Journal of Chemical Ecology(2020) 46:30-39 https:/ /doi.org/10.1007/s 10886-019-01128-7

Checkfor Pheromone Composition and Chemical Ecology of Six Species updates of Cerambycid Beetles in the Subfamily Lamiinae

1 2 3 1 3 1 Linnea R. Meier ' • Yunfan Zou • Judith A. Mongold-Diers • Jocelyn G. Millar • Lawrence M. Hanks CI:)

Received: 27 July 2019 /Revised: 15 November 2019 / Accepted: 27 November 2019 / Published on line: 6 December 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract Cerambycidbeetles of the subfamilyLamiinae use male-producedaggrega tion-sex pheromones thatare attractiveto bothsexes. Te1penoid pheromones have been identified from species in the tribes Acanthoderini and Acanthocinini native to North and South America, comprisedof (E)-6, 1 0-dimethyl-5,9-undecadien-2-one (geranylacetone), the structurally related 6-methylhept-5- en-2-one (sulcatone), and/or specific enantiomers or nomacemic ratios of enantiomers of the related compounds (E)-6, 10- dimethyl-5,9-undecadien-2-ol (fuscumol), its acetate ester, (E)-6, 10-dimethyl-5,9-undecadien-2-yl acetate (fuscumol acetate), and 6-methylhept-5-en-2-ol (sulcatol). Here, we present new infonnation about thechemical ecology of si.x acanthoderine and acanthocininespecies nativeto the easternUSA. The pheromoneof Astyleiopus variegatus (Haldeman) previously wasidentified as a blend of (S)-fuscumoland (S)-fuscumol acetate, and we report here thatgeranyl acetone is a synergistic component. Males of

Aegomorphus modestus (Gyllenhal), Lepturges angulatus (LeConte), and Lepturges confluens (Haldeman) were fow1d to pro­ duce similar blends composed of the enantiomers of fuscumolacetate and geranylacetone, whereas males of Astylidius parvus (LeConte) and Sternidius alpha (Say) produced both enantiomers of fuscumol together with (R)-fuscumol acetate and geranylacetone. Field experiments with synthesized chemicals revealed that species with similar pheromone composition nev­ e1theless differed in their responses to individual components, and to variousblends of components, and in how attraction was influenced by chemicals thatwere pheromonecomponents of other species. Sulcatone and/or sulcatol antagonizedattraction of some species to pheromones of the geranylacetone class, suggesting that there is an adaptive advantage in an ability to detect these heterospecific compounds, such as in avoiding cross attraction to other cerambycid species, as yet w-1known, that use pheromones composed of both chemical classes.

Keywords Longhomed beetle · Attractantpheromone · Terpenoids · Antagonism · Geranylacetone · Sulcatone

Introduction sex pheromones,produced by males and attractive to both sexes, that are either hydroxyethers or terpenoids (reviewed by Hanks Cerambycid beetles of the largest subfamily, the Lamiinae and Millar 2016). An example of thehydroxyether pheromone (Svacha and Lawrence 2014), are known to use aggregation- class is 2-(undecyloxy)ethanol (common name monochamol), the pheromone for several species in the genus Jvfonochamus (tribe Monochamini) that are native to Nmih America and The online version of this article Eurasia (e.g., Ryall et al. 2015). Te1penoid pheromones of (https://doi.org/10.1007/s10886-019-01128-7) contains supplementary material,Electronicwhich supplementary is available tomaterial authorized users. lamiines were first reported for the South American Hedypathes betulinus (Klug) (tribe Acanthoderini) whose [8l Lawrence M. Hanks male-produced pheromone is composed of (E)-6,10-dimethyl- [email protected] 5,9-undecadien-2-one (geranylacetone) and enantiomers of the con-esponding alcohol, (E)-6, 1 0-dimethyl-5,9-undecadien-2-ol 1 t Department of Entomology, Universiy of Illinois at (fuscumol), and acetate, (E)-6, 1 0-dimethyl-5,9-undecadien-2-yl Urbana-Champaign, 505 S. Goodwin Ave., 320 Morrill Hall, acetate(fuscumol acetate; Fonseca et al. 2010; Vidal al.et 2010). Urbana, IL61801, USA (S)-Fuscumol is also a pheromone component of cerambycid Present address: USDA-APHIS-PPQ-CPHST, 1398 W. Truck Rd., species in the subfamily Spondylidinae (Silk et al. 2007; OtisAir National Guard Base, BuzzardsBay, MA 02542, USA Sweeney et al. 2010). Recent research has shown that phero­ 3 Department of Entomology, University of California. mones of several North American species in the tribes Riverside, CA 92521, USA

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Acanthoderini and Acanthocinini are composed of compounds Identification of Pheromones of the geranylacetone class (Meier et al. 2016), or of (S)-6- methylhept-5-en-2-ol ([S]-sulcatol),an analogof the stmctu.rally Live adults of A. modestus, A. variegatus, L. conjluens, and related 6-methylhept-5-en-2-one (sulcatone; Meier etal. 2019). S. alpha, for collection of headspace volatiles, were caught Here, weprovide new information about thechemical ecol­ with cross-vane panel traps (black conugated plastic, Alpha ogy ofsi_x lamiine species nativeto theeastem USA, including Scents,Portland, OR, USA) withinterior surfaces coated with the acanthoderine Aegomorphus modestus (Gyllenhal), and the lubricantFluon® (fluoropolymer dispersion, 10% aqueous the acanthocinines Astyleiopus variegatus (Haldeman), dilution; NorthernProducts, Woonsocket, RI, USA). The trap Astylidius parvus (LeConte), Lepturges angulatus collection buckets were replaced with plastic jars having alu­ (LeConte), Lepturges confluens (Haldeman), and Sternidius minum screenbottoms that allowed rainwater to drain. Traps alpha (Say). All si_x species are broadly distributed in the were suspended -1 m above groundfrom inveited L-shaped eastem USA (Lingafelter 2007) andare among the most abun­ framesof polyvinylchloride pipe mounted on steel reinforcing dant lamiines in the area of our studies, east-central Illinois bar posts. Lures were polyethylene sachets (5.1 x 7.6 cm, (Hanks et al. 2014). The adult beetles are activeduring sum­ Bagettes® model 14,770, Cousin Corp., Largo, FL, USA) mer (Hankset al. 2014) and crepuscular to noctumal (LMH, loaded with 50 mg ofracemic fuscumol and/orfuscumol ac­ unpub. data). Larvae of all si_x species develop in woody tis­ etate dissolved in 1 ml isopropanol. Lures contained a cotton sues of hardwood trees and shrubs of many families roll (1 x 4 cm dental wick, Patterson Dental Supply, Inc., St. (Lingafelter 2007). Paul, MN, USA) that absorbed the solutions and minimized Pheromone components of some ofthe study species have leakage. Lures were replaced every 10-14 d. Single trapswere been identified previously. That is, males of A. parvus have deployed during summer months of 2013 to 2017 at all si_x been repmted to produce geranylacetone and both enantio­ study sites,and beetles werecollected eve1y 1-2 d. mers of fuscumol and (R)-fuscumol acetate, whereas males Captured beetles were sexed by themorphology ofthe fifth ofL. angulatus produce a blend of geranylacetone with both abdominal stemite (longer and more emarginate in females; enantiomersof fuscumol acetate (Meier et al. 2016). The pher­ Linsley and Chemsak 1995). Males and females were caged omone ofA. variegatus originally was identified asa blend of separately in the laboratory (ambient conditions: (S)-fuscumol and (S)-fuscumol acetate (Hughes et al. 2013), -12:12 h L:D, -20 °C). Because the adult beetles may feed but we rep01t here that geranylacetone is a synergistic phero­ on the tender bark ofoak or maple branchlets(pers. obs.), they mone component. Pheromones of A. modestus, L. confluens, were provided pieces of twigs freshly cut from oak or maple and S. alpha areidentified here for the firsttime. trees (Quercus alba L., Q. rubra L., Acer saccharum Marshall) at the same field sites where beetles had been trapped. Beetles also wereprovided sugar water(10% aque­ Methods and Materials ous sucrose solution in a glass vial with a cotton wick) as a sourceof moisture. Beetles were allowed to acclimate for 24 h Sources of Chemicals prior to the first headspace collections, and betweei1 subse­ quent collections. Compounds from commercial sources included racemic (E)­ Volatiles producedby beetleswere collected frombeetles held fuscumol and (E)-fuscumol acetate (Bedoukian Research, in glass Ma.son-stylecanning jars near closedexterior windows Danbury, CT, USA), (E)-geranylacetone (Sigma-Aldrich, St. (natural photoperiod, -14:10 h L:D, -20 °C) using previously Louis, MO, USA), sulcatone and racemic sulcatol (Alfa describedmethods (Meieret al. 2016, seeSupplementaty online Aesar, Haverhill, MA, USA). (S)-Fuscumol (98% e.e.), information). Chemicals were tentatively identified by gas (R)-fuscumol acetate (96.6% e.e.), and(S)-fuscumol acetate chromatography-mass spectrometry and stmctu.res were con­ (98% e.e.) were synthesized as described in Hughes et al. finnedby comparingretention times and massspectra with those (2013). of authentic standards(for details, see Supplementaryonline in­ fonnation). Extracts of headspace volatiles were prepared from Study Sites 14 males and 5 females of A. modestus, 15 males and 5 females ofA. va,iegatus, 5 males and O females ofL. conjluens, and 13 Field bioassays were conducted at si_x study sites in eastern males and 5 females of S. alpha. Illinois (Table 1), most of which were mature second-growth or successional forests dominated by species of deciduous Field Bioassays trees, including species of hickory (Carya), oak (Quercus), maple (Acer), and ash (Fra:xinus). The private residence was Attraction of beetles to synthesized pheromone components, in a suburban urban forest (-60 yearsold) of mature decidu­ presented individually and in blends, was tested with field ous and coniferous trees of many native and exotic species. bioassays using cross-vane panel traps as described above,

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t t Table 1 Sudy sies for field bioassays conducted in east­ Name County GPS coordinates(lat., long.) Area(ha) cenlral Illinois during 2013-2018 Allerton Park" Piatt 39.996, -88 .651 600 BrownfieldWoods" Champaign 40.145, -88.165 26 Forest Glen Preserveb Vermillion 40.0152, -87.568 728 Private residence (city of Urbana) Champaign 40.0971, -88.2032 NIA Trelease Woods" Champaign 40.132, -88.141 29 Vermillion River Observatory" Vermillion 40.066, -87.561 192

• Property of University ofIllinois naturalarea (h ttp://research.illinois.edu/cna/) b VerrnilionCounty ConservationDistrict but with thesupplied trap basins partly filled with saturated pheromone components ofa pmticular species, but werepher­ aqueous NaCl solution to kill andprese1ve captured beetles. omone components of other sympatric species (hereafter re­ Traps werepositioned 10 m apmt in lineai·transects, andtreat­ fen-edto as "heterospecific"compow1ds), as follows: ments were randomly assigned to traps on the day of setup. Traps were se1viced eve1y 1-3 d, at which time treatments 1) Experiment 1 was a follow-up to the field screeningtrials were shifted one position along transects to compensate for of Mitchell et al. (2011 ), but with treatments added to small-scale positional effects. assess the influence of geranylacetone (Table 2). Lmes Experiments 1-4 tested 19 w1ique combinations ofracemic were sachets made fromheat-sealed polyethylene tubing and chiral compow1ds of the germ1ylacetone and sulcatone sections (5 x 4 cm; Associated Bag, Milwaukee, WI, classes (Table 2), including several combinations of com­ USA) loaded with 1 ml of w1diluted chemical. Release pow1ds that had not been tested in earlier experiments (see rates were standardized to �20 mg/d by using tubing of Hughes et al. 2016; Meier et al. 2016, 2019; Mitchell et al. differentwall thicknesses and adding a cotton roll to some 2011). Some treatmentswere included specificallyto confum lures, as follows: fuscumol (1.5 mil wall thickness, with attractionof targetedspecies to synthesizedreconstmctions of cotton roll); fuscumol acetate (3 mil wall thickness, with their pheromones, others assessed the contribution of individ­ cotton roll), geranylacetone(3 mil wallthickness, without ual pheromone components to attraction, while still others cotton roll) (authors'w1pub. data). Separatelmes contain­ tested for antagonistic effects by compounds that were not ing differentchemicals were groupedon traps to produce

Table 2 Experimental treatments forfour field experiments that Treatment Abbreviation Expt. 1 Expt. 2 Expt. 3 Expt 4 tested attractionofa dult beetles of lamiine species to various blends Solvent control control X X X X ofracernic fuscurnol and Fuscurnol F X X fuscurnol acetate, the (R)-or (S)- Fuscurnol acetate Fa X X X enantiorners ofthese compounds, gcranylacetone, sulcatone, and Geranylacetone Ga X X X racernic sulcato1, and Sulcatone Sone X abbreviations of treatments used F+Fa FFa X X X in figures F +(R)-Fa FRFa X (S)-F + (S)-Fa SFSFa X F+Ga FGa X Fa+Ga FaGa X X F+Fa+Ga FFaGa X X F +(R)-Fa+Ga FRFaGa X (S)-F + (S)-Fa +Ga SFSFaGa X Fa+sulcatone FaSone X Ga+sulcatone GaSone X Fa+Ga+ sulcatone FaGaSone X Sulcatone + sulcatol SoneSol X F + sulcatone + sulcatol FSoneSol X Fa+sulcatone + sulcatol FaSoneSol X F +Fa +sulcatone + sulcatol FFaSoneSol X

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blends. Empty sachets (3 mil wall thickness) se1ved as dropped from analyses. For each analysis, greater weight blank controls. Lures were replaced every ~14 d. The was given to replicates with the largest numbers of speci­ experiment was conducted during three years at four mens (i.e., replicates that represented the independent re­ study sites (Table 3). sponses of multiple beetles to experimental treatments) by 2) Experiment 2 assessed the role of geranylacetone in at­ dropping those having fewer than a threshold number of traction ofA variegatus with the treatments including a beetles. Threshold numbers were chosen so as to maximize blend of (S)-fuscumol and (S)-fuscumol acetate, alone the number of beetles captured per replicate while main­ and combined with geranylacetone, as well as blends with taining a robust statistical test (at least 12 replicates; range (R)-enantiomers to test for antagonistic effects(Table 2). ofthreshold numbers 2 to 5). Type I enors were controlled Lures were the samepolyeth ylenesachets as were used to by setting the significancelevel ofP to 0.0025, so as to not collect beetles for headspace sampling, containing a cot­ be excessively conservative (Quinn and Keough 2002). ton wick loaded with individual compow1ds diluted in Assuming a significant overall statistical test, pairs oftreat­ isopropanol in doses of 50 mg for racemic fuscumol and ment means were compared using the Ryan-Einot-Gabriel­ fuscumolacetate (i.e., 25 mg perenantiomer) or 25 mg of Welsch Q multiple comparison test (REGWQ; SAS geranylacetone andspecific en antiomers offuscumol and Institute 2011 ). fuscumol acetate. The experiment was conducted during three years at five study sites (Table 3). 3) Experiment 3 tested for attraction of W1Specified species to various combinations of geranylacetone-type com­ Results pow1ds with sulcatone (Table 2), w1der the assumption that certain species might have pheromones comprised Extracts of volatiles emitted by males of A. modestus, ofboth basic structures. Trap lures consisted of the same A. variegatus, L. confluens, and S. alpha contained com­ polyethylene sachets mentioned above, loaded with pow1ds ofthe geranylacetone class in significant amounts, as 50 mg of racemic fuscumol or fuscumol acetate and confumed by matching mass spectra and retentiontimes with 25 mg of geranylacetone or sulcatone, diluted in 1 ml thoseof authentic standards.There were detectable quantities isopropanol. The experiment was conducted during ofthese compoundsin 10ofl4 extracts (~71%) frommales of 2017 at two study sites (Table 3). A. modestus, 11 of 15 extracts (~73%) from males of 4) Experiment 4 tested fuitherwuque combinations ofcom­ A. variegatus, 4 of 5 extracts (~80%) from males of pow1ds of the geranylacetone and sulcatone classes L. conjluens, and 4 of 13 extracts (~31%) from males of (Table 2). Lures wereconstructed of heat-sealed polyeth­ S. alpha. None of these compow1ds were detected in head­ ylene tubing (1.5 mil), loaded with 50 mg of racemic space extracts from femalesnor in system controls. sulcatol, fuscumol, or fuscumol acetate, or with 25 mg Headspace extracts from adult males of A variegatus of sulcatone, diluted in 1 ml ofisopropanol. The expeli­ containedtraces of geranylacetone (Table 4), and analysis of ment was conducteddwing two years at two study sites extracts on a chiral stationary phase Cyclodex B GC column (Table3). revealed that males also produce the (S)-enantiomers of fuscumol and fuscumol acetate, as reported previously Taxonomy of captured beetles follows Mom1eand Hovore (Hughes et al. 2013). Males of A. modestus, L. confluens, (2005). Representative specimens are available from the lab­ and S. alpha produced greater amow1ts of geranylacetone orato1y collection ofLMH, andvoucher specimens have been and specific enantiome1s of fuscumol and fuscumol acetate placed in the collection ofthe Illinois Natural Histo1y Survey, in combinations apparently identical to those produced by Champaign, IL. other species. That is, males ofA modestus and L. conjluens appeared to produce the same blend as malesof L. angulatus, Statistical Analysis consisting of both enantiomers of fuscumol acetate + geranylacetone, whereas males ofS. alphaproduced thesame Data from the four field experiments were analyzed sepa­ blend as males of A. parvus, i.e., both enantiomers of rately foreach ofthe study species that were represented by fuscumol + (R)-fuscumol acetate + geranylacetone. Within at least 10 specimens. The data violated homoscedasticity species, ratios ofenantiomers were in some cases difficult to assumptionsof ANOVA (Sokal and Rohlf1995), so differ­ estimatebecause compow1dswere present only in trace quan­ ences between treatment means, blocked by site and date, tities, for which peak area could not be measured reliably. were tested using the nonparametric Friedman's Test Nevertheless, there did appear to be some variability in the (PROC FREQ, option CMH; SAS Institute 2011). ratios offuscumol or fuscumolacetate enantiomers produced Replicates with zero specimens ofthe species of interest, by individual males of A. modestus, L. confluens, and for example as a consequence of inclement weather, were S. alpha.

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Table 3 Study sites and timing of four field experimentsthat tested Experiment Site (s) Time period attraction of cerambycid beetles to blends of synthesized AllertonPark, Brownfiel d Woods 22June - 6 August 2014 pheromone components Forest Glen 15 July -16 September 2015 Vermilion River Observatory 5 August-5 September 2013 2 Allerton,Fore st Glen, private 25 July- 10 September 2014; 23 July- 6 September residence 2018 Brownfield Woods, Trelease Woods 12June-23 August 2017 3 BrownfieldWoods, Trelease Woods 12June - 23 August 2017 4 BrownfieldWoods 22 April -4 August 2016 Forest Glen 15 July-15 September 2015

Field Bioassays reconstruction of the pheromone, but not to either compo­ nent alone, and attraction was antagonized by the A total of 1484 beetles of the six species were captured during heterospecific compow1d fuscumol. However, in experi­ the field experiments(Table 5). During experiment 1, adults of ment 3, beetles of this species were attracted to fuscumol A. variegatus were significantly attracted only to the complete acetate alone (Fig. 3A), and attraction was not significantly blendof racemic fuscumol,fuscumol ace tate, and geranylacetone influenced by either geranylacetone or the heterospecific

(overall Friedman's Q7,72 =42.2, P <0.001), with the blend compow1d sulcatone. In experiment 4 the only significant attracting a mean(± SE) of 3.9 ±0.8 beetles per replicate com­ attractant was the blend of fuscumolacetate + sulcatone + paredto means of fewer than1.1 beetlesfor traps baited with the sulcatol (Fig. 4A), an w1expected enhancing effect by the individual components, binmy combinations, and the control latter two heterospecific compow1ds. Addition of fuscumol (REGWQ,P <0.05). This finding suggested thatall the compo­ to this blend again resulted in antagonism. Earlier research nents were necessaiy for optimal attraction and that the non­ had confirmed attraction of A. modestus to (S)-fuscumol natural (R)-enantiomers were not strongly antagonistic. acetate alone, and not to the (R)-enantiomer, but also to However,previous studieshave shown thatadults of thisspecies racemic fuscumol acetate, and racemic fuscumol acetate respond to racemic fuscumol acetatealone (i.e., attraction to [S]­ + fuscumol, despite the antagonistic propeities of the latter fuscumol acetate inthe racemate) or blended with fuscumol, in compow1d (Hanks et al. 2012, 2018; Hughes et al. 2016; the absence of geranylacetone (e.g., Hanks and Millar 2013; Millar et al. 2018; Mitchell et al. 2011). Millar et al. 2018; Mitchell et al. 2011). Other species in the Adults of L. angulatus were attracted to traps baited with present study also were attractedby such incompletereco nruuc­ fuscumol + fuscumol acetate in experiment 2 (Fig. 2A) which tions ofpherom one blendswhen the optimal blendwas not in­ lacked a treatment with fuscumol acetate alone, and cludedas a treatmentin a particularbioassay, as explainedbelow. ger-anylacetone was not an attractant, but 1-ather· antagonized at­ We next consider the species whose pheromones were traction. The lack of attraction to racemic fuscumol + (R)­ composed of both enantiomers of fuscumol acetate + fuscumol acetate, and to (S)-fuscumol + (S)-fuscumol acetate, geranylacetone: A. modestus, L. angula tus, and suggested that the individual enantiomers of fuscumol acetate L. confluens. Adults of A. modestus were attracted only were insufficient for at11-action. In expe1imer1t 3, beetles were to the blend of racemic fuscumol acetate + geranylacetone at11-acted by fuscumol acetate alone (Fig. 3B), and apparently during experiment 1 (Fig. IA), i.e., the complete not ililluer1ced by either ger·anylacetone or the heter·ospecific

Table 4 Average relative percentages of compounds detected in ordered so as to emphasize similarities in pheromone composition. headspace samples from males of six species of cerarnbycid beetles of Chirality of fuscurnoland fuscurnol acetate is indicated by R and S and the subfamily Larniinae native to eastern North America. Species are ratios of enantiornerswithin brackets

Species Fuscurnol (RJS) Fuscurnolacetate (RJS) Geranyl-acetone Reference

Astyleiopus wiriegatus 33 (0/100) 67 (0/100) trace Hughes et al. 2016, presentarticle Aegomorphus modestus 75 (25/75) 25 present article Lepturgesangulatus 75 (70/30) 25 Meier et al. 2016 Lepturges conjluens 10 (71/29) 90 present article Astylidius pannis 60 (50/50) 20 (100/0) 20 Meier et al. 2016 Stemidius alpha 56 (60/40) 36 (100/0) 8 present article

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Table S Numbers of six species ofcerambycid beetles captured during Males of both A. parvus and S. alpha produced both enan­ four field experiments conducted in east-central Illinois during 2013- tiomers of fuscumol, (R)-fuscumol acetate, and 2018 geranylacetone, and adults of A. parvus were significantly Species Experiment Total attracted to traps baited with the complete reconstmction of

this blend during experiment 2 (Fig. 2C). However, significant 2 3 4 numbers of beetles also were attracted to traps baited with Aego11101phus modestus 70 7 45 29 151 racemic fuscumol + fuscumol acetate without geranylacetone. Astyleiopus variegatus 67 60 21 24 172 These findingssuggest that the latter compound was at most a Astylidius pmvus 338 56 8 65 467 weak synergist, and that the non-natural (S)-fuscumol acetate Leptwges angulatus 229 45 108 29 411 was not antagonistic. Experiment 4 showed that this species Leptwges conf/uens 37 52 26 3 118 was attracted by racemic fuscumolalone (Fig. 4C), a treatment Sternidius alpha 139 8 0 18 165 missing in the previous experiment, and attraction was not Total 880 228 208 168 1484 influenced by the heterospecific compounds sulcatone +

A 4 a compound sulcatone, but antagonized by the blend of these two OI ,g 3 compounds. The inconsistent effect of geranylacetone between Q. experiments 2 and 3 suggests that it is antagonistic only when a, 2 paired with the heterospecific compounds fuscumol or sulcatone. ia, .c Lastly, experiment 4 again showed significant attraction to 'It C OI fiJScumol acetate alone (Fig. 4B), and antagonism by sulcatone a, ::!! + sulcatol. There is a wealth of information about the chemical b ecology ofthis common species, withseveral earlierstudies hav­ F Fa Ga FFa FGa FaGa FFaGa Control ing demonstrated attraction to fuscumol acetate, that both enan­ tiomers are necessary, and that attractionis not influencedby the B 1.5 heterospecific compound fuscumol (e.g., Hanks and Millar .21 2013; Hankset al. 2018; Meier et al. 2016; Mitchell et al. 2011). -� a Adults of L. co,?fluens were significantlyattracted only to the g. 1.0 ab blend of racernic fuscumol + fuscumol acetate + geranylacetone a, in experiment 1 (Fig. lB), although weak statistical power limit­ a, � 0.5 ed interpretationof other treatment effects. Beetles were not sig­ C ab OI nificantly attracted to the same blend in experiment 2 (Fig.2B ), a, but rather only to (S)-fuscumol + (S)-fuscumol acetate + b b geranylacetone, suggesting that (S)-fuscumol acetate + F Fa Ga FFa FGa FaGa FFaGa Control geranylacetone was a sufficient attractant given that this species C s.o does not produce either enantiomer of fiJScumol, and that (R)­ fiJScmnol wasantagonistic. Earlier studies have shown attraction .21 .2 of L. co,?fluens to racernic fiJScmnol acetate alone, or combined with racernic fuscumol (Hanks and Millar 2013; Millar et al. �o a, 2018). This prior attractionto racernic fuscumol acetate, but only I to the blend containing (S)-fuscumol acetate in experiment 2 of � 2.0 C the present article, suggests that (R)-fiJScumol acetate is not nec­ OI a, essary for attraction, even though it was present in the insect­ ::!! b b b b produced volatiles. Despite apparently having pheromones of similar composi­ F Fa Ga FFa FGa FaGa FFaGa Control Treatment tion, A. modestus, L. angulatus, and L. co,?fluens showed quite Fig. 1 Mean (± SEJ number of beetles (sexes combined) per replicate different responses to the same experimental treatments. For ex- captured during experin1ent I offue species: AJ Aego11101phus modestus A. ample, there were marked differences between modestus and (Friedman's Q7,64=36.0, P<0.0001), BJ Lepturges confluen s L. co,?fluens in their responses during experiment 1 (Fig. lA,B), (Friedman's Q7,72 = 24.9, P= 0.0008), and CJ Stemidius alpha (Q7,80 = 58.8, P<0.0001). Chemical abbreviations: F=racemic fuscumol, Fa= between L. angulatus and L. co,?fluens during experiment 2 A. racemic fuscumol acetate, Ga=geranylacetone (see Table 2 for complete (Fig. 2A,B), and between modestus and L. angulatus during list oftreatments). Means wifuin figures wifu different letters are signif­ experiment 3 (Fig.3A,B ) and experiment 4 (Fig.4A,B ). icantly different (REGWQ test, P < 0.05)

� Springer 36 J Chem Ecol (2020) 46:30-39 sulcatol. Racemic fuscumol acetate was not inherently attrac­ racemic fuscumol acetate, even though males produce (R)­ tive, farther suggesting that (R)-fuscumolacetate was not like­ fuscumol acetate (see above). The latter finding suggests that ly to be attractive as a single component. Previous reports the heterospecific (S)-fuscumol acetate is antagonistic. have shown that adults ofA parvus were attracted by racemic Attraction to racemic fuscumol again was evident in experi­ fuscumoL but not to the individual enantiomers, further ment 4 (Fig. 4D), as was antagonism by racemic fuscumol supporting the case for synergism between enantiomers, and acetate, and also by the heterospecific compounds sulcatone were not influenced by (R)-fuscumol acetate even though it + sulcatol. Earlier studies have shown attraction ofthis species was present in headspace volatiles from males (e.g., Meier to racemic fuscumol,but not to fuscumol acetate (Hanks et al. et al. 2016; Millar et al. 2018). 2018; Millar et al. 2018; Mitchell et al. 2011). Adults of S. alpha were significantly attracted by racemic As with the above mentioned three species with similar fuscumol in experiment 1 (Fig. 1C) , and attraction was not pheromones, adults of S. alphaand A parvus differed in their influenced by geranylacetone, but was antagonized by responses to treatments in the one experiment they had in common, experiment 4 (compare Fig. 4C,D), despite their A 2.0 apparently having pheromones of similar composition. .s"' a .!:! Discussion CII 1.0 b .cCII Our research revealed a third compound in the blend pro­ 'II, C duced by males of A. variegatus, geranylacetone, which CII"' ::!: synergized attraction to the two components identified pre­ 0.0 viously, (S)-fuscumol and (S)-fuscumol acetate (Hughes Ffa FFaGa SFSFaGa FRFa Ga FRFaGa Control et al. 2013). Geranylacetone also was detected in head­ B 3.5 space extracts from males of the other five species, and

! 3.0 "' A 1.5 � a 2.5 !"' 2.0 .!:! CII g- 1.0 CII 1.5 .c CII 'II, C 1.0 ab CII"' ::!: 0.5 � 0.5 b C 0.0 CII"' ::!: FRFa Ga FRFaGa Control C C C C 2.0 Fa Sone FaSone FaGaSone Ga FaGa GaSone .s Control "' B 3.o .!:! 1.5

! a ab CII -� :;::, 1.0 ii CII abc f! 2.0 .cCII 'II, abc C 0.5 CII "' CII CII be ::!: � 1.0 C 0.0 CII"' Ffa SFSFaGa ::!: FRFa Ga FRFaGa Control C

Treatment Fa Sone FaSone FaGaSone Ga FaGa GaSone Control Fig. 2 Mean (± SEJ number of beetles (sexes combined) per replicate captured during experiment 2 of the species: AJ Lepturges angulatus Treatment (Q7,112=46.3, P<0.0001), BJ Lepturges confluens (Q7,78= 19.0, P= Fig. 3 Mean (± SEJ number of beetles (sexes combined) per replicate 0.008), and CJ Astylidius parvus (Q7,88=33.7, P<0.0001). Chemical captured during experin1ent3 of the species: AJ Aego11101phus modestus abbreviations: F= racemic fuscumol, Fa= racemic fuscumol acetate, (Q7,96, = 38.2, P<0.0001), and BJ Lepturges angulatus (Q7,96=47.7, Ga= geranylacetone; R and S indicate individual enantiomers of P<0.0001). Chemical abbreviations: Fa=racemic fuscumol acetate, fuscumol and fuscumol acetate (see Table 2 for complete list of Ga= geranylacetone, Sane=sulcatone (see Table 2 for complete list of treatmentsJ. Means within figures with different letters are significantly treatmentsJ. Means within figures with different letters are significantly different (REGWQ test, P < 0.05 J different (REGWQ test, P< 0.05)

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2.5 A ◄ Fig. 4 Mean (± SEJ number of beetles (sexes combined) per replicate .!l captured during experin1ent4 of the species: AJ Aego11101phus modestus � 2.0 a - (Q7,64=30.4, P<0.0001). BJ Lepturges angulatus (Q7,96=32.7, C. P<0.0001), CJ Astylidius parvus (Q7,88=37.8, P<0.0001), and DJ !!! 1.5 "iii Sternidius alpha (Q7,103 =57.3, P< 0.0001). Chemical abbreviations: a, F= racemic fuscumol, Fa=racemic fuscumol acetate, SoneSol= _! 1.0 sulcatone + racemic sulcatol (see Table 2 for complete list of 'II: treatmentsJ. Means within figures with differentletters are significantly C 0.5 different (REGWQ test, P< 0.05) ::!E=

F SoneSol FSoneSol FFaSoneSol Fa FFa FaSoneSol Control A. parvus, and S. alpha, but apparently antagonizing at­ traction of L. angulatus when in the presence of the B 1.5 heterospecific compounds fuscumol or sulcatone. The varying role of geranylacetone is consistent withearlier .!:! a research which has shown that some compounds released by C. !!! 1.0 male cerambycids appear not to be involved in attraction of Hi conspecifics (Hanks et al. 2019). For example, the pheromone :; a, of the South American lamiine Psapharochrus maculatissimus � 0.5 C (Bates)is composed of (S)-fiJScumolacetate with a small propor­ tion of the (R)-enantiomer, and the minor component appears to ::!E= contributenothing to attraction(Silva et al. 2019). However, such

F SoneSol FSoneSol FFaSoneSol apparently inactive compounds may play a role in mediating Fa FFa FaSoneSol Control interactions among sympatric species, such as by antagonizing attraction to a shared pheromone component. For example, the C 2.0 pheromone of the NorthAmericanXylotrechus colonus (F.) (sub­ .!l., a family Cerambycinae) is composed primarily of (R)-3- ,l:l 1.5 hydroxyhexan-2-one, a necessary and sufficient attractant. but !!! enantiomers of the related 2,3-hexanediols also are produced in "iii ab � 1.0 trace quantities. The diols apparently have no effect on attraction a, a, of conspecifics, but rather antagonize attraction of the sympatric 'II: 0.5 Neoclytus mucronatus mucronatus (F.)to the shared ketol com­ a,i ::!E ponent (Hankset al. 2019). Thus, at least some male cerambycids C C 0.0 appear to produce pheromone blends containing components F FSoneSol FFaSoneSol which affect the behavior of both conspecifics and Fa FFa FaSoneSol Control heterospecifics. In the latter example, adults of N m. mucronatus clearly D 1.0 a are capable of detecting the antagonistic 2,3-hexanediols of .!l ., X colonus, even though these compounds were not pro­ .!:! C. duced by males of N m. mucronatus. Antagonism by com­ !!! "iii pounds produced by heterospecifics provides further evi­ � 0.5 a, dence of behavioral mechanisms that have evolved to limit a, .c b 'II: interspecific attraction. As a further example, males of A. variegatus produced (S)-fuscumol + (S)-fuscumol ace­ a,., ::!E tate + geranylacetone, but attraction of A. modestus to the b b b b 0.0 fuscumol acetate component would be antagonized by the F SoneSol FSoneSol FFaSoneSol Fa FFa FaSoneSol Control fuscumol component, and attraction of S. alpha to Treatment fuscumol would be antagonized by the fuscumol acetate component. in fact is a pheromone component of the first lamiine spe­ The interplay of attractants and antagonists for the six spe­ cies reported to have pheromones of the geranylacetone cies in our study is summarized in Table 6. In addition to the class, H betulinus (Vidal et al. 2010). However, among species specificity imparted by antagonistic compounds pro­ the other five study species, geranylacetone appeared to duced by heterospecifics, interspecific attraction between sev­ play a variable role, enhancing attraction of L. ca,�fluens, eral other pairs of the study species is likely to be averted due having little if any effect on attraction of A. modestus, to the absence of critical components, as follows:

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Table 6 Summary of chemically mediated interactions among the study species Species Compounds produced by each species Antagonists Interspecificsynergist

Fuscurnol Fuscurnol Geranyl-acetone acetate

(R) (S) (R) (S)

Astyleiopus variegatus p p p Aegomorphus modestus + p -+ Fuscurnol Sulcatone + sulcatol blend Lepturgesangulatus p p + Geranylacetone + sulcatone blend Lepturges conjluens + p p (R)-fuscurnol Astylidius pannis p p + + Stemidius alpha p p -+ -+ (S)-fuscurnol acetate, sulcatone + sulcatol blend Compoundsunderlined in bold are reported for the fasttime; olhercompounds have been previouslyrepor ted. P = pheromone, +=present, but withno clear activity

1) Adults of A. variegatus, A. modestus, L. angulatus, and differing diel activity periods, may also play a role in repro­ L. confluens all require (S)-fuscumol acetate, which is ductive isolation (e.g., Meier et al. 2019). Other factors that absentin thepheromones of A. parvus and S. alpha; could serve to segregate the species include vaiyingresponses 2) Adults of L. angulatus require both enantiomers of to pruticular host plant volatiles or preferences for different fuscumolacetate, but males ofA. variegatus only produce vertical strata within forests (reviewed by Hanks and Millar the (S)-enantiomer; 2016; Millai· andHanks 2017). 3) Adults of A. pa1vus and S. alpha require enantiomers of The pheromone of Astylopsis macula (Say) provided the fuscumolthat are absent in the pheromones of the other firstevidence that pheromones oflamiines may be composed fom species. of compow1ds of both the geranylacetone andsulcatone clas­ ses (Meier et al. 2019). Although none of thesi_x study species in the present article produced compounds of the sulcatone It also should be notedfrom these examples that the mech­ class, the field experiments nevertheless revealed that anisms that prevent interspecificattraction can be complimen­ sulcatone and/or sulcatol influenced attraction of tmy, for example preventing attraction betweenA. variegatus L. angulatus and S. alphato theirreconstructed pheromones, andA parvus in both directions. There also is evidence that indicating that these two compow1ds ai·e indeed perceived by these mechanisms can be redundant. For example, attraction these species. This in tumsuggests thatthese species may be ofA. modestus to callingmales of A parvus would be ave1ted sympatric with asyet w1kt1owt1species which produce blends both due to antagonism by fuscumol and the absence of the of geranylacetone- and sulcatone-type compounds. critical attractant (S)-fuscumolacetate. The research presentedhere has not identified factors that Acknowledgements We thankJodie A. Ellis for assistance with labora­ would prevent cross attraction between species that apparently tory work. We also thank S. Buck and the University of Illinois Committee on Natural Areas for access to field sites. Thisresearch was have pheromones composed of the same components. supported by a grant to L.M.H. from The Alphawood Foundation of Specifically, males of A. modestus, L. angulatus, and Chicago, and grants to J.G.M. and L.M.H. from the United Stated L. confluens produce blends consisting of both enantiomers Department of Food and Agriculture, National Institute of Food and of fuscumol+ geranylacetone, while males of A pan,us and Agriculture (USDA-NIFA, grant numbers 2012-67013-19303 and 2015-67013-23173), and USDA-Animal and Plant Health Inspection S. alpha produce both enantiomers of fuscumol + (R)­ Service grant#15-8130-1422-CA. fuscumol acetate+ geranylacetone.For other types of insects in which multiple related species have similar pheromones, in particularmoths and barkbeetles (Curculionidae, Scolytinae), References species specificityoften is impartedby relative ratiosof com­ ponents(e.g., Borden et al. 1980; Heathet a!. 1990). However, BordrnJH, Handley JR McLeanJA, Silverstein RM, Chong L, Slessor previous research has suggested that conspecific males of KN, JohnstonBD, Schuler HR(1980) Enanliorner-basedspecificity lamiines may vary considerably in the ratios of pheromone in pheromonecommunication by twosyrnpatric Gnathot,ichiis spe­ components they produce (Meier et al. 2016, 2019), suggest­ cies (Coleoptera: Scolytidae). J Chern Ecol 2:445-456 Fonseca MG, Vidal DM, Zarbin PHG (2010) Male-produced sex phero­ ing that ratios of components may not be reliable indicators of mone of the cerarnbycid beetle Hedypathes betulinus: chemical species specificity, and that other mechanisms, such as identificationand biological activity. J ChernEcol 36:1132-1139

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Hanks LM, Millar JG (2013) Field bioassays of cerambycid pheromones Millar JG, Hanks LM (2017) Chemical ecology of cerambycids. In: reveal widespread parsimony of pheromone structures, enhance­ Wang Q (ed) Cerambycidae ofthe world: biology and pestmanage­ ment by host plant volatiles, and antagonism by components from ment. CRC Press/faylor & Francis, Boca Raton, pp 161-208 heterospecifics. Chernoecol23:21-44 Millar JG, Mitchell RF, Mongold-Diers JA, Zou Y, Bogran CE, Fierke Hanks LM, Millar JG (2016) Sex and aggregation-sex pheromones of MK,Ginzel MD, Johnson CW, Meeker JRPoland Thf, Ragenovich cerambycid beetles: basic science and practical applications. J IR, Hanks LM (2018) Identifying possible pheromones of Chern Ecol 42:631-654 cerarnbycid beetles by field testing knownpheromone components HanksLM, MillarJG, Mongold-Diers JA, Wong JCH, Meier LR, Reagel in four widely separated regions of the United States. J Econ PF, Mitchell RF (2012) Using blends of cerambycid beetle phero­ Entornol 111:252-259 mones and host plant volatiles to simultaneously attracta diversity Mitchell RF, Graham EE, Wong JCH, Reagel PF, StrirnanBL, Hughes ofcerambycidspecies. Can J For Res 42:1050-1059 GP, Paschen MA, Ginzel MD, Millar JG, Hanks LM (2011) HanksLM, Reagel PF, Mitchell RF, Wong JCH, Meier LR Silliman CA, Fuscurnol and fuscumol acetate are general attractants for many GrahamEE, Slrirnan BL, Robinson KP, Mongold-Diers JA, Millar species of cerarnbycid beetles in the subfamily Lamiinae. Entornol JG (2014) Seasonal phenology of the cerarnbycid beetles of east­ Exp Appl 141:71-77 Central Illinois. Ann Entornol Soc Arn 107:211-226 Monne MA, Hovore FT (2005) Checklist of the Cerambycidae Hanks LM, Mongold-Diers JA, Atkinson TII, Fierke MK, Ginzel MD, (Coleoptera) of the Western hemisphere. BioQuip, Rancho GrahamEE, Poland Thf, RichardsAB, RichardsonML, Millar JG Dominguez (2018) Blends of pheromones, with andwi thout host plantvolatiles, QuinnGP, KeoughMJ (2002) Experimental design and data analysis for can attractmult iple species of cerambycid beetles simultaneously. J biologists. Cambridge UniversityPress, Cambridge, UK Econ Entornol 111 :716-724 Ryall K., Silk P, Webster RP, GutowskiJM, Meng Q, Li Y, Gao W, Fidgen Hanks LM, Mongold-Diers JA, Mitchell RF, Zou Y, Wong JCH, Meier J, Kimoto T, Scarr T, Mastro V, Sweeney JD (2015) Further evi­ LR Johnson TD, Millar JG (2019) The role of minor pheromone dence thatmonocharnol is attractive to Monochamus (Coleoptera: components in segregating 14 species of longhomed beetles Cerambycidae) species, withattraction synergised by host plant vol­ (Coleoptera: Cerambycidae) of the subfamily Cerambycinae. J atiles andbark beetle (Coleoptera:Curculionidae) pheromones. Can Econ Entornol 112:2236-2252 Entornol 147:564-579 HeathRR, Mitchell ER Tovar JC (1990) Effectofrelease rateand ratioof SAS Institute(2011) SAS/STAT 9.3 user's guide. SAS Institute Inc., Cary (Z)-11-hexadecen- l-ol from synthetic pheromone blends on trap Silk PJ, Sweeney J, Wu J, Price J, Gutowski JM, Kettela EG (2007) capture of Heliothis subflexa (Lepidoptera: Noctuidae). J Chern Evidence for a male-produced pheromone in Tetropium fascum Ecol 16:1259-1268 (F.) and Tetropium cinnamopterum (Kirby) (Coleoptera: Hughes GP, Zou Y, Millar JG, Ginzel MD (2013) (S)-fuscurnoland (S)­ Cerambycidae). Naturwissenschaften 94:697-701 fuscumol acetate produced by a male Astyleiopus variegatus Silva \VD, Zou Y, HanksLM, BentoJMS, Millar JG (2019) Enantiorners (Coleoptera: Cerambycidae). Can Entornol 145:1-{5 of fuscurnolacetate comprise the aggregation-sex pheromone of the Hughes GP, Meier LR Zou Y, Millar JG, HanksLM, Ginzel MD (2016) south American cerambycidbeetle Psapharochrus maculatissimus, Stereochernislryoffuscurnol and fuscumol acetateinfluences attrac­ and likely pheromones ofthe cerarnbycidsEu promerellaplawnann i tion of longhomed beetles (Coleoptera: Cerambycidae) of the sub­ andHylettus seniculus. Entomol Exp Appl 167:915-921 family Lamiinae. Environ Entornol45:1271-1275 Sokal RR Rohlf FJ (1995) Biometry, 3rd edn. WH Freeman and Co, Lingafelter SW (2007) Illustrated key to the longhorned wood-boring New York beetles of the eastern United States. Special publication no. 3. Svacha P, Lawrence JF (2014) Chapter 2.4 Cerambycidae Latreille, 1802, Coleopterists Society,North Po tomac, p 206 pp. 77-177. In: Leschen RAB, Beutel RG (eds) handbook of zool­ Linsley EG, Chernsak JA (1995) The Cerambycidae of North America, ogy: Arthropoda: Insecta: Coleoptera,beetl es. Vol. 3: morphology part VII, no. 2: taxonomy and classification of the subfamily and systematics (Phytophaga), Walter de Gruyter, Berlin/Boston Lamiinae, tribes Acanthocinini through Hernilophini. Univ Calif Sweeney JD, Silk PJ, Gutowski JM, Wu J, Lemay MA, Mayo PD, Magee Pub! Entomol 114:1-292 DI (2010) Effectof chirality, release rate, and host volatiles on re­ Meier LR, Zou Y, Millar JG, Mongold-Diers JA, Hanks LM (2016) sponse of Tetropiumfiiscum (F.), Tetropium cinnamopterum Kirby, Synergism betweenenantiomers creates species-specific pheromone and Tetropium castaneum (L.) to the aggregation pheromone, blends andminimizes cross-attraction for two species of cerambycid fuscurnol. J Chern Ecol 36:1309-1321 beetles. J Chern Ecol 42: 1181-1192 Vidal DM, Fonseca MG, Zarbin PH (2010) Enantioselective synthesis MeierLR Millar JG, Mongold-Diers JA, Hanks LM (2019) (S)-Sulctola and absolute configuration of the sex pheromone of Hedypathes is a pheromone component for two species of cerambycid beetles in betulimis (Coleoptera: Cerarnbycidae). Tetrahedron Lett 51:6704- the subfamily Lamiinae. J Chern Ecol 45:447-454 6706

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