CHEMICAL ECOLOGY (R)-3-Hydroxyhexan-2-one Is a Major Pheromone Component of Anelaphus inflaticollis (Coleoptera: Cerambycidae)

1,2 3 4 4 1,5 A. M. RAY, I. P. SWIFT, J. A. MOREIRA, J. G. MILLAR, AND L. M. HANKS

Environ. Entomol. 38(5): 1462Ð1466 (2009) ABSTRACT We report the identiÞcation and Þeld bioassays of a major component of the male- produced aggregation pheromone of Anelaphus inflaticollis Chemsak, an uncommon desert ceram- bycine beetle. Male A. inflaticollis produced a sex-speciÞc blend of components that included (R)-3-hydroxyhexan-2-one, (S)-2-hydroxyhexan-3-one, 2,3-hexanedione, and (2R,3R)- and (2R,3S)- 2,3-hexanediols. Field trials with baited bucket traps determined that the reconstructed synthetic pheromone blend and (R)-3-hydroxyhexan-2-one alone attracted adult A. inflaticollis of both sexes, with signiÞcantly more beetles being attracted to the blend. We conclude that (R)-3-hydroxyhexan- 2-one is a major pheromone component of A. inflaticollis, and our results suggest that one or more of the minor components may further increase attraction of conspeciÞcs. Scanning electron microscopy showed that male A. inflaticollis have pores on the prothorax that are consistent in structure with sex-speciÞc pheromone gland pores in related species. Males also displayed stereotyped calling behavior similar to that observed in other cerambycine species. This study represents the Þrst report of volatile pheromones for a cerambycine species in the tribe Elaphi- diini.

KEY WORDS Mojave desert, longhorned beetle, mating behavior, sex pheromone, (R)-3-hydroxy- hexan-2-one

Sex or aggregation pheromones produced by males areas in the western Mojave Desert (Cope 1984, So- have been identiÞed for 14 species in four tribes of the lomon 1995). It is rarely collected, being represented cerambycid beetle subfamily Cerambycinae to date by only two specimens in the insect collection of the (reviewed by Ray et al. 2006, 2009; Hanks et al. 2007). Smithsonian National Museum of Natural History The pheromone components of most of these species (S. W. Lingafelter, personal communication), one share a similar motif, consisting of compounds that are specimen in the California Academy of Sciences, and 6, 8, or 10 carbons long with hydroxyl or carbonyl no specimens in either the Entomology Research Mu- groups at C2 and C3 (Hanks et al. 2007). Furthermore, seum at the University of California, Riverside, or the males of these species have sex-speciÞc gland pores in Los Angeles County Museum of Natural History the prothorax that release the pheromone (Ray et al. (A.M.R. and I.P.S., unpublished data). Larvae of A. 2006; Hanks et al. 2007; Lacey et al. 2008, 2009; and inflaticollis bore within and eventually girdle branches references therein). When releasing pheromone, of box thorn (Lycium cooperi Gray, Solanaceae) and males adopt a characteristic calling posture, termed black greasewood [Sarcobatus vermiculatus (Hook) the “pushup stance,” in which they remain still with Torrey, Chenopodiaceae; Hovore et al. 1978, Cope only their prothoracic legs extended, elevating the 1984]. In the process, they produce a linear series of anterior portion of the body (Lacey et al. 2007a, b). small holes through which they expel frass (Hovore This posture may enhance release of pheromone into and Giesbert 1976, Solomon 1995). Larvae may re- the airstream (Lacey et al. 2007b). quire 1Ð2 yr to complete development (Solomon We report on the chemical ecology of the ceram- 1995). The crepuscular adults are cryptically colored bycine species Anelaphus inflaticollis Chemsak. A. in- and have been collected in May (Linsley 1963). There flaticollis apparently is limited in distribution to a few is no published information on the behaviors of adult A. inflaticollis (see Solomon 1995). We provide evidence that (R)-3-hydroxyhexan-2- 1 Department of Entomology, University of Illinois at Urbana- one, the most abundant sex-speciÞc compound pro- Champaign, Urbana, IL 61801. 2 Current address: Department of Entomology, University of Cal- duced by male A. inflaticollis, attracted both sexes in ifornia, Riverside, CA 92521. Þeld trials. The addition of a blend of the minor com- 3 Department of Watershed and Lands, Contra Costa Water Dis- ponents [(S)-2-hydroxyhexan-3-one, 2,3-hexanedi- trict, Concord, CA 94520. one, and (2R,3R)- and (2R,3S)-2,3-hexanediol] to 4 Department of Entomology, University of California, Riverside, CA 92521. (R)-3-hydroxyhexan-2-one further increased attrac- 5 Corresponding author, e-mail: [email protected]. tion of beetles.

0046-225X/09/1462Ð1466$04.00/0 ᭧ 2009 Entomological Society of America October 2009 RAY ET AL.: AGGREGATION PHEROMONE OF A. inflaticollis 1463

Materials and Methods 6 ␮m of gold-palladium, and imaging them with an en- vironmental scanning electron microscope equipped We reared adult A. inflaticollis from branches of box with a Þeld-emission electron gun (Philips model thorn that were collected on 16 March 2007 from a site XL30; FEI Company, Hillsboro, OR) operated at 5.0 in the Mojave Desert (Ϸ56 km northeast of Mojave, kV. Remnants of specimens have been retained by Kern Co., CA, State Hwy. 14; 35Њ28Ј12Љ N, 117Њ57Ј43Љ A.M.R. at the University of California, Riverside. W, 964-m elevation). Other dominant perennial plants We tested the response of adult A. inflaticollis to the at the site were creosote bush [Larrea tridentata (de speciÞc blend of compounds released by males (see Candolle) Coville, Zygophyllaceae], Joshua tree Results) and to (R)-3-hydroxyhexan-2-one alone in (Yucca brevifolia Engelman, Agavaceae), and cholla Þeld bioassays from 26 April to 17 June 2008 (daily air cactus (Cylindropuntia sp., Cactaceae). Branches of temperatures: maxima, 16Ð39ЊC; minima, 4Ð21ЊC; box thorn were placed in a 40-liter plastic storage Њ maximum daily sustained wind speeds 19Ð71 km/h). container on a laboratory bench (12:12-h L:D, 20 C, We included the treatment with only (R)-3-hydroxy- Ϸ50% RH). Adult beetles that emerged were caged 3 hexan-2-one because that compound commonly is the individually in the laboratory in 0.1-m cylindrical most active component in pheromone blends of cer- cages of aluminum screen with glass petri dishes at top ambycine species (Hanks et al. 2007). The prolonged and bottom. Beetles were provided 10% sucrose so- trapping period was necessary because literature re- lution in 8-ml vials plugged with cotton rolls. Males in ports of the ßight period of A. inflaticollis and collec- cages were observed to adopt a body posture analo- tion dates of museum specimens range from April gous to the pushup stance of calling males of other through June (Linsley 1963; A.M.R., unpublished data). cerambycine species (see Lacey et al. 2007a, b). We conducted bioassays at the desert study site where Headspace volatiles were collected from three fe- infested host material previously had been collected male and three male A. inflaticollis held individually in (see above). We initially used ßight-intercept panel Ϸ 0.3-liter glass vacuum traps lined with aluminum traps (black, 1.2 by 0.30 m, model PT Intercept; screen to provide a perch. One nipple of each chamber APTIV, Portland, OR) suspended from branches of was connected with Teßon tubing to a collector con- Joshua trees, but they often were knocked down by sisting of a glass tube (6 cm by 4 mm ID) that con- wind. We therefore used bucket traps after 13 May, tained 100 mg of 80/100 mesh SuperQ (Alltech Asso- partially burying the traps to prevent their being dis- ciates, DeerÞeld, IL) held between plugs of silanized lodged by wind. Bucket traps were constructed from glass wool. Charcoal-puriÞed air was pulled through 19-liter plastic buckets Þtted with 30-cm-diameter alu- the apparatus with a water aspirator (0.7 liter/min). minum funnels (Catalog 2851B; BioQuip Products, One male and one female were aerated simulta- Rancho Dominguez, CA). Each trap was buried such neously on a laboratory windowsill for 24 h, with that the lip of the bucket was Ϸ8 cm above ground different beetles aerated on 3 consecutive d. Collec- level to exclude vertebrates. Traps were placed Ϸ10 m tors were eluted with three 0.5-ml aliquots of meth- apart in three northeasterly transects that were Ϸ0.4Ð ylene choride (CH2Cl2). The resulting extracts were 0.8 km apart. analyzed on a Hewlett-Packard (HP; Sunnyvale, CA) Lures consisted of clear self-seal polyethylene bags 5973 mass selective detector interfaced to an HP 6890 (Bagette model 14770, 5.1 by 7.6 cm, 0.05-mm wall gas chromatograph, Þtted with a DB5-MS column (30 thickness; Cousin, Largo, FL) that were sealed and m by 0.25 mm ID, 0.25-␮m Þlm thickness; J&W Sci- suspended with wire from the handle of the bucket entiÞc, Folsom, CA); temperature was programmed over the center of the funnel. Lures were loaded with from 40 (1-min hold) to 250ЊCat10ЊC/min. Injector (1) 1 ml of an ethanol solution of the reconstructed temperature was 250ЊC, and injections were made in blend of pheromone components found in aeration splitless mode. Absolute conÞguration of the insect- extracts from male A. inflaticollis (see Results), in- produced compounds was determined by analysis of cluding (per ml of solution) 2,3-hexanedione (19 ␮l), an extract on a Cyclodex-B GC column (30 m by 0.25 mm (R)-3-hydroxyhexan-2-one (100 ␮l), (S)-2-hydroxy- ID, 0.25-␮m Þlm thickness; J&W ScientiÞc) pro- hexan-3-one (6 ␮l), (2R,3R)-2,3-hexanediol (11 ␮l), grammed from 50 (1-min hold) to 200ЊCat5ЊC/min; and (2R,3S)-2,3-hexanediol (6 ␮l); (2) 1 ml of a so- injector and detector temperatures were 100 and 200ЊC, lution of 100 ␮lof(R)-3-hydroxyhexan-2-one in eth- respectively. IdentiÞcations of peaks were conÞrmed by anol; or (3) 1 ml of ethanol (solvent control). Each coinjections of extracts with authentic standards. transect included one trap for each treatment. 2,3-Hexanedione was purchased from Aldrich (Mil- We checked traps for beetles every 3Ð4 d, at which waukee, WI). (R)-3-hydroxyhexan-2-one was prepared time lures were replaced and treatments were rotated by kinetic resolution of the racemate as described by one position along the transect to control for posi- Lacey et al. (2007a). (S)-2-Hydroxyhexan-3-one and tional effects. Differences between treatments in (2R,3R)- and (2R,3S)-2,3-hexanediols were prepared as numbers of beetles captured were tested with the described by Lacey et al. (2008). two-way nonparametric FriedmanÕs test (blocked by Specimens of both sexes of A. inflaticollis were pre- day; PROC FREQ with CMH option; SAS Institute pared for scanning electron microscopy using meth- 2001) because assumptions of analysis of variance ods described by Ray et al. (2006). We used the same (ANOVA) were violated by heteroscedasticity (Sokal beetles that had been used to collect headspace vola- and Rohlf 1995). Differences between pairs of means tiles (see above), coating the excised prothoraces with were tested with the REGWQ means-separation test 1464 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 5

Table 1. Retention times/elution order of hydroxyhexanones of extract with the racemic hydroxyhexanones or di- and 2,3-hexanediols on a Cyclodex-B GC column, programmed ols, to show conclusively that one of the two peaks in from 50°C/1 min, 5°C/min, to 200°C a racemate was enhanced by coinjection of the ex- Compound Retention time (min) tract. In total, these analyses unambiguously identiÞed the Þve male-speciÞc compounds as (R)-3-hydroxy- (R)-2-hydroxyhexan-3-one 10.49 Ϸ (R)-3-hydroxyhexan-2-one 10.56 hexan-2-one ( 70%), (S)-2-hydroxyhexan-3-one (4%), (S)-2-hydroxyhexan-3-one 10.67 2,3-hexanedione (14%), (2R,3R)-2,3-hexanediol (8%), (S)-3-hydroxyhexan-2-one 11.10 and (2R,3S)-2,3-hexanediol (4%). Extracts of headspace (2S,3S)-2,3-hexanediol 15.12 aerations of female A. inflaticollis did not contain any of (2R,3R)-2,3-hexanediol 15.31 (2R,3S)-2,3-hexanediol 15.77 these compounds in detectable quantities. (2S,3R)-2,3-hexanediol 15.87 The prothoraces of male and female A. inflaticollis are strongly dimorphic in punctation and shape of the pronotum (Hovore and Giesbert 1976). Pronota and to control maximum experimentwise error rates (SAS pleura of males had groups of pores within indenta- Institute 2001). We include in the analysis data from tions, whereas females had indentations but lacked the only two trapping periods during which we captured pores (Fig. 1). adult A. inflaticollis (see Results). We captured adult A. inflaticollis during only two Voucher specimens of A. inflaticollis have been de- 2-d trapping periods (17Ð19 and 19Ð21 May 2008) of posited in the Entomology Research Museum at the the 34 d that bucket traps were in place in the Mojave University of California, Riverside, and the Smithsonian Desert (with the exception of one female that was National Museum of Natural History, Washington DC. captured on 1 June). A total of 27 beetles were cap- tured (13 females, 13 males; one specimen was dam- aged and could not be sexed), with a maximum of Results seven adults captured in one trap that was baited with Comparisons of extracts of headspace volatiles col- the blend of pheromone components. Treatment means were signiÞcantly different from one another lected from male and female A. inflaticollis showed ϭ ϭ that there were Þve male-speciÞc compounds. These (Fig. 2; sexes combined, FriedmanÕs Q2,17 11.7, P compounds were tentatively identiÞed (in order of 0.003). Traps baited with the A. inflaticollis–speciÞc elution) as 2,3-hexanedione, 3-hydroxyhexan-2-one, blend and those baited with (R)-3-hydroxyhexan-2- 2-hydroxyhexan-3-one, (2R*,3R*)-2,3-hexanediol, and one as a single component captured signiÞcantly more (2R*,3S*)-2,3-hexanediol by matches of their mass beetles than did controls, and the blend attracted spectra with spectra from our library of known cer- more than twice as many beetles as (R)-3-hydroxy- ambycid pheromone components. The identiÞcations hexan-2-one alone (Fig. 2). Traps baited with syn- were conÞrmed by matching the mass spectra and thetic pheromone did not capture any other insect retention times with those of authentic standards on species in consistent or signiÞcant numbers. DB-5 and Cyclodex B columns (see below). Having determined the gross structures of the two hydroxy- Discussion hexanones and the two 2,3-hexanediols, the absolute conÞgurations of the insect-produced compounds (R)-3-hydroxyhexan-2-one is known to be an im- were determined by analysis of the extracts on a chiral portant component or the sole component of male- stationary phase Cyclodex-B column (Table 1). Iden- produced pheromones of 14 species of cerambycine tiÞcations were conÞrmed by coinjection of aliquots beetles (Hanks et al. 2007). In fact, adults of many

Fig. 1. Scanning electron micrographs (dorsal view) of the prothorax of adult female (A) and male (B) A. inflaticollis. The prothorax of the male has pores lying within indentations (inset in B); females also have indentations, but lack pores (inset in A). October 2009 RAY ET AL.: AGGREGATION PHEROMONE OF A. inflaticollis 1465

in males of other cerambycine species that are also known to produce hydroxyketone and/or 2,3-al- kanediol pheromones (Ray et al. 2006, Hanks et al. 2007, Lacey et al. 2009). It therefore seems likely that these pores have the same function in male A. infla- ticollis. Male A. inflaticollis displayed the characteris- tic “pushup” stance that has been associated with call- ing behaviors of other cerambycines (Lacey et al. 2007a, b). The small numbers of A. inflaticollis that were cap- tured at our Þeld site during only two trapping periods during the 48 d that intercept or bucket traps were deployed is consistent with literature reports that A. inflaticollis is uncommon and has a short adult activity period (Cope 1984, Solomon 1995). Attraction of both Fig. 2. Mean (Ϯ SE, N ϭ 6 replicates) number of adult A. sexes of A. inflaticollis in Þeld bioassays to lures baited inflaticollis (sexes combined) that were captured on 17Ð19 and with the species-speciÞc blend, or (R)-3-hydroxy- 19Ð21 May 2008 in the Mojave desert with bucket traps baited hexan-2-one alone, suggests that males produce an with ethanol solutions of the blend of male-speciÞc components aggregation rather than a sex pheromone according to [(R)-3-hydroxyhexan-2-one, (S)-2-hydroxyhexan-3-one, 2,3- hexanedione, (2R,3R)- and (2R,3S)-2,3-hexanediols], (R)-3- the accepted deÞnitions of these terms (Wyatt 2003), hydroxyhexan-2-one alone (R-ketone), or ethanol (control). as seems to be true for many other cerambycine spe- Bars with different letters are signiÞcantly different (REGWQ cies (Hanks et al. 2007). However, it should be pointed test P Ͻ 0.05). out that the male-produced pheromone may actually be a sex pheromone that attracts females, but which is exploited by other males as an indirect method cerambycine species respond as strongly to (R)-3- of Þnding females. This “satellite male” strategy, hydroxyhexan-2-one alone as they do to more com- whereby noncalling males intercept females respond- plete reconstructions of blends of headspace volatiles ing to calling males, has been reported for other types collected from live males (Hanks et al. 2007). In this of insects [e.g., the cockroach Nauphoeta cinerea context, the markedly stronger behavioral response of (Olivier); Moore et al. 1995]. Alternatively, the pher- adult A. inflaticollis to the blend compared with their omone signal may be increased when calling males responses to (R)-3-hydroxyhexan-2-one alone is note- aggregate, thereby improving the chances of attract- worthy and indicates that one or more of the minor ing receptive females and allowing females to choose components also forms part of the pheromone blend among a number of males. Analogous types of mating for A. inflaticollis. The 3-hydroxyhexan-2-one, 2-hy- aggregations are known for insects as diverse as arctiid droxyhexan-3-one, and 2,3-hexanediol structures of moths (Wunderer et al. 1986) and true fruit ßies the major and minor components produced by male A. (Aluja and Norrbom 2000). inflaticollis (tribe Elaphidiini) match the structural In summary, we identiÞed and conducted bioassays motif shared by the pheromones identiÞed for cer- of a male-produced aggregation pheromone blend for ambycine species in the tribes Anaglyptini, Callidiini, a rare desert-dwelling cerambycid beetle. The most and Clytini (Lacey et al. 2004, 2007a, 2008, 2009; Ray abundant component of the pheromone, (R)-3-hy- et al. 2006; Hanks et al. 2007). droxyhexan-2-one, conforms to the structural motif We have not attempted to further optimize the characteristic of pheromones for related species in blend and narrow down the speciÞc minor compo- several tribes of the Cerambycinae, lending further nent(s) that contribute to the increased attraction support to our working hypothesis that male-pro- because of the scarcity of the species and the very duced pheromones are common and may be con- short time window that adult beetles apparently are served across this large and diverse subfamily, which active every year (e.g., we captured beetles during is represented by nearly 5,000 species and 65 tribes in only two trapping periods). Thus, Þeld trials to deter- the New World alone (Monne´ and Hovore 2006). mine the role of each of the minor components and the exact nature of the full pheromone blend could re- Acknowledgments quire many Þeld seasons. It also is important to note that the minor component (S)-2-hydroxyhexan-3-one We thank D. Erskine-Hellrigel for assistance with Þeld was not simply an artifact of isomerization of the work, S. Lingafelter for helpful comments and assistance with rather thermolabile (R)-3-hydroxyhexan-2-one (Leal museum specimens, and two anonymous reviewers whose et al. 1995), because isomerization would have re- constructive comments helped to improve the manuscript. sulted in approximately equivalent amounts of (R)- Scanning electron microscopy was made possible through collaboration between A.M.R. and Bugscope, The Imaging and (S)-2-hydroxyhexan-3-one, as well as lesser Technology Group, Beckman Institute for Advanced Science amounts of (S)-3-hydroxyhexan-2-one from isomer- and Technology (http://bugscope.beckman.uiuc.edu/), ization in the reverse direction. University of Illinois at Urbana-Champaign (UIUC). This The pores on the prothorax of male A. inflaticollis project was supported by a Francis M. and Harlie M. Clark are similar in structure to the pheromone gland pores Research Support Grant (UIUC, to A.M.R.), the Alphawood 1466 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 5

Foundation (to L.M.H.), and the National Research Initiative bycid beetles Xylotrechus colonus and Sarosesthes fulmi- of the USDA Cooperative State Research, Education and nans. J. Chem. Ecol. 35: 733Ð740. Extension Service, Grant 2006-35302-17457 (to J.G.M. and Leal, W. S., X. Shi, K. Nakamuta, M. Ono, and J. Meinwald. L.M.H.). This research was in partial fulÞllment of a PhD 1995. Structure, stereochemistry, and thermal isomeriza- degree for A.M.R. from UIUC. tion of the male sex pheromone of the longhorn beetle Anaglyptus subfasciatus. Proc. Natl. Acad. Sci. U.S.A. 92: 1038Ð1042. References Cited Linsley, E. G. 1963. The Cerambycidae of North America, Aluja, M., and A. L. Norrbom (eds). 2000. Fruit ßies (Te- part IV. Taxonomy and classiÞcation of the subfamily phritidae): phylogeny and evolution of behavior. CRC, Cerambycinae, tribes Elaphidionini through Rhinotra- Boca Raton, FL. gini. Univ. Calif. Publ. Entomol. 21: 105. Cope, J. 1984. Notes on the ecology of western Ceramby- Monne´, M. A., and F. T. Hovore. 2006. Checklist of the cidae. Coleop. Bull. 38: 27Ð36. Cerambycidae (Coleoptera) of the Western Hemi- Hanks, L. M., J. G. Millar, J. A. Moreira, J. D. Barbour, E. S. sphere. BioQuip Publications, Rancho Dominguez, CA. Lacey, J. S. McElfresh, F. R. Reuter, and A. M. Ray. 2007. Moore, A. J., N. L. Reagan, and K. F. Haynes. 1995. Condi- Using generic pheromone lures to expedite identiÞcation tional signaling strategiesÑeffects of ontogeny, social of aggregation pheromones for the cerambycid beetles experience, and social status on the pheromonal signal of Xylotrechus nauticus, Phymatodes lecontei, and Neoclytus male cockroaches. Anim. Behav. 50: 191Ð202. modestus modestus. J. Chem. Ecol. 33: 889Ð907. Ray, A. M., E. S. Lacey, and L. M. Hanks. 2006. Predicted Hovore, F. T., and E. F. Giesbert. 1976. Notes on the ecol- taxonomic patterns in pheromone production by long- ogy and distribution of Western Cerambycidae (Co- horned beetles. Naturwissenschaften 93: 543Ð550. leoptera). Coleop. Bull. 30: 349Ð360. Ray, A. M., J. G. Millar, J. S. McElfresh, I. P. Swift, J. D. Hovore, F. T., R. L. Penrose, and E. F. Giesbert. 1978. Notes Barbour, and L. M. Hanks. 2009. Male-produced aggre- on North American Cerambycidae (Coleoptera). Ento- gation pheromone of the cerambycid beetle Rosalia fu- mol. News 89: 95Ð100. nebris. J. Chem. Ecol. 35: 96Ð103. Lacey, E. S., M. D. Ginzel, J. G. Millar, and L. M. Hanks. SAS Institute. 2001. SAS/STAT userÕs guide for personal 2004. Male-produced aggregation pheromone of the computers, release 8.01. SAS Institute, Cary, NC. cerambycid beetle Neoclytus acuminatus acuminatus. Sokal, R. R., and F. J. Rohlf. 1995. Biometry, 3rd ed. Free- J. Chem. Ecol. 30: 1493Ð1507. man and Co., New York. Lacey, E. S., J. A. Moreira, J. G. Millar, A. M. Ray, and L. M. Solomon, J. D. 1995. Guide to insect borers in North Amer- Hanks. 2007a. Male-produced aggregation pheromone ican trees and shrubs. U.S. Dep. Agric. Forest Service of the cerambycid beetle Neoclytus mucronatus mucrona- Agriculture Handbook 706. USDA, Washington, DC. tus. Entomol. Exp. Appl. 122: 171Ð179. Wunderer, H., K. Hansen, T. W. Bell, D. Schneider, and J. Lacey, E. S., A. M. Ray, and L. M. Hanks. 2007b. Calling Meinwald. 1986. Sex pheromones of two Asian moths behavior of the cerambycid beetle Neoclytus acuminatus (Creatonotos transiens, C. gangis; Lepidoptera-Arctiidae): acuminatus (F.). J. Insect Behav. 20: 117Ð128. behavior, morphology, chemistry and electrophysiology. Lacey, E. S., J. A. Moreira, J. G. Millar, and L. M. Hanks. 2008. Exp. Biol. 46: 11Ð27. A male-produced aggregation pheromone blend consist- Wyatt, T. D. 2003. Pheromones and animal behaviour: com- ing of alkanediols, terpenoids, and an aromatic alcohol munication by smell and taste. Cambridge University from the cerambycid beetle Megacyllene caryae. J. Chem. Press, Cambridge, United Kingdom. Ecol. 34: 408Ð417. Lacey, E. S., J. G. Millar, J. A. Moreira, and L. M. Hanks. 2009. Male-produced aggregation pheromones of the ceram- Received 24 November 2008; accepted 2 July 2009.