J Chem Ecol (2016) 42:631–654 DOI 10.1007/s10886-016-0733-8

Sex and Aggregation-Sex Pheromones of Cerambycid Beetles: Basic Science and Practical Applications

Lawrence M. Hanks1 & Jocelyn G. Millar2

Received: 6 May 2016 /Revised: 25 June 2016 /Accepted: 21 July 2016 /Published online: 8 August 2016 # Springer Science+Business Media New York 2016

Abstract Research since 2004 has shown that the use of vol- Introduction atile attractants and pheromones is widespread in the large bee- tle family Cerambycidae, with pheromones now identified from The family Cerambycidae, or the longhorned beetles, is more than 100 species, and likely pheromones for many more. among the largest insect families, with approximately 35,000 The pheromones identified to date from species in the subfam- described species in the eight subfamilies currently recognized ilies Cerambycinae, Spondylidinae, and Lamiinae are all male- (Švácha and Lawrence 2014). The larvae are endophytic, with produced aggregation-sex pheromones that attract both sexes, the vast majority of species developing in woody plants whereas all known examples for species in the subfamilies (Haack 2016). As such, the beetles perform valuable ecosys- Prioninae and Lepturinae are female-produced sex pheromones tem services by initiating the degradation of woody materials, that attract only males. Here, we summarize the chemistry of and species can be useful indicators of ecosystem health (e.g., the known pheromones, and the optimal methods for their col- Maeto et al. 2002). However, many cerambycid species are lection, analysis, and synthesis. Attraction of cerambycids to important pests in their native regions, damaging and killing host plant volatiles, interactions between their pheromones trees in natural and managed habitats, and degrading wood and host plant volatiles, and the implications of pheromone destined for lumber (e.g., Solomon 1995). The larvae fre- chemistry for invasion biology are discussed. We also describe quently are long-lived, and consequently are transported read- optimized traps, lures, and operational parameters for practical ily by global commerce, concealed in wooden products and applications of the pheromones in detection, sampling, and shipping materials (Haack et al. 2010). As a result, management of cerambycids. cerambycids are among the most common exotic phytopha- gous species intercepted in international quarantine (e.g., Keywords Semiochemical . Attractant . Host plant volatile . Brockerhoff et al. 2006;Haack2006;Liebholdetal.1995; Invasive species . Detection Nowak et al. 2001). Exotic cerambycids have the potential to become devastat- ing pests when introduced into new regions of the world. For Submitted to: Journal of Chemical Ecology example, the invasion of North America, and the more recent invasion of Europe by the Asian longhorned beetle, Electronic supplementary material The online version of this article (doi:10.1007/s10886-016-0733-8) contains supplementary material, Anoplophora glabripennis (Motschulsky) (Meng et al. which is available to authorized users. 2015), represent examples of the invasive potential of cerambycids, and the enormous difficulty and cost in detect- * Lawrence M. Hanks ing, controlling, or eradicating them from regions they have [email protected] invaded. On both continents, we are about to witness the huge economic and environmental impact that exotic cerambycids 1 Department of Entomology, University of Illinois at can have on natural and urban forests as A. glabripennis ex- Urbana-Champaign, Urbana, IL 61801, USA pands its ranges, attacking and killing deciduous trees of many 2 Departments of Entomology, University of California, species (Haack et al. 2010). In the context of these and other Riverside, CA 92521, USA invasions by cerambycid beetles, pheromone-baited traps that 632 J Chem Ecol (2016) 42:631–654 are effective for detecting and sampling the insects would be stereoisomers can be used in lures. Although it has been pos- valuable additions to the otherwise meager tools available for sible only to show that prionic acid is present in extracts from their surveillance, eradication, or management. This review females of one other species (Prionus laticollis [Drury], JGM summarizes the rapid progress that has been made since unpub. data), the mixture of stereoisomers is attractive to 2004 in the identification and synthesis of cerambycid phero- males of all six North American Prionus species with which mones, the patterns of pheromone and host plant volatile use it has been tested, as well as the European Prionus coriarius by species in the various subfamilies, the development of (L.) (Agnello et al. 2011; Barbour et al. 2011). Furthermore, practical applications of their pheromones, and the implica- prionic acid was found to be highly attractive to males of the tions of pheromone chemistry for invasion biology. Asian prionine Dorysthenes granulosus (Thomson) (Wickham et al. 2016a), suggesting that the pheromone struc- Types of Pheromones Used by Cerambycids ture is conserved across at least these two genera. It remains to be seen whether this compound will attract any of the many Despite the economic importance of many cerambycid species other species of Dorysthenes that are native to Asia (Löbl and in their native countries, and the threat they pose as potential Smetana 2010). invaders worldwide, remarkably little is known about the basic A second structural class of female-produced sex phero- biology of even common and well-known species (e.g., see mones, consisting of 2,3-alkanediols, has been discovered in Solomon 1995). Until recently, even less was known about other prionine genera (Tables 1, S1). Thus, females of several their chemical ecology, with attractant pheromones identified North American species of Tragosoma seem to use various from fewer than 10 species (Allison et al. 2004). However, stereoisomers of 2,3-hexanediol as sex attractant pheromones during the past decade, pheromones or likely pheromones have (Ray et al. 2012a), whereas the sex pheromone of the Asian been identified from more than 100 species (Tables 1, S1; prionine Megopis costipennis (White) consists of (2R,3S)-2,3- Millar and Hanks 2016). The volatile attractant pheromones octanediol (Wickham et al. 2016c). Surprisingly, the same 2,3- are of two types: aggregation pheromones (or aggregation-sex alkanediol structures are used as aggregation-sex pheromones pheromones, sensu Cardé 2014), that are produced by males by many species in the Cerambycinae (see below), and even and attract both sexes, or sex pheromones that are produced by more remarkable is that the pheromone-producing prothoracic females and attract only males (Millar and Hanks 2016). glands seem to be similar between the female prionines and Research to date suggests that the use of these two types of the male cerambycines that produce these types of phero- pheromones breaks out along subfamily lines, with species in mones (see Ray et al. 2006; Wickham et al. 2016c). the Cerambycinae, Lamiinae, and Spondylidinae using Furthermore, both the chemistry of the pheromones and the aggregation-sex pheromones and species in the Prioninae and sites of production are entirely different between species that Lepturinae using sex pheromones. In general, the chemistries produce prionic acid and those that produce 2,3-alkanediols of the two types of pheromones are markedly different, but (discussed below). within each type, there is often remarkable biosynthetic parsi- Female-produced sex pheromones also have been identi- mony, with the same or similar pheromone components being fied from several species in the Lepturinae (Tables 1, S1). The shared by species across genera, tribes, and in at least two first such pheromone, identified from Ortholeptura valida cases, different subfamilies (Tables 1, S1, discussed below). (LeConte), consists of cis-vaccenyl acetate. By a biological Moreover, closely related cerambycid species on different con- coincidence, this chemical also is a well-known pheromone tinents often have similar, or even identical pheromones, indi- component of male fruit flies (Drosophila species; Ray et al. cating that at least some of these pheromones have persisted 2011). Another chemical, given the common name (R)- unchanged for millions of years. desmolactone, is a sex pheromone, or at least sex attractant for several lepturine species in the genus Desmocerus,includ- Sex Attractant Pheromones The first female-produced sex ing the endangered D. californicus dimorphus Fisher (Ray attractant pheromone in the Cerambycidae was identified et al. 2012b, 2014). The authors have evidence from coupled from Prionus californicus Motschulsky (Prioninae) as gas chromatography-electroantennogram detection (GC- (3R,5S)-3,5-dimethyldodecanoic acid, given the common EAD) that several other lepturine species have female- name prionic acid (Rodstein et al. 2009, 2011). The phero- produced sex pheromones (unpub. data), suggesting that use mone is emitted in very small amounts, likely from an eversi- of sex pheromones is common in the subfamily, but no other ble gland on the ovipositor (Barbour et al. 2006). The phero- structures have been identified yet. mone is highly attractive, with lures containing submilligram doses remaining attractive for many weeks, and attracting Aggregation-Sex Pheromones To date, all volatile phero- male beetles from hundreds of meters away. Fortunately, the mones known from species in the Cerambycinae, Lamiinae, three “non-natural” stereoisomers are not inhibitory (Rodstein and Spondylidinae are male-produced aggregation-sex phero- et al. 2011), so the relatively inexpensive mixture of all four mones (Tables 1, S1). These were the first type of pheromones J Chem Ecol (2016) 42:631–654 633

Table 1 Sources of cerambycid pheromone components, and references to recommended methods of synthesis

identified from cerambycids, by Iwabuchi and coworkers, et al. 1986; Sakai et al. 1984). The congener X. chinensis who showed that males of the Asian cerambycine Chevrolat subsequently was shown to produce the same two Xylotrechus pyrrhoderus Bates produced (S)-2- compounds (Iwabuchi et al. 1987). Over the next 15 years, hydroxyoctan-3-one and (2S,3S)-2,3-octanediol (Iwabuchi several related structures were identified from males of other 634 J Chem Ecol (2016) 42:631–654

Table 1 (continued)

cerambycine species, including (R)-3-hydroxyhexan-2-one, borer, and Pyrrhidium sanguinium (L.) (Fettköther et al. 1995; (2R,3R)-2,3-hexanediol, and (2S,3R)-2,3-hexanediol from Schröder et al. 1994); (R)-3-hydroxyhexan-2-one and (R)-3- the European Hylotrupes bajulus (L.), known as the old house hydroxyoctan-2-one from the Asian Anaglyptus subfasciatus J Chem Ecol (2016) 42:631–654 635

Table 1 (continued)

Pic (Leal et al. 1995); and (S)-2-hydroxydecan-3-one and 3- cerambycine species from different regions of the world hydroxydecan-2-one from the Asian Xylotrechus quadripes (Table S1). The corresponding 2,3-alkanediones are found Chevrolat (Jayarama et al. 1998; Rhainds et al. 2001). frequently in headspace volatiles from male beetles, but to The use of these closely related structures by species in date, it seems likely that they are artifacts, because there is different genera and tribes, and from different continents, sug- no evidence that they are attractants or synergists. In addition, gested that the hydroxyketone and 2,3-alkanediol type phero- the first variations on the hydroxyketone motif recently have mones were highly conserved within the subfamily, and this been discovered, such as (2S,4E)-2-hydroxyoct-4-en-3-one has since been borne out by the identification of these types of and (3R,4E)-3-hydroxyoct-4-en-2-one, which were discov- structures as pheromones or likely pheromones for numerous ered from Tylonotus bimaculatus Haldeman (Zou et al. 636 J Chem Ecol (2016) 42:631–654

2015), i.e., unsaturated analogs of hydroxyoctanone. The first The second class of pheromone structures found from examples of acetylated hydroxyketone structures also have lamiine species consists of hydroxyethers and related com- been tentatively identified from cerambycine species (JGM, pounds. The first example, a blend of 4-(heptyloxy)butan-1- unpub. data). ol and the corresponding aldehyde, was identified from the In addition to the hydroxyketone and 2,3-alkanediol type notoriously invasive A. glabripennis (Zhang et al. 2002). pheromones, evidence is accumulating that the pheromones The alcohol component has now been identified from the of cerambycine species actually may be very diverse Asian congener Anoplophora chinensis (Forster) as well (Table S1). For example, very small and simple molecules (Hansen et al. 2015). A related pheromone, 2-(undecyloxy) such as 1-butanol and the enantiomers of 2-methylbutanol ethanol (known as monochamol) was identified originally have been identified from several different species. Other from the European Monochamus galloprovincialis Olivier species such as Megacyllene caryae (Gahan) have phero- (Pajares et al. 2010), and has since been shown to be a pher- mones consisting of terpenoids such as geranial and neral omone or likely pheromone for another 13 congeners native to (Lacey et al. 2008), whereas Rosalia funebris Motschulsky Eurasia and North America (Table S1). Furthermore, during produces (Z)-3-decenyl (E)-2-hexenoate (Ray et al. 2009a), field bioassays in China, monochamol was found to attract which resembles compounds from true bugs. Recently, the species in four other lamiine genera (Acalolepta formosana novel pyrrole structure 1-(1H-pyrrol-2-yl)-1,2-propanedione [Breuning], Pharsalia subgemmata [Thomson], was found in the headspace volatiles from male beetles in Pseudomacrochenus antennatus [Gahan], and Xenohammus several different cerambycid genera, either alone or in com- bimaculatus Schwarzer; Wickham et al. 2014), suggesting bination with 3-hydroxyhexan-2-one (Table S1;JGM,LMH, that it represents another highly conserved pheromone motif. and coworkers, unpub. data), and in field trials, it has proven However, the African Monochamus leuconotus (Pascoe) to be a crucial component of attractant blends for at least seems to be an exception, because the males produce a differ- two invasive Callidiellum species from Asia, C. rufipenne ent hydroxyether, 2-(heptyloxybutoxy)ethanol (Hall et al. (Motschulsky) (Zou et al. 2016)andC. villosulum 2006; Pajares et al. 2010). It remains to be seen what other (Fairmaire) (Wickham et al. 2016b). Other recently identi- pheromone structures will be found from species in the fied pheromones or likely pheromones include the unsatu- Lamiinae, and whether they will be as diverse as those turning rated straight-chain aldehyde (6E,8Z)-6,8-pentadecadienal up among species in the Cerambycinae. (Silva et al. 2016a) and the saturated methyl-branched alde- To date, all known or suspected pheromones from species hyde 10-methyldodecanal (Silva et al. 2016b), as well as a in the Spondylidinae are of the geranylacetone structural class pyrone, a tetrahydropyran, and several cyclic terpenoid de- shared with some lamiines (Table S1). In fact, fuscumol was rivatives (JGM, LMH, and coworkers, unpub. data). Thus, it first identified from the Palearctic spondylidine Tetropium seems that cerambycine pheromones may be characterized fuscum (F.) and the North American T. c. cinnamopterum by widely divergent structural types, but with widespread Kirby (Silk et al. 2007; Sweeney et al. 2010). Fuscumol has sharing of particular structural types among more closely since been implicated as a pheromone for several other related species. spondylidine species (Table S1). Overall, pheromones or like- In contrast, the known or suspected pheromones of species ly pheromones have been identified from only a few species of in the Lamiinae fall into two main structural classes. The first this relatively small subfamily (see Švácha and Lawrence class consists of geranylacetone; the corresponding alcohol 2014), and so it is impossible to predict whether this subfamily (E)-6,10-dimethylundeca-5,9-dien-2-ol (known as fuscumol); will turn out to have a diversity of pheromone structures. and its acetate ester (E)-6,10-dimethylundeca-5,9-dien-2-yl No pheromones have been identified from species in the acetate (fuscumol acetate), either as single components or as remaining three currently recognized subfamilies in the blends (Table S1). The latter two components each have two Cerambycidae — the Parandrinae, Necydalinae, and enantiomeric forms, providing additional opportunities for Dorcasominae — all of which are relatively small (Švácha evolution of species-specific blends. For example, a recent and Lawrence 2014). study found evidence in North American lamiines of the rel- atively rare phenomenon of enantiomeric synergism (Meier Conundrums among the Volatile Pheromones of et al. 2016): males of Astylidius parvus (LeConte) produce Cerambycidae Over the past decade of studying cerambycid (R)- and (S)-fuscumol + (R)-fuscumol acetate + pheromones, the authors and their coworkers have encoun- geranylacetone, whereas males of Lepturges angulatus tered several examples of two currently unexplainable phe- (LeConte) produce (R)- and (S)-fuscumol acetate + nomena. First, males of several cerambycine species produce geranylacetone. The fact that several other lamiine species chemicals that are typical pheromone components of are known to produce nonracemic blends of fuscumol enan- cerambycines, but all attempts at attracting the producing spe- tiomers (LMH, unpub. Data) suggests that enantiomeric syn- cies with these compounds have failed. For example, males of ergism may be common within this subfamily. thesugarmaplepestGlycobius speciosus (Say) and the J Chem Ecol (2016) 42:631–654 637 important outbreak pest Enaphalodes rufulus (Haldeman) departure from the hydroxyalkanone-alkanediol motif. both produce two isomers of 2,3-hexanediol, but all attempts However, Chlorida festiva (L.) also has the male-specific to attract beetles with racemic or chiral compounds, emitted pores (Ray et al. 2006), but males produce a pheromone of singly or as blends, in traps placed at ground level or in the very different structure, (6E,8Z)-6,8-pentadecadienal (Silva forest canopy, have failed. Males of Tragidion armatum et al. 2016a). Linsley produce copious quantities of (R)-3-hydroxyhexan- The pheromone glands of cerambycines are in the 2-one, but no beetles have been attracted to traps by lures endocuticle of the prothorax, and connect by ducts to pores containing either the chiral or racemic compound. Finally, on the cuticular surface (Hoshino et al. 2015). The fact that males of the very common and abundant locust borer, the prothoracic gland pores are sex specific in cerambycines Megacyllene robiniae (Forster), produce an isomer of 2,3- suggests that they can be used as reliable evidence that a spe- hexanediol, (R)-3-hydroxyhexan-2-one, and (R)-1- cies uses an aggregation-sex pheromone. For example, in a phenylethan-2-ol, but attempts to trap beetles with all possible morphological study of cerambycine species, Ray et al. blends of components, over trials spanning a decade, have (2006) reported that 49 of the 65 species had the male- provided no evidence of even the slightest attraction. In all specific pores, and indeed, 11 of those species subsequently four cases, the compounds are produced sex specifically by were found to use aggregation-sex pheromones (Lacey et al. males, and their structures are identical to common phero- 2008, 2009; Mitchell et al. 2013, 2015; Ray et al. 2015;Zou mone components of related species, so it seems unlikely that et al. 2016). In contrast, the absence of male-specific gland the compounds might be, for example, defensive compounds pores may indicate either that a species does not use a volatile instead of pheromones. pheromone, or that the pheromone glands are elsewhere on the Conversely, large numbers of both sexes of the invasive body. For example, the cerambycine Rosalia funebris lacks the species Callidiellum rufipenne were serendipitously attracted male-specific gland pores (Ray et al. 2006), and males produce to a blend of 3-hydroxyhexan-2-one and 1-(1H-pyrrol-2-yl)- a pheromone with a unique structure ([Z]-3-decenyl [E]-2- 1,2-propanedione during a field trial in Japan that targeted a hexenoate), from as yet unidentified glands (Ray et al. 2009a). different species (Zou et al. 2016). Although we have found 3- Prothoracic gland pores also have been associated with pro- hydroxyhexan-2-one in numerous extracts of headspace vol- duction of female-produced sex pheromones in some species in atiles from male C. rufipenne, we have never detected the the Prioninae, with the pores being specific to females. pyrrole in any extracts (Zou et al. 2016). Furthermore, 3- Remarkably, the 2,3-alkanediols used as sex pheromones in hydroxyhexan-2-one alone is not attractive. Thus, it is unclear these prionine species are the same compounds as are produced why both compounds are necessary for attraction of this spe- by males of many cerambycine species (Table S1). For exam- cies when all analyses done to date indicate that the males ple, females of the North American Tragosoma depsarium “sp. produce only one of the two components. However, it does nov. Laplante” (tribe Meroscelisini) have prothoracic gland suggest that it may be worth field testing the pyrrole as a pores, and they produce (2R,3R)-2,3-hexanediol as a sex pher- possible “cryptic” pheromone component for the three cur- omone that attracts only males (Ray et al. 2012a). Similarly, rently intractable species described in the previous paragraph. females of the Asian prionine Nepiodes (formerly Megopis) c. costipennis White (tribe Megopidini) have prothoracic gland Morphological Structures and Behaviors Associated with pores and produce (2R,3S)-2,3-octanediol as a sex pheromone Pheromone Use To date, two types of pheromone-producing (Wickham et al. 2016c). structures have been identified in various cerambycid species: Another group of species in the Prioninae uses sex phero- glands in the prothorax, or glands associated with the ovipos- mones that apparently are produced by an eversible gland on itor (Millar and Hanks 2016). Prothoracic glands are common the ovipositor. In female Prionus californicus,extractsofthis in many species of cerambycines that produce pheromones of gland contained prionic acid, as well as several analogs and the hydroxyalkanone-alkanediol motif, such as Xylotrechus homologs, although prionic acid alone is both necessary and pyrrhoderus (Iwabuchi 1986), Anaglyptus subfasciatus sufficient for attraction of males (Maki et al. 2011b; Rodstein (Nakamuta et al. 1994), Hylotrupes bajulus (Noldt et al. et al. 2009, 2011). 1995), and Neoclytus a. acuminatus (F.) (Lacey et al. There is little information about biosynthesis of 2007b). Nevertheless, some cerambycines that have the cerambycid pheromones. Kiyota et al. (2009)reasoned male-specific prothoracic pores have aggregation-sex phero- that the structural similarity between the (S)-2- mones with different structures. For example, males of hydroxyoctan-3-one and (2S ,3S)-2,3-octanediol phero- Tylonotus bimaculatus have prominent pores which are absent mone components of Xylotrechus pyrrhoderus indicated in females (LMH, unpub. data), and produce a blend of that one was the biosynthetic precursor of the other. (2S,4E)-2-hydroxyoct-4-en-3-one with smaller amounts of They tested this hypothesis by synthesizing deuterium- three isomers. This compound is an unsaturated analog of 2- labeled standards of each compound, topically applied hydroxyoctan-3-one, and so does not represent a significant them to the prothoraces of males, and subsequently 638 J Chem Ecol (2016) 42:631–654 collected the chemicals that males produced. This study that are pheromone components of other cerambycine species, demonstrated that the ketol was converted to the diol, consistent with the important role of antagonism by compo- pinpointing the ketol as the precursor (also see Iwabuchi nents in the pheromone blends of sympatric congeners in et al. 2014). averting interspecies attraction (e.g., Mitchell et al. 2015). For the Spondylidinae, Mayo et al. (2013) suspected that geranylacetone was a biosynthetic precursor of fuscumol for Nonvolatile Contact Pheromones Once the sexes are in con- male Tetropium fuscum based on its structural relationship and tact, male cerambycids recognize females by contact chemore- presence as a minor component in headspace aerations. This ception, detecting species- and sex-specific contact phero- was confirmed by topically applying deuterium-labeled mones in the cuticular wax layer of females (Hanks and geranylacetone to males, resulting in them producing labeled Wang 2016). Several of these contact pheromones have been fuscumol. A suspected key biosynthetic enzyme was identified, and they consist of a subset of the cuticular lipids expressed primarily in the midgut, suggesting that the phero- that make up the wax layer (reviewed by Ginzel 2010; Millar mone was produced there. Zarbin et al. (2013)alsoapplied and Hanks 2016). Moreover, there is now evidence from sev- labeled geranylacetone to different body parts of males of the eral cerambycid species that non-volatile chemicals (possibly lamiine Hedypathes betulinus (Klug), demonstrating that the lipids) that females deposit on the substrate while walking may chemical was converted to labeled fuscumol and fuscumol act as a chemical trail that males can follow to locate females acetate by tissues in the prothorax of males, but there was no (Galford 1977; Hoover et al. 2014;Wang1998, 2002). such effect in females. Calling behaviors of cerambycids vary with the type of Implications of Pheromone Chemistry for Invasion pheromone they produce and the location of their pheromone Biology glands. Males of cerambycines that have the prothoracic gland pores commonly adopt a characteristic “push-up stance” that is The cumulative body of research on cerambycid chemical thought to aid in dissemination of pheromone: the front legs are ecology has shown that the pheromone chemistry of related extended, elevating the prothorax above the substrate (Lacey species often is highly conserved, with many species sharing et al. 2007a, b, 2009; Ray et al. 2009b). Males of the pheromone components, or even having identical pheromones spondylidine Tetropium fuscum display a similar behavior that (Table S1). Moreover, pheromone components may be shared is associated with release of the aggregation-sex pheromone by species on different continents, despite their having di- fuscumol (Lemay et al. 2010), although the location of the verged many thousands of years ago, notably: 1) pheromone glands has not yet been verified (Mayo et al. 2013). hydroxyalkanones and alkanediols among cerambycines; 2) Female Prionus californicus adopt a very different posture fuscumol, fuscumol acetate, and monochamol among when calling, with the head down, the tip of the abdomen lamiines; 3) fuscumol among spondylidines; and 4) prionic raised, and the ovipositor extended (Barbour et al. 2006). acid and alkanediols among prionines (Table S1). These close The presumed pheromone gland, on the dorsum of the ovi- similarities in pheromone composition are supported further positor, then is everted. Additional reports of similar postures by the recently discovered 1-(1H-pyrrol-2-yl)-1,2- in female prionines of other species (e.g., Benham and Farrar propanedione, which is at least an attractant or synergist for 1976; Gwynne and Hostetler 1978; Paschen et al. 2012), is five species of Asian cerambycines (Wickham et al. 2016b; further evidence that use of sex pheromones is likely wide- Zou et al. 2016), and a confirmed pheromone component of spread among the prionines, and that pheromones are pro- the North American cerambycine Dryobius sexnotatus duced by a gland on the ovipositor. Linsley (LMH, unpub. data). The antennae have been demonstrated to be the organs that This pheromonal parsimony among cerambycids in differ- cerambycids use to perceive volatile pheromones. The sexu- ent parts of the world has profound implications for the suc- ally dimorphic, more heavily branched antennae of prionine cessful colonization of exotic species. Specifically, new pop- males suggested that they use sex pheromones to find females ulations of invaders usually will be founded by a small num- (Linsley 1959), in analogy to male Lepidoptera. The role of ber of adults emerging from wood products or wooden pack- the antennae as pheromone detectors has been supported by ing materials. Asynchronous emergence of adults will result in several studies using electroantennography (e.g., Álvarez et al. their being widely separated as they disperse into the new 2015a;Iwabuchietal.1985;MacKayetal.2015; Ray et al. region. The dispersion of these few founding adults will be 2011; Rodstein et al. 2009). To our knowledge, the molecular even greater for lamiine species, because adults require a pe- basis of pheromone reception has been explored only once to riod of maturation feeding before mating. Thus, initial popu- date, by Mitchell et al. (2012), who functionally characterized lation densities will be very low and it will be difficult for olfactory receptors for three of the nine pheromone compo- males and females to find one another to mate, the so-called nents produced by males of the cerambycine Megacyllene Allee effect (Courchamp et al. 1999). Under these conditions, caryae. Two of the receptors also were sensitive to isomers pheromonal communication for the invading species may be J Chem Ecol (2016) 42:631–654 639 significantly disrupted by calling adults of much more abun- has proven possible to simply concentrate and replace the dant native species that use the same pheromone components, methylene chloride in the extract containing headspace vola- posing a major barrier to reproduction and establishment. tiles with deuterated methylene chloride, and run the resulting Thus, at least some exotic cerambycid species may fail to concentrated extract with microprobe NMR (e.g., Silva et al. establish because their pheromone channel overlaps with 2016a, b). In contrast, female-produced sex pheromones seem those of native species. Conversely, the exotic species that to be produced in only nanogram amounts per individual, so have the best chance of establishing may be those whose all identifications to date have relied on combinations of mass pheromone chemistries do not overlap with those of any na- spectral interpretations, microchemical tests, and use of reten- tive species, allowing for efficient and unhindered mate loca- tion indices, because the amounts available were not sufficient tion. In short, the latter species are able to colonize and invade for NMR analysis. Below, we describe the methods used to a “pheromone-free space”. It should be noted that the concept obtain crude pheromone extracts, followed by the specific of pheromone-free space is by no means exclusive to conditions used for analyses of those extracts. cerambycids, but likely applies to any invasive insect species that relies on volatile pheromones to bring the sexes together. Pheromone Collection Methods The first cerambycid pher- An example of a successful invader that may have benefit- omones identified, the aggregation-sex pheromones of ed from pheromone-free space is Asian longhorned beetle, the Xylotrechus pyrrhoderus and X. chinensis, were collected by highly polyphagous Asian longhorned borer, Anoplophora confining beetles in glass containers and then rinsing the con- glabripennis (Haack et al. 2010). The two-component aggre- tainers with solvent, or by extracting whole insects or body gation-sex pheromone of this species seems to be unique in parts with solvent (Iwabuchi 1986;Iwabuchietal.1987; North America, based on two lines of evidence (Table S1): 1) Kuwahara et al. 1987; Sakai et al. 1984). These methods also no native species has yet been found to produce either com- were used during identification of the pheromone of pound, and 2) no native species have been shown to be sig- Hylotrupes bajulus (Fettköther et al. 1995). Crude extracts nificantly attracted by the synthesized pheromones during of most other cerambycid pheromones have been obtained trapping programs conducted in multiple areas of the eastern by what has become the standard methodology for collecting USA, and for extended periods of time (DiGirolomo and odor samples, that is, by passing clean air through a closed Dodds 2014; Nehme et al. 2014; unpub. data). Thus, estab- chamber containing the insects (with or without host plant lishment of A. glabripennis may have been favored by its material), and trapping the headspace volatiles on an adsor- having a unique pheromone, allowing it to independently col- bent such as Tenax (Supelco, Bellefonte, PA, USA), Porapak onize multiple regions of North America (Haack et al. 2010). Q (now Hayesep Q; Sigma-Aldrich, St. Louis, MO, USA), or In contrast, the European Tetropium fuscum has established activated charcoal (Millar and Simms 1998). The trapped vol- in Canada, despite the fact that it uses the same pheromone, atiles then are eluted with small volumes of a solvent such as fuscumol, as is used by several native spondylidines, including methylene chloride. For the aggregation-sex pheromones, aer- T. c. cinnamopterum. Moreover, it seems that many North ation samples of a single beetle, if the beetle has produced American lamiine species share fuscumol as a pheromone com- pheromone at all, usually are dominated by the male- ponent (Table S1). This overlap in pheromone chemistry may produced compound(s), so that they are unmistakable, even explain why the invasion of North America by T. fuscum has if the beetle is aerated along with host plant material. stalled, with minimal spread during the 20 years since its intro- In contrast, aeration extracts from species with female- duction, possibly due to low population densities and reproduc- produced sex pheromones usually are entirely different, with tive failure (Dearborn et al. 2016; Rhainds et al. 2011). It seems the pheromone peak being a minor or trace component. Thus, possible, and even likely that the geographic range of this in- careful comparison of extracts with the corresponding controls, vader has been constrained by interference in its pheromone with the analogous extracts from male beetles, or both, is re- communication by the much more abundant native quired to locate pheromone candidates. This process is made spondylidines and lamiines that use the same pheromone. much more reliable and efficient if the extracts are analyzed by coupled gas chromatography-electroantennography (GC- Collection, Analysis, and Identification of Cerambycid EAD), because the antennae of males respond strongly and Pheromones selectively to even trace amounts of the pheromone compo- nents (e.g., Ray et al. 2011, 2012b, 2014). In the case of The methods used to identify volatile pheromones of Prionus californicus, collections of headspace odors from fe- cerambycids depend on the amounts available, which in turn males did not contain enough material to elicit a detectable GC depend to a large extent on the type of pheromone being peak. Instead, a composite sample was finally obtained by produced. Specifically, aggregation-sex pheromones general- inserting a solid phase microextraction fiber into the opening ly are produced in microgram or larger amounts, i.e., amounts from which the presumed pheromone gland was everted, on the that are sufficient to obtain NMR spectra. In optimal cases, it ovipositors of 14 females, followed by thermal desorption of 640 J Chem Ecol (2016) 42:631–654 the entire sample directly into the GC column (Rodstein et al. isomers (i.e., the pairs of enantiomers of each positional 2009). This method produced a sample with detectable peaks, isomer) are readily separable on either a Cyclodex B col- from which mass spectra were obtained (Rodstein et al. 2009). umn (elution order 2R:3R:2S:3S) or a Betadex 225 col- The identification of the pheromone of P. californicus was par- umn (retention order 3R:2R:2S:3S). Remarkably, the en- ticularly difficult because of the high polarity and poor chro- antiomers of 3-hydroxyhexan-2-one are separated by sev- matographic properties of the critical compound, the carboxylic eral minutes on a Mega-Dex DAC-Beta column (Mega acid 3,5-dimethyldodecanoic acid (Rodstein et al. 2009). SNC, Legnano, Italy; Andres Gonzalez, pers. comm.). If a chiral stationary phase column is not available, the Analysis of Cerambycid Pheromone Components GC- hydroxyketones can be esterified with (R)- or (S)- EAD, to detect potential pheromone compounds, and coupled methoxy(trifluoromethyl)phenyl acetyl chloride to form gas chromatography-mass spectrometry (GC-MS), to provide diastereomeric derivatives that are separable on a standard mass spectra and retention indices, are typically the methods GC column (e.g., Leal et al. 1995). used to analyze extracts of insect pheromones, because of the excellent resolution of GC, and the nanogram or less sensitiv- For the corresponding eight-carbon analogs, the positional ity of both methods. However, there are two potential pitfalls. isomers again do not separate well on a nonpolar DB-5 col- First, some cerambycid pheromone components, such as 2- umn, but are completely separated on a polar DB-Wax col- methylbutan-1-ol, are very volatile, and so the GC tempera- umn, with the 2-hydroxyoctan-3-one eluting first. On a ture program must have a low initial temperature to ensure that Cyclodex B column, the elution order is 2R:2S:3R:3S, but they are not missed, or hidden under the solvent peak. the 2S and 3R-stereoisomers nearly overlap. However, by se- Alternatively, the presence of a very volatile compound can quential analysis on DB-Wax to determine which positional be verified by a solventless collection method, such as solid isomer is present, followed by analysis on Cyclodex B, each phase microextraction. isomer can be identified unequivocally. Second, the most common types of cerambycine phero- Similarly, the positional isomers of the 10-carbon homo- mones, the 3-hydroxyalkan-2-ones and the corresponding 2- logs are not well resolved on a DB-5 column, but they are hydroxyalkan-3-ones, are thermally labile and readily inter- easily separable on polar GC phases, or they can be readily convert between the two regioisomers in hot GC injector ports identified from their mass spectra. The enantiomers of 2- or columns (Leal et al. 1995; Sakai et al. 1984). Thus, for hydroxydecan-3-one resolve on a Cyclodex B column (2R analyses of these types of compounds, it is essential to use a elutes first; Rhainds et al. 2001), or on a CP-Chirasil-DexCB relatively low injector temperature (e.g., 100 °C), particularly column (2R elutes first; Chrompack, Middleberg, when running samples in splitless mode where the dwell time TheNetherlands;Halletal.2006). Similarly, racemic 3- of the sample in the injector is many seconds. Alternatively, hydroxydecan-2-one resolves on Cyclodex B, but we have cool on-column injection (e.g., Rhainds et al. 2001), or pro- not yet made enantiomerically enriched standards to deter- grammed temperature vaporization can be used for sample mine which peak corresponds to which enantiomer. introduction. In parallel, a slow oven temperature program helps to minimize isomerization. 3. The stereoisomers of 2,3-hexanediol and 2,3-octanediol Some cerambycid pheromones, such as the hydroxyether are identified readily in two steps. First, the syn-andanti- compounds produced by species of Monochamus and diastereomers of the 2,3-alkanediols are well separated on Anoplophora, are achiral and do not have stereoisomers, and DB-5 and DB-17 columns, or on polar phases (e.g., so can be completely identified on the basis of GC retention Fettköther et al. 1995), with the syn-diastereomers eluting time and mass spectral matches with standards. However, oth- first. Second, all four stereoisomers of each compound er types of pheromones may have positional and/or stereoiso- separate well on Cyclodex B (elution order for mers, and so several sequential analyses are required to fully hexanediols and octanediols: 2S,3S:2R,3R:2R,3S: identify the structure, as follows: 2S,3R). The syn-enantiomers also resolve well on a β- cyclodextrin column (SGE, Milton Keynes, UK), with 1. The enantiomers of 2-methylbutan-1-ol are resolved on a the (2S,3S)-enantiomer eluting first (Hall et al. 2006). chiral stationary phase Cyclodex B column (J&W 4. The E-andZ-isomers of fuscumol are readily separable Scientific, Folsom, CA, USA), with the (R)-enantiomer by GC; so far, only the E-isomer has been reported as a eluting first (e.g., Hanks et al. 2007). They also resolve cerambycid pheromone (Table S1). Fuscumol is not re- on a Betadex 225 column (J&W Scientific). solved on Cyclodex B or Betadex 225 columns, and is 2. The positional isomers 2-hydroxyhexan-3-one and 3- only partially resolved on permethylated cyclodextrin col- hydroxyhexan-2-one do not separate well on a nonpolar umns (HP-Chiral 20B, Agilent, Santa Clara, CA, USA, DB-5 column (J&W Scientific), but they can be distin- Vidal et al. 2010;orβ-Dex 120, Supelco, Sweeney et al. guished by their mass spectra. Alternatively, all four of the 2010). Fuscumol acetate is resolved on Cyclodex B, and J Chem Ecol (2016) 42:631–654 641

partially resolved on HP-Chiral-20B (Vidal et al. 2010). species. Nevertheless, 2,3-hexanedione is commercially Alternatively, fuscumol can be derivatized with available, and analytical standards of the 8- and 10- enantiomerically pure (R)- or (S)-2-acetoxypropionyl carbon compounds are readily made by oxidation of the chloride (Slessor et al. 1985). The resulting diastereomer- corresponding hydroxyketones (e.g., Hall et al. 2006)or ic derivatives separate easily on a standard GC column permanganate oxidation of 2-alkenes (Rhainds et al. 2001). (e.g., Meier et al. 2016; Sweeney et al. 2010). 3. The hydroxyketone pheromones have been prepared as a 5. The enantiomers of desmolactone are not resolved mixture of all four stereo- and regioisomers by partial

on Cyclodex B, but do resolve on β-Dex 225 reduction of 2,3-alkanediones using NaBH4 in ethanol (Ray et al. 2012b). (Hanks et al. 2007), or more cleanly by reduction with

6. Prionic acid (3,5-dimethyldodecanoic acid) has four ste- zinc and ZnCl2 (Millar et al. 2009). Racemic 3- reoisomers (two diastereomeric pairs of enantiomers). hydroxyalkan-2-ones can be prepared by reaction of the The syn-andanti-diastereomers separate easily on a non- lithium anion of 2-methyl-1,3-dithiane with the appropri- polar DB-5 column, with syn eluting first. The corre- ate aldehydes, followed by deprotection (Fettköther et al. sponding methyl esters elute in the same order, with the 1995;Schröderetal.1994), or by alkylation of added advantage that the peak shapes are better, and the trimethylsilyl-protected cyanohydrins with methyllithium retention times are more reproducible than those of the followed by hydrolysis of the imine intermediates free acids. However, neither of the enantiomeric pairs of (Rhainds et al. 2001). However, the recommended route

methyl esters was resolvable on a Cyclodex B chiral sta- is the one-pot reaction of 3-hydroxy-1-alkynes with BF3 tionary phase column. Thus, the insect-produced enantio- in MeOH with HgO catalyst, followed by aqueous acidic mer had to be deduced from bioassays of the two anti hydrolysis of the intermediate methyl enol (Imrei et al. enantiomers (i.e., 3R,5S and 3S,5R; Rodstein et al. 2011). 2013;Lealetal.1995). The reaction also can be done in

one step using aqueous H2SO4, but larger amounts of the highly toxic HgO catalyst apparently are required Synthesis of Cerambycid Pheromones (Noseworthy et al. 2012). This reaction uses relatively inexpensive and readily available intermediates, is easily Table 1 lists all cerambycid pheromones reported to date, scalable to >100-g quantities; and when reaction condi- where they can be obtained, and/or their recommended tions are optimized, yields are good to excellent. methods of synthesis, with the exception of some minor com- Enantiomerically enriched 3-hydroxyalkan-2-ones ponents such as 2-nonanone and some monoterpenes that are have been prepared by Wacker oxidation of OH- readily available from numerous commercial sources. Some protected chiral 1-alken-3-ols (Schröder et al. 1994), or further discussion on the syntheses of several of these com- by ring-opening of protected epoxyalcohols and oxida- pounds is warranted. In particular, for practical applications, tion, with the epoxyalcohol intermediates being generated syntheses of the aggregation-sex pheromones must yield from kinetic resolution of allylic alcohols by Sharpless multigram and even kilogram quantities because lures must asymmetric epoxidation (Leal et al. 1995). Alternatively, contain 25–100 mg or more of pheromone components to HgO-catalyzed reaction of chiral 1-alkyn-3-ol substrates, allow release of several milligrams per day for periods of a as described above, produces the desired hydroxyketones few weeks. Thus, syntheses must be short, efficient, and if without epimerization (Leal et al. 1995). However, the possible, involve no expensive chromatographic purifications simplest and recommended route to both enantiomers is of intermediates or final products. by kinetic resolution of the racemate with Amano lipase AK and vinyl acetate, resulting in highly selective acety- 1. Racemic and (S)-2-methylbutan-1-ol are readily avail- lation of the (S)-enantiomer. The unchanged (R)-3- able, but the (R)-enantiomer apparently is not. In our ex- hydroxyalkanone and the acetylated (S)-enantiomer are perience, smaller companies purporting to sell the (R)- separated easily on multigram scale chromatographically enantiomer actually provided either the (S)-enantiomer (Lacey et al. 2007a). The (S)-enantiomer is recovered by or the racemate, i.e., caveat emptor. However, the (R)- careful alkaline hydrolysis, to minimize epimerization. enantiomer is readily available by enzymatic resolution Alternatively, racemic 3-O-acetylalkan-2-one can be of the racemate (Bello et al. 2015), or it can be made from enantioselectively hydrolyzed with the same enzyme in commercial (S)-3-bromo-2-methylpropan-1-ol (Mitchell neutral phosphate buffer to produce the (S)-3- et al. 2012). hydroxyalkan-2-one (Lacey et al. 2007a). 2. 2,3-Hexanedione and the corresponding 8- and 10-carbon analogs are seen frequently in aeration extracts of species Racemic 2-hydroxyalkan-3-ones can be made by reaction that produce 2-hydroxyalkan-3-ones or 3-hydroxyalkan-2- of 2-alkyl-1,3-dithianes with acetaldehyde (Fettköther et al. ones, but to date, have not been shown to be active for any 1995; Schröder et al. 1994), or reaction of the trimethylsilyl- 642 J Chem Ecol (2016) 42:631–654 protected cyanohydrin from acetaldehyde with alkyllithiums (Novozyme 435) from the yeast Pseudozyma (Rhainds et al. 2001). However, syntheses of the racemates are (=Candida) antarctica (Goto, Sugiy. & Iizuka) probably unnecessary because of the ease with which the en- Boekhout (Sigma-Aldrich; Hughes et al. 2013; Sweeney antiomers can be prepared (see below). The enantiomers of 2- et al. 2010; Vidal et al. 2010). Choice of the lipase was hydroxyalkan-3-ones have been synthesized by kinetic reso- important, because other lipases produced much poorer lution of allylic alkenols to chiral epoxides followed by ring resolution (Sweeney et al. 2010). opening and oxidation (Mori and Otsuka 1985; Rhainds et al. 6. All of the hydroxyether-type pheromones (e.g., 2001). However, the simplest and cheapest method for syn- monochamol) are readily prepared by standard thesis of 2-hydroxyalkan-3-ones is via alkylation of the lithi- Williamson ether syntheses, reacting an alkoxide with um salts of tetrahydropyran-protected lactic acid enantiomers, an alkyl halide (e.g., Zhang et al. 2002). a total of four straightforward steps from commercially avail- able (R)- or (S)-ethyl lactates (Hall et al. 2006; Lacey et al. Only single syntheses of most of the other pheromones 2008; Sakai et al. 1984). The tetrahydropyran protecting shown in Table 1 have been reported, and the citations to group was found to be best when compared to benzyl or silyl those syntheses are listed in the table. protecting groups (Hall et al. 2006). Host Plant Volatiles and Bark Beetle Pheromones 4. Mixtures of all four stereoisomers of 2,3-alkanediols can as Kairomones

be made by NaBH4 reduction of the corresponding 2,3- alkanediones (Hanks et al. 2007) or LiAlH4 or NaBH4 Many cerambycid beetles mate on the host plants on which the reduction of racemic 2-hydroxyalkan-3-ones or 3- adults feed, or on the host plants of the larvae, suggesting to hydroxyalkan-2-ones (Fettköther et al. 1995; Lacey Linsley (1959) that finding mates and finding host plants are et al. 2008). The racemates of the syn-andanti-diastereo- intimately associated. It had long been suspected that the adult mers can be prepared in large scale and with good yield in beetles primarily use volatile cues to locate host plants (e.g.,

one step by OsO4-catalyzed oxidation of the correspond- Beeson 1930), which encouraged some authors to conclude ing (E)- or (Z)-2-alkenes, respectively, with inexpensive that various species did not use long-range attractant phero- N-methylmorpholine N-oxide as the terminal oxidant mones (e.g., Allison et al. 2004; Hanks et al. 1996;Luetal. (Lacey et al. 2004). 2007). Much of the research on attraction of cerambycids by The individual stereoisomers of 2,3-alkanediols can be plant volatiles has focused on species whose larvae develop in prepared in several ways. Historically, several multistep coniferous trees that have been stressed by environmental con- routes were developed based on chiral starting materials ditions (e.g., Fan et al. 2007;Hanks1999;Wood1982). (Sakai et al. 1984; Schröder et al. 1994) or the Sharpless Because many cerambycid species are polyphagous, it seems asymmetric epoxidation (Mori and Otsuka 1985; likely that the volatile cues used by adults to locate hosts are Schröder et al. 1994). Sharpless asymmetric common to all the host species. Two such compounds, which dihydroxylation of stereochemically pure 2-alkenes also are broadly attractive to saprophytic insects in general, are eth- has been used to provide a one-step synthesis, but the anol and the monoterpene α-pinene (Millar and Hanks 2016). resulting diols are only ~80–90 % enantiomerically pure Volatiles emitted by conifers primarily consist of monoter- (Hall et al. 2006; Lacey et al. 2004). Alternatively, reduc- penes and sesquiterpenes, with α-pinene being a dominant tion of enantiomerically enriched starting hydroxyketones component (Millar and Hanks 2016). Consequently, α- produces a chromatographically separable mixture of the pinene also is a dominant component of the distilled essential corresponding syn- and anti-diols (e.g., [R]-3- oils of conifers known as turpentines (Byers and Zhang 2011; hydroxyhexan-2-one produces a mixture of [2R,3R]- and Fan et al. 2007; Kozlowski and Pallardy 1997). In contrast, [2S,3R]-2,3-hexanediols; Hall et al. 2006; Lacey et al. ethanol is commonly produced by plants that are stressed, 2008). For multigram scale, kinetic resolution of the race- damaged, or dying (e.g., from water deficit or fire), and it mic anti-diols with Amano lipase PS and vinyl acetate in attracts adults of cerambycid species whose larvae require methyl t-butyl ether is the preferred method, with the such hosts (Joseph et al. 2001; Kelsey and Joseph 2003). (2R,3S)-enantiomer being selectively acetylated Ethanol synergizes attraction to α-pinene for some (Wickham et al. 2016c). In contrast, the enantiomers of cerambycid species, but not others (e.g., Allison et al. 2001; syn-2,3-alkanediols can be prepared in a few steps in Chénier and Philogène 1989; Ibeas et al. 2007; Sweeney et al. multigram amounts from commercially available D-or 2004). Thus, traps baited with either ethanol, α-pinene, or the L-threonine (Mitchell et al. 2012). combination typically attract only a few cerambycid species in 5. The enantiomers of fuscumol and fuscumol acetate are large numbers (e.g., Byers 2004; Gardiner 1957;Milleretal. prepared easily in multigram scale by kinetic resolution 2015a, b;MontgomeryandWargo1983). Nevertheless, the of racemic fuscumol with an immobilized lipase combination is commonly, but probably somewhat J Chem Ecol (2016) 42:631–654 643 erroneously, considered to be a general “plant volatile” attrac- simulated the specific blends of floral volatiles that cue attrac- tant for trapping saprophytic insects (e.g., Campbell and tion of these species. Borden 2009; Chénier and Philogène 1989; Costello et al. Pheromones of scolytine bark beetles also serve as 2008; Kobayashi et al. 1984). kairomones for cerambycids (reviewed by Ibeas et al. 2007), The limited attraction of many cerambycids to blends of guiding them to trees that have had their defenses weakened ethanol and α-pinene is no doubt due to these compounds by being infested by the scolytine larvae. As a bonus, the representing a crude approximation of the complex volatile scolytine larvae are preyed upon facultatively by the profiles of conifers (e.g., Kimmerer and Kozlowski 1982; cerambycid larvae, providing additional concentrated nutri- Yatagai et al. 2002). Ethanol and α-pinene also may fail to ents (e.g., Allison et al. 2001, 2003; Billings and Cameron attract cerambycids that infest deciduous trees, which have 1984; Dodds et al. 2001). Ipsenol and ipsdienol are among profiles of volatiles that are not dominated by α-pinene. For the most common bark beetle pheromone components, and these and other hosts, other classes of plant volatiles are likely the most studied (e.g., Seybold et al. 1995; Wood 1982). A to be more reliable cues for locating larval hosts, and the particular bark beetle pheromone may act as a kairomone for particular host trees that are in a suitable physiological condi- one cerambycid species, but antagonize attraction or be entire- tion for larval development (see Phillips et al. 1988; Schroeder ly neutral for other species (reviewed by Ibeas et al. 2007). and Lindelöw 1989). For example, larvae of some cerambycid The response of a cerambycid species to the pheromone of a species develop in fire-damaged conifers, and the adults are particular scolytine species probably is determined by whether attracted to such hosts by the blend of volatiles in smoke they share host species, and by their coevolutionary history. (reviewed by Allison et al. 2004;Sucklingetal.2001). For That is, pheromones of a scolytine beetle may serve as these species, attraction may be antagonized by chemicals that kairomones for sympatric cerambycid species, but have no are characteristic of healthy as opposed to fire-damaged trees, effect on cerambycids native to other parts of the world, or i.e., hosts that are not in a condition suitable for the larvae that infest hosts other than those attacked by the particular (Suckling et al. 2001; Yamasaki et al. 1997). Similarly, vola- scolytine species. The response to scolytine pheromones tiles from tree species that are not natural hosts also may may be influenced further by plant volatiles such as ethanol inhibit attraction (Yatagai et al. 2002). and α-pinene, which may synergize attraction of some spe- Deciduous hosts do indeed produce volatiles that at- cies, but have no effect on others (e.g., Miller et al. 2015a, b). tract adult cerambycids (e.g., Barata and Araújo 2001; Coyle et al. 2015; Fujiwara-Tsujii et al. 2012; Hanks Host Plant Volatiles and Bark Beetle Pheromones et al. 1996; Liendo-Barandiaran et al. 2010), but there as Synergists or Antagonists of Pheromones has been little progress in identifying the specific attrac- tive components. Contributing to the problem is the com- Plant volatiles and bark beetle pheromones may influence plexity of the blend of volatile chemicals released by host attraction of cerambycids to the aggregation-sex pheromones plants. For example, Barata et al. (2000) demonstrated produced by their males, perhaps by providing an ecological that antennae of the cerambycine Phoracantha “context” to the chemical signal, indicating that the calling semipunctata (F.) can detect more than 40 chemicals in male has discovered a suitable host for the larvae. Again, the odor blends from their eucalypt hosts, but the researchers effects of host plant volatiles and bark beetle pheromones on could not identify two thirds of these chemicals. responses of cerambycids to their pheromones vary widely Adult cerambycids also may use chemical cues to locate (Table S1). Some cerambycid species are not attracted to ei- host plants for their own feeding. For example, adults of the ther their pheromones or host plant volatiles alone, but re- lamiine Anoplophora malasiaca (Thomson) are attracted to spond strongly when the two are combined. Others are mod- volatiles released from host plants as a result of feeding by erately attracted to their pheromones, with the responses in- conspecifics (see Yasui 2009; Yasui et al. 2007, 2008). Some creased additively or synergistically by host plant volatiles. of these volatiles become absorbed in the beetles’ cuticular For a third group, and particularly those species using lipids, and in turn serve to attract mates. Thus, the host plant female-produced sex pheromones, the pheromone alone is volatiles directly and indirectly mediate location of both host necessary and sufficient for strong attraction, with host plant plants and mates. Adults of the cerambycine Anaglyptus or other volatiles apparently playing no role. For example, subfasciatus feed on nectar and pollen, and use floral volatiles volatiles associated with spruce trees critically synergize at- to locate their hosts (Ikeda et al. 1993). In a screening trial of traction of Tetropium species (Spondylidinae) to their phero- floral volatiles, Sakakibara et al. (1998) attracted a few mones (Silk et al. 2007; Sweeney et al. 2004). Similarly, eth- lepturine and cerambycine species to different chemicals, al- anol and α-pinene and/or bark beetle pheromones critically though the vast majority of species in the local community synergize attraction of some Monochamus species were not attracted by any of the baits. This limited response (Lamiinae) to their pheromones, whereas for other suggests that the chemicals that were tested may not have Monochamus species they merely enhance attraction, or seem 644 J Chem Ecol (2016) 42:631–654 to have no activity (Allison et al. 2012;Fierkeetal.2012; Furthermore, to work effectively, it is essential that all trap Hanks et al. 2012; Macias-Samano et al. 2012; Pajares et al. surfaces be coated with the Teflon dispersion Fluon, or other 2010, 2013; Ryall et al. 2015; Teale et al. 2011). For species lubricants, which render trap surfaces so slippery that the bee- whose larvae develop in deciduous hosts, ethanol may en- tles cannot alight such that they immediately fall into the col- hance attraction to aggregation-sex pheromones, whereas lection bucket (Allison et al. 2011, 2014, 2016; Álvarez et al. monoterpenes such as α-pinene (common volatiles of coni- 2015b;Grahametal.2010, 2012). This single factor can in- fers) may be either neutral or repellent (e.g., Collignon et al. crease trap capture more than 10-fold (Graham et al. 2010). 2016; Hanks et al. 2012). Floral volatiles from host plants of Similarly, the inner surface of the collection bucket should be the adults also may influence attraction to pheromone. For coated with Fluon so that trapped beetles cannot escape, or example, floral volatiles critically synergized attraction of else the collection bucket should contain a killing and preserv- Anaglyptus subfasciatus to the aggregation-sex pheromone ing solution such as soapy water, saturated brine, or aqueous of males, but they were not attractive when tested alone propylene glycol (Allison et al. 2014). The Fluon treatment is (Nakamuta et al. 1997). durable enough to last for several seasons (Allison et al. 2016; Álvarez et al. 2015b). We cannot overemphasize the impor- Optimization of Pheromone Lures and Pheromone-Baited tance of these factors for effective trapping of cerambycids. Traps Developing lures that can release milligrams of pheromone per day was a second crucial factor in efficient attraction of The development of pheromone-baited traps as effective prac- cerambycids that use aggregation-sex pheromones. In partic- tical tools for detection and monitoring of cerambycid beetles ular, males of these species can release >100 micrograms per has been contingent upon several interlinked factors, includ- male per day of pheromone (e.g., Hanks et al. 2007). ing design of efficient traps; development of lures with release However, because males usually release pheromone only for rates of several milligrams per day; and in some cases, the a few of hours per day (during their normal activity period) release of host plant volatiles with the pheromones. The fact whereas passive pheromone release devices emit pheromone that some species require a period of maturation feeding be- continuously, the release devices must emit several milligrams fore they produce or respond to pheromones (Hanks and per day to match or exceed the release rate from a single male. Wang 2016) also may have contributed to earlier failures to Thus, pheromone lures for these species typically are made recognize that those species were indeed using pheromonal from permeable plastic sachets or similar devices that can attractants. The iterative optimization of trap and lure design achieve these release rates. For example, for research pur- has contributed to the radical paradigm shift that has occurred poses, the authors use small, low-density polyethylene reseal- since 2004, when it was suggested that cerambycids made able sachets loaded with ethanol or isopropanol solutions of limited use of volatile pheromones (Allison et al. 2004), to the pheromone as release devices (e.g., Hanks et al. 2014). the current situation, where it seems likely that most Adding a cotton wick (1 × 4 cm dental wick) to sachets helps cerambycid species use volatile sex or aggregation-sex pher- to minimize leakage and to stabilize release rates. These sa- omones. As mentioned above, volatile pheromones have been chets seem to have adequate release rates for many identified from more than 100 species, with likely phero- cerambycid pheromones, as judged by trap catches, and they mones known from many more species based on trap catches are cheap and readily available almost anywhere in the world. in pheromone screening trials, and incidental catches in field However, these sachets are impractical for operational pur- experiments targeting other species (Table S1). A more de- poses or commercialization because they must be used imme- tailed discussion of each of these points is useful in under- diately after they have been loaded, and so are not suitable for standing why the elucidation of the chemical ecology of the shipping, or storage for extended periods. Nevertheless, for cerambycids has lagged far behind that of many other eco- relatively short term field experiments, their simplicity and nomically important insects. convenience are major assets. Trap design is crucial for effective trapping and retention of As already discussed, lures must be loaded with tens to cerambycids. During our earliest experiments with hundreds of milligrams of pheromone so that they will emit cerambycid pheromones, we soon discovered that sticky cards several milligrams of pheromone per day for periods of a week and similar traps were ineffective because the relatively large to several months. Lures that release racemic 3- cerambycids either would not walk on the adhesive, or were hydroxyhexan-2-one, fuscumol, and other common phero- able to extricate themselves from it (Lacey et al. 2008). mone components of cerambycid beetles are available from Numerous studies have now determined that multiple funnel commercial sources (Synergy Semiochemical Corp., Burnaby, traps or cross-vane panel traps, both of which mimic the ver- BC, Canada; Sylvar Technologies Inc., Fredericton, New tical silhouette of tree trunks, are the traps of choice for Brunswick, Canada; ChemTica Internacional SA, San Jose, cerambycids (discussed in Allison et al. 2014; Álvarez et al. Costa Rica; Alpha Scents, West Linn OR), and in some cases 2015b;Grahametal.2012; Millar and Hanks 2016). have been tested under field conditions (Miller et al. 2015b; J Chem Ecol (2016) 42:631–654 645

Ryall et al. 2015; Sweeney et al. 2014). Lures that target spe- only one height may result in serious underestimation of the cies of Monochamus with the pheromone monochamol also local population density. It follows that studies that aim to are available from commercial sources (Alpha Scents, West detect the maximum number of species possible, either for Linn OR; Econex, Murcia, Spain; SEDQ, Barcelona, Spain; estimating species richness or for detecting invasive species, Witasek, Feldkirchen in Kärnten, Austria). should deploy traps at several different heights. Failure to do The discussion above of high release rates and consequent so may result in missing some species altogether. lure design is primarily applicable to cerambycid species that Another useful discovery with major implications for prac- use aggregation-sex pheromones. The female-produced sex tical applications of pheromone-baited traps was that phero- pheromones of species in the Prioninae and Lepturinae are mone components can be blended to create “generic” lures for emitted in much smaller amounts, so that for at least some cerambycids, and these lures attract multiple species simulta- species, lure loadings of a fraction of a milligram remain ef- neously. This effect first became clear during field bioassays fective for weeks. In fact, higher loadings may not result in of blends consisting of fewer than 10 cerambycid pheromone improved trap catch (Rodstein et al. 2011). For species using components, in which traps baited with these blends in some sex pheromones, the authors have used dilute solutions of the cases caught many thousands of cerambycid beetles of more pheromones in plastic sachets as described above, but other than 100 species (Table S1; e.g., USA: Graham et al. 2012; devices, such as heat-sealed plastic tubes, also have proven Handley et al. 2015; Hanks and Millar 2013; Hanks et al. effective (Alston et al. 2015; Meier et al. 2016). In general, it is 2007, 2012; Australia: Hayes et al. 2016). Ongoing field likely that other commonly used pheromone release devices screening trials conducted by the authors’ collaborators in such as rubber septa would work for these sex pheromones, Asia, Europe, and South America are yielding similar results. but this may not always be the case. For example, rubber septa Thus, in at least some cases, a series of traps, each of which did not work well as release devices for prionic acid, the sex would have been baited with a single pheromone, now can be pheromone of Prionus californicus (Rodstein et al. 2009). replaced by a single trap baited with a blend of pheromones of The height at which pheromone-baited traps are deployed many species, resulting in cost savings in terms of both mate- is known to affect catches of lepidopteran species in orchards, rials and labor. Obviously, these generic lures have major im- where trees are typically no more than a few meters high (e.g., plications when the goal is to attract as many species as pos- Riedletal.1979). This effect may be exacerbated for sible, for example in surveillance efforts to detect incursions cerambycids in forests, where the trees are typically much of invasive species or in surveys of species richness (e.g., taller, in part as a result of the dynamics of the pheromone Graham et al. 2012; Handley et al. 2015; Hanks and Millar plume. However, there is a rapidly growing body of evidence 2013). For the majority of the known pheromone components, that stratification of different cerambycid species at different it also is useful that their chemistry is such that they are un- heights within forests may be a much more important effect. likely to react chemically with each other when mixed in For example, in field bioassays in east-central Illinois, USA, blends, particularly if they are formulated in a diluent such five cerambycid species were caught in significantly greater as isopropanol numbers by traps that were positioned in the forest canopy The use of multispecies pheromone blends is most effec- (~12 m above the ground) than in the understory, whereas tive when there is the least likelihood of the blend of compo- another three species were caught mostly by traps in the un- nents producing heterospecific behavioral antagonism, which derstory (1.5 m above the ground; Schmeelk et al. 2016;also would otherwise reduce attraction and trap catch of many of see Wong and Hanks 2016). Dodds (2014)reportedsimilar the targeted species. Optimal multispecies attraction is there- differences among species of cerambycids (and scolytines) as fore achieved when the components are from more distantly to whether they were caught in greater numbers by traps in the related species in different subfamilies (i.e., the chemistry of forest canopy versus the understory. Additional indications of the pheromones is completely different), and/or when the stratification were reported in trapping studies in Michigan pheromones are of different types (i.e., aggregation-sex pher- (Graham et al. 2010) and New Brunswick, Canada (Webster omones versus sex pheromones). Using blends of closely re- et al. 2016). This stratification in the flight patterns of lated species’ pheromones for multispecies trapping is ill- cerambycids is presumably a consequence of the preferred advised because closely related species frequently share pher- feeding, mating, and oviposition sites of various species. For omone components; recent studies have shown that analogous example, it would be predicted that adults of species that infest to the pheromone blends of other insect taxa, the attractiveness dead wood or roots would be found primarily in the understo- of cerambycid pheromone blends can depend strongly on the ry, whereas species that feed on foliage of trees and/or whose presence of minor components, either as synergists for con- larvae develop in the branches of living trees would likely specifics or as behavioral antagonists reducing the attraction spend much of their lives within the canopy (Hanks and of heterospecifics (e.g., Hanks and Millar 2013; Meier et al. Wang 2016). Overall, these studies suggest that unless the 2016;Mitchelletal.2015; Ray et al. 2012b, 2015). These preferred height of a target species is known, traps hung at synergistic and antagonistic effects provide mechanisms for 646 J Chem Ecol (2016) 42:631–654 maintaining the species specificity of signals among sympatric Neoclytus a. acuminatus (Lacey et al. 2004) and several spe- species whose pheromones are dominated by the same single cies of Monochamus that have invaded various parts of the compound. Thus, if the goal is to attract a single species most world (Table S1). Furthermore, at the time of writing, the effectively, then an accurate reconstruction of its pheromone authors and their collaborators also have tentatively identified blend should be used. Conversely, if the goal is to attract the pheromones for several other potentially invasive Asian spe- maximum number of species with the minimum investment in cies, including Anoplophora chinensis (Hansen et al. 2015), materials and labor, then formulating mixtures of the phero- Callidiellum villosulum (Wickham et al. 2016b), Xylotrechus mone blends of many species will be most cost effective. rufilius Bates (Narai et al. 2015), as well as A. bungii and However, it must be clearly understood that use of such blends Trichoferus campestris (Faldermann) (unpub. data). may limit or prevent the attraction of species that are strongly Semiochemically baited traps also are finding increased inhibited by one or more components. The roles of, and inter- use for monitoring populations of species within their native actions between, minor components of pheromone blends range, such as Monochamus galloprovincialis, the primary merit further study to assess the extent to which such inhibi- vector of the lethal pinewood nematode in Europe (Álvarez tion may affect trap catches. et al. 2015b; Rassati et al. 2012). Historically, cerambycids that could cause damage to timber and cut logs have been Applications of Semiochemicals in Detection, Monitoring, monitored with traps baited with common host plant volatiles and Management of Cerambycid Beetles such as α-pinene or turpentine (reviewed in Allison et al. 2004; Millar and Hanks 2016). However, the recent and on- Pheromones and other semiochemicals have several important going identifications of pheromones for several economically uses in insect management, including detection of one or more important species coupled with concurrent improvements in particular species, monitoring population densities or seasonal trap design have improved the efficiency of trapping many phenology of a particular species, and control of a particular fold, and so it seems likely that pheromone-baited traps will species (Millar 2007). The first of these two applications over- rapidly supplant use of traps baited only with host plant com- lap considerably and are thus considered together. pounds or other kairomonal attractants. In addition, pheromone traps may be the most powerful Use of Pheromones for Detection and Monitoring of tools available for detecting and monitoring populations of Cerambycids Two of the major advantages of pheromone- endangered or threatened cerambycid species, such as the baited traps are their sensitivity and their selectivity, both of iconic Rosalia alpina (L.), a European species that has been which have been exploited in the detection of cerambycids. red-listed by the International Union for Conservation of There are several different scenarios in which pheromone- Nature (http://www.iucnredlist.org/details/19743/0), or the baited traps can be used to advantage in detecting low- valley elderberry longhorned borer, Desmocerus californicus density populations. First and foremost is the detection of dimorphus, in the western USA (Ray et al. 2014). For the exotic species. The problem of exotic species being inadver- latter species, a small survey of several populations with tently introduced into new areas of the world is particularly pheromone-baited traps collected more beetles than the entire pernicious for cerambycids because their long-lived larvae, number held in collections when the species was first listed as concealed within the wood of packing cases, dunnage, pallets, threatened by the United States Fish and Wildlife Service (Ray or other wooden materials, are readily transported by interna- et al. 2014). Similarly, field studies using pheromone-baited tional commerce. Thus, species such as the Asian traps frequently result in new state records for cerambycid Anoplophora glabripennis have invaded North America and species. For example, of the 69 cerambycid species caught Europe, with the potential for causing devastating losses of during a study in Delaware, USA using traps baited with a hardwoods. Similarly, the Asian species Aromia bungii generic pheromone blend, seven species represented new state (Faldermann) has recently invaded Europe (EPPO 2014)and records (Handley et al. 2015). Japan (Kiriyama et al. 2015), where it represents a major threat More generally, cerambycid species perform valuable eco- to fruit trees. For these and numerous other species of system services as primary decomposers of woody biomass cerambycids, semiochemically baited traps may be powerful and they can be important indicators of ecosystem health (e.g., tools for detecting new incursions as well as monitoring range Maeto et al. 2002). In this context, semiochemically baited expansions and the success of eradication efforts. For exam- traps are powerful tools for conducting surveys of species ple, pheromonal attractants are now known for richness and abundance (e.g., Dodds et al. 2015;Graham A. glabripennis (Zhang et al. 2002), Callidiellum rufipenne et al. 2012; Handley et al. 2015; Hanks and Millar 2013; (Zou et al. 2016), and Tetropium fuscum (Sweeney et al. Hayes et al. 2016; Sweeney et al. 2014; Wickham et al. 2010), all of which have invaded North America. 2014). They may be particularly useful for surveying species Conversely, pheromonal attractants are available for North in the forest canopy because these species could easily be American species that have invaded Europe, such as missed in ground level surveys conducted by other means. J Chem Ecol (2016) 42:631–654 647

Use of Pheromones for Control of Cerambycid Beetles For 2009; Petersen-Silva et al. 2015, Sweeney et al. 2013)ornem- insects in general, three main pheromone-based control strat- atodes (e.g., Li et al. 1989) for control of cerambycids, includ- egies have been developed. These include mating disruption ing experimenting with attract-and-kill techniques using host with sex pheromones, attract-and-kill using attractants in as- plant compounds as the attractant (Li et al. 2007). However, to sociation with insecticides or pathogens, and mass trapping our knowledge, attract-and-kill using pheromones as attrac- using traps baited with pheromones, often in combination tants and pathogens as the killing agent has not yet been re- with host volatiles. The choice of which method to use is ported for any cerambycid species, although such experiments dictated by the natural history of the target species, and of may be in progress (e.g., see Álvarez-Baz et al. 2015). This course, economics. For example, mating disruption may be technique could prove to be useful for auto-disseminating ei- most suitable for managing species in which the females pro- ther fungal conidia or nematodes throughout a population, duce powerful sex pheromones, because it could prevent them using either sex or aggregation-sex pheromones as the attrac- from attracting mates. Nevertheless, mating disruption may tants. In the latter case, both sexes would be contaminated still be effective with species that use male-produced aggre- with pathogens upon being attracted to traps, whereas in the gation-sex pheromones, as demonstrated in a study in which former case, dissemination of pathogens to females would aerial application of fuscumol-impregnated dispensers over occur only during mating with contaminated males. forested areas resulted in significant reductions in populations Mass trapping has been explored for control of cerambycid of Tetropium fuscum (Sweeney et al. 2011a). pests in several systems. In a preliminary trial using the In terms of economics, the cost of synthesizing, formulat- female-produced sex pheromone of Prionus californicus, ing, and deploying pheromone release devices area-wide for deploying pheromone-baited traps around a central baited sen- mating disruption often may not be competitive with control tinel trap decreased by 88 % the number of males caught by using insecticides or deployment of plants engineered to pro- the sentinel trap (Maki et al. 2011a). Even more promising, a duce insecticidal toxins, particularly in crops grown on very longer term experiment testing control of P. californicus in large acreages with low profit margins. When viewed in this sweet cherries showed that after five consecutive years of light, it seems unlikely that pheromone-based mating disrup- mass trapping, trap catches in monitoring traps were reduced tion would be a feasible strategy for use over huge areas, such from 265 to 2 male beetles (Alston et al. 2015). These data as in controlling cerambycid species that are pests of forest suggest that exertion of continuous trapping pressure on this trees. However, mating disruption could indeed be feasible, univoltine species, which reproduces relatively slowly due to and possibly even crucial, for suppression or eradication of its long life cycle of several years, can indeed drive popula- newly established invasive species that are limited to small tions to low levels, as occurs in mass trapping of large tropical geographic areas. weevils (Faleiro 2006; Oehlschlager 2016, this issue). Several pheromone-based strategies have now been tested Furthermore, mass trapping may be a viable strategy for other for control of cerambycid species, with promising results. For prionine species that have long life cycles, such as the sugar instance, mating disruption has been tested twice with Prionus cane pest Dorysthenes granulosus that is readily caught by californicus, the females of which produce the powerful sex using simple pheromone-baited pitfall traps consisting of pheromone prionic acid in nanogram quantities that can attract vertical-sided holes dug in the ground (Wickham et al. 2016a). males from hundreds of meters away. In a preliminary test in Mass trapping trials also have been conducted with two hop yards, the number of males that reached a pheromone- species that have aggregation-sex pheromones. Grids of baited sentinel trap was reduced by 84 % when the sentinel pheromone-baited traps resulted in lower infestation rates was encircled by pheromone lures at distances of 18 or 36 m, of the invasive species Tetropium fuscum in bait logs compared to the number of males reaching a similar sentinel (Sweeney et al. 2011b). However, this method might only encircled by lures containing only solvent (Maki et al. 2011a). be useful for treating spot infestations because the A similar trial in a sweet cherry orchard was even more prom- 10 × 10 m spacing of the traps would likely be prohibitive- ising, with pheromone lures reducing by 94 % the number of ly expensive to deploy over larger areas. The European males reaching a pheromone-baited sentinel trap (J. D. lamiine Monochamus galloprovincialis, a primary vector Barbour, pers. comm.). Prionus californicus has a long and of pine wood nematode, also may be targeted for mass asynchronous life cycle (3–5 years), so it may take several trapping using lures consisting of a blend of the pheromone years for the effects of mating disruption to be fully realized. monochamol and bark beetle pheromone synergists, opti- Nevertheless, in the latter case, it was noted that after two years, mized over many years by Pajares and coworkers (e.g., catches of males in monitoring traps had decreased more than Álvarez et al. 2015b). The lures currently are widely used seven-fold, suggesting that the strategy was indeed effective in in Europe to monitor for this pest (e.g., Rassati et al. 2012), suppressing the local population (Alston et al. 2015). but are so effective that trap densities of less than one trap Researchers have been experimenting for several decades per hectare may reduce beetle population densities by 95 % with using entomopathogenic fungi (e.g., Hajek and Bauer (Sanchez-Husillos et al. 2015). As an added bonus, 648 J Chem Ecol (2016) 42:631–654 because the pheromone is highly conserved within the ge- family will increase our ability to predict whether a species nus Monochamus (as discussed above), monochamol is the uses volatile pheromones, and if so, whether they are male- aggregation-sex pheromone of congeners in Europe that produced aggregation-sex pheromones or female-produced also may vector the pinewood nematode. Thus, these spe- sex pheromones, and what their general structures are likely cies also are strongly attracted tothelure,sothesinglelure to be. This information would be invaluable for rapidly devel- and trap should be effective against multiple species. oping pheromone-based tools for detecting, tracking, and eradicating new exotic invaders before they have a chance to spread widely. Summary Second, the explosion of identifications of cerambycid se- miochemicals is rapidly outpacing our understanding of the Based on the studies described herein, it seems likely that roles of these compounds in the ecology of the beetles and the cerambycid pheromones will find use in numerous applications, interactions of species with other members of their communi- including ecological studies assessing insect communities in ties. For example, it remains unclear why the three subfamilies different habitats, detection and estimation of the ranges of rare Cerambycinae, Lamiinae, and Spondylidinae seem to have or threatened species, detection and eradication of invasive spe- only aggregation-sex pheromones, whereas based on the rel- cies, and management of pest species, be they native or exotic. atively few pheromones identified from the Prioninae and To date, the development of practical applications for Lepturinae, these two subfamilies may have only female- cerambycid pheromones has lagged behind that of many other produced sex pheromones. Numerous other biological and insect groups, for several reasons. For example, it is only in the ecological questions abound, such as why some species are last decade that it has become clear that, as with so many other attracted to the pheromones of congeners, or why some spe- insect species, volatile pheromones are crucial mediators of cies produce what seem to be pheromones, but there is no cerambycid reproduction, and these pheromones should be evidence that they are attracted by these compounds. readily exploitable for management of pest species. This para- Third, although there have been major advances in trap digm shift has come about in part as a consequence of the design, the pheromone lures may still be a weak link in at “explosion” in the identifications of cerambycid pheromone least some cases. To be widely adopted, pheromone lures must structures, with sex or aggregation-sex pheromones now known have effective lifetimes of several weeks, and they must be from all five of the major subfamilies within the Cerambycidae. robust, with reasonable shelf life, and be suitable for shipping. In tandem with the rapidly growing database of pheromone This is clearly an area in which improvements can be made. structures (Table S1), great improvements in methods for their We also suggest that the implications of pheromone practical application have shown that semiochemically based chemistry in the success or failure of invasion biology war- tools can be far more effective than the detection, monitoring, rants careful investigation. That is, if indeed pheromonal or control methods that they could replace. communication by an exotic species is essentially blocked Few cerambycids are crop pests, so sources of grant funds by similar or identical pheromones produced by much more for studying these insects are less obvious and less available abundant native species, this natural form of mating disrup- than for important pests of major crops such as corn or soy- tion will reduce the chances of establishment and spread of beans. However, we suggest that the work summarized in this an invader. Conversely, an exotic species that does not over- review makes a compelling case for the continued develop- lap in pheromone chemistry with native species, which ment and incorporation of semiochemicals into programs for therefore is free to invade a “pheromone-free space”, will detecting, controlling, and where necessary, eradicating exotic be more likely to become established. Thus, we suggest that cerambycid species. the pheromone chemistry of an exotic species and its over- We also suggest several areas for future research. First, it is lap in pheromone chemistry with native species should be a clear that there are numerous additional pheromone structures factor to consider in developing risk assessments for poten- waiting to be identified, and there are still no pheromones tially invasive exotic species. identified from the small subfamilies Parandrinae, Overall, the rapid expansion of research on cerambycid Necydalinae, and Dorcasominae. Further identification of semiochemicals over the past decade has opened a new win- new pheromones has obvious practical implications, but in dow into the biology and ecology of this large and diverse addition, the new structures will help to flesh out the develop- insect family. This new expansion has been most timely be- ing picture of pheromone types and pheromone use within the cause of the increasing problems worldwide with exotic family, and it is likely that pheromone structures can be used cerambycids as devastating invasive pests. We hope that this in systematic studies of the evolutionary relationships among review serves as a useful reference for the increasing number species, genera, and tribes. Furthermore, because of the con- of researchers and regulatory personnel working to understand servation of structures within closely related taxonomic the chemical ecology of cerambycids, and as needed, to man- groups, a more detailed plan of pheromone use within the age their populations. J Chem Ecol (2016) 42:631–654 649

Acknowledgments The authors are grateful for the many students, Barata EN, Pickett JA, Wadhams LJ, Woodcock CM, Mustaparta H technicians, postdoctoral and visiting scholars, and collaborators who (2000) Identification of host and nonhost semiochemicals of euca- contributed so extensively to much of the research described herein. We lyptus woodborer Phoracantha semipunctata by gas chromatogra- also appreciate helpful comments on the manuscript from reviewer Jon phy-electroantennography. J Chem Ecol 26:1877–1895 Sweeney. We thank the numerous funding sources which have supported Barbour JD, Cervantes DE, Lacey ES, Hanks LM (2006) Calling behav- our work on cerambycid semiochemistry over the past 12 years, including ior in the primitive longhorned beetle Prionus californicus Mots. J the United States Department of Agriculture (USDA) - Animal and Plant Ins Behav 19:623–629 Health Inspection Service, the USDA CSREES National Research Barbour JD, Millar JG, Rodstein J, Ray AM, Alston DG, Rejzek M, Initiative, National Institute of Food and Agriculture, Western Regional Dutcher JD, Hanks LM (2011) Synthetic 3,5-dimethyldodecanoic Integrated Pest Management Fund, North Central Region Integrated Pest acid serves as a general attractant for multiple species of Prionus Management Center, and the Alphawood Foundation of Chicago. JGM (Coleoptera: Cerambycidae). Ann Entomol Soc Am 104:588–593 also gratefully acknowledges ongoing funding from Hatch Act project Beeson CFC (1930) Sense of smell of longicorn beetles. Nature 126:12 CA-R*ENT-5181-H. Bello JE, McElfresh JS, Millar JG (2015) Isolation and determination of absolute configurations of insect-produced methyl-branched hydro- carbons. 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