were (S)-fuscumol acetate, (R)- fuscumol, and (S)-fuscumol alone, respectively. Thus, significant Fig. 1. Mean (6 1 SE) number of beetles captured per trap and count period during Experiment 1. Means with different letters within species are signifi- treatment effects of racemic fuscumol and fuscumol acetate, and cantly different (REGWQ means-separation test; P < 0.05). their individual enantiomers in field experiments should be inter- preted with caution, because lamiine beetles may be attracted to During Experiment 2, adults of A. variegatus were not caught in traps by incomplete reconstructions of their pheromones, or blends that include components which are not produced by their conspe- sufficient numbers for analysis, but three other lamiine species cific males, such as the enantiomers of their pheromone showed significant treatment effects, each to a different treatment, components. as follows (Fig. 2): 1) adults of Aegomorphus modestus (Gyllenhal) Attraction to pheromones also may be augmented by host vola- were attracted only by (S)-fuscumol acetate (Q ¼2.6; 24,115 tiles, as is the case with T. fuscum, adults of which were much more P ¼ 0.0004); 2) adults of Astylidius parvus (LeConte) were attracted attracted by a combination of fuscumol and spruce volatiles than by only by (R)-fuscumol (Q ¼5.75; P ¼ 0.032); and 3) adults of 14,5 fuscumol alone (Sweeney et al. 2010). In fact, fuscumol-baited traps Astylopsis macula (Say) were attracted only by (S)-fuscumol captured no more T. fuscum than control traps in the latter study. In (Q ¼5.14; P ¼ 0.0087). 9,10 the Lamiinae, H. betulinus females were more attracted to a com- bination of the male-produced pheromone with host volatiles than Discussion to the pheromone alone (Fonseca et al. 2010). Thus, host plant vola- tiles may play an important role in the attraction of spondylidine Attraction of A. variegatus by the specific blend of the (S)-enantio- and lamiine longhorned beetles, and future studies should investi- mers of fuscumol and fuscumol acetate which simulated the blend gate whether host volatiles may act additively or synergistically with produced by males confirmed the pheromone chemistry of this spe- the pheromones of the species captured in the present study. cies, and demonstrated that the two compounds are both necessary Although the pheromones of A. modestus, A. macula, A. parvus, and sufficient for trap capture. Mitchell et al. (2011) previously had and G. fasciatus have yet to be formally identified (i.e., by confirming reported that adults of A. variegatus were significantly attracted to that the compounds to which they are attracted are indeed produced racemic fuscumol acetate during field experiments, but the blend of by males), the present study provides evidence that all five species, racemic fuscumol þ racemic fuscumol acetate was more attractive including A. variegatus, differ as to which combinations of fuscumol when also present as another treatment. Moreover, G. fasciatus had and fuscumol acetate stereoisomers attract their adults. These findings previously been shown to be attracted to racemic fuscumol acetate lend support to the hypothesis that the chirality of fuscumol and Environmental Entomology, 2016, Vol. 45, No. 5 1275 fuscumol acetate allows for a sufficient number of distinct stereoiso- Hanks, L. M., J. G. Millar, J. A. Mongold-Diers, J.C.H. Wong, L. R. Meier, P. meric blends to provide species-specific pheromones for a community F. Reagel, and R. F. Mitchell. 2012. Using blends of cerambycid beetle of lamiine species. Interaction of the pheromones with host volatiles pheromones and host plant volatiles to simultaneously attract a diversity of also may serve to maintain reproductive isolation among sympatric cerambycid species. Can. J. For. Res. 42: 1050–1059. species with similar pheromone compositions. Thus, in the present Hansen, L., T. Xu, J. Wickham, Y. Chen, D. Hao, L. M. Hanks, J. G. Millar, and S. A. Teale. 2015. Identification of a male-produced pheromone compo- study we may have missed some species because we tested only a lim- nent of the citrus longhorned beetle, Anoplophora chinensis. PLoS ONE 10: ited number of combinations of stereoisomers and components. For e0134358. example, adults of Sternidius alpha (Say) were reportedly attracted by Hughes, G. P., Y. Zou, J. G. Millar, and M. D. Ginzel. 2013.(S)-fuscumol and racemic (E/Z)-fuscumol, and not affected by (E/Z)-fuscumol acetate (S)-fuscumol acetate produced by a male Astyleiopus variegatus (Mitchell et al. 2011, Hanks and Millar 2013), and the low numbers (Coleoptera: Cerambycidae). Can. Entomol. 145: 327–332. caught in our studies may be due to the absence of treatments that Lacey, E. S., M. D. Ginzel, J. G. Millar, and L. M. Hanks. 2004. Male-pro- included both enantiomers of fuscumol, or the absence of the duced aggregation pheromone of the cerambycid beetle Neoclytus acumina- (Z)-isomer. Overall, the cumulative results to date suggest that lam- tus acuminatus. J. Chem. Ecol. 30: 1493–1507. iine species in the tribes Acanthocinini and Acanthoderini, which Millar, J. G., and L. M. Hanks. 2016. Chemical ecology of cerambycid beetles. overlap broadly in seasonal flight period, may remain segregated by In Q. Wang (ed.), Cerambycidae of the world: biology and pest manage- differences in the stereochemistry of their pheromones. ment. CRC Press/Taylor & Francis, Boca Raton, FL (in press). Millar, J. G., L. M. Hanks, J. A. Moreira, and E. S. Lacey. 2009. Pheromone Downloaded from chemistry of cerambycid beetles, pp. 52–79. In K. Nakamuta and J. Millar Acknowledgments (eds.) Chemical ecology of wood-boring insects. Forestry and Forest Products Research Institute, Ibaraki, Japan. We thank the Northern Indiana Citizens Helping Ecosystems Survive Mitchell, R. F., E. E. Graham, J.C.H. Wong, P. F. Reagel, B. L. Striman, G. P. (NICHES) Land Trust and Tippecanoe County Parks & Recreation Hughes, M. A. Paschen, M. D. Ginzel, J. G. Millar, and L. M. Hanks. 2011. Department for allowing us to conduct experiments on their properties, and

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