CHEMICAL ECOLOGY
(R)-3-Hydroxyhexan-2-one Is a Major Pheromone Component of Anelaphus inflaticollis (Coleoptera: Cerambycidae) A. M. RAY,1,2 I. P. SWIFT,3 J. A. MOREIRA,4 J. G. MILLAR,4
AND
L. M. HANKS1,5
Environ. Entomol. 38(5): 1462Ð1466 (2009)
ABSTRACT We report the identiÞcation and Þeld bioassays of a major component of the maleproduced aggregation pheromone of Anelaphus inflaticollis Chemsak, an uncommon desert cerambycine 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-hydroxyhexan2-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 Elaphidiini. KEY WORDS Mojave desert, longhorned beetle, mating behavior, sex pheromone, (R)-3-hydroxyhexan-2-one
Sex or aggregation pheromones produced by males have been identiÞed for 14 species in four tribes of the cerambycid beetle subfamily Cerambycinae to date (reviewed by Ray et al. 2006, 2009; Hanks et al. 2007). The pheromone components of most of these species share a similar motif, consisting of compounds that are 6, 8, or 10 carbons long with hydroxyl or carbonyl groups at C2 and C3 (Hanks et al. 2007). Furthermore, males of these species have sex-speciÞc gland pores in the prothorax that release the pheromone (Ray et al. 2006; Hanks et al. 2007; Lacey et al. 2008, 2009; and references therein). When releasing pheromone, males adopt a characteristic calling posture, termed the “pushup stance,” in which they remain still with only their prothoracic legs extended, elevating the anterior portion of the body (Lacey et al. 2007a, b). This posture may enhance release of pheromone into the airstream (Lacey et al. 2007b). We report on the chemical ecology of the cerambycine species Anelaphus inflaticollis Chemsak. A. inflaticollis apparently is limited in distribution to a few 1 Department of Entomology, University of Illinois at UrbanaChampaign, Urbana, IL 61801. 2 Current address: Department of Entomology, University of California, Riverside, CA 92521. 3 Department of Watershed and Lands, Contra Costa Water District, Concord, CA 94520. 4 Department of Entomology, University of California, Riverside, CA 92521. 5 Corresponding author, e-mail:
[email protected].
areas in the western Mojave Desert (Cope 1984, Solomon 1995). It is rarely collected, being represented by only two specimens in the insect collection of the Smithsonian National Museum of Natural History (S. W. Lingafelter, personal communication), one specimen in the California Academy of Sciences, and no specimens in either the Entomology Research Museum at the University of California, Riverside, or the Los Angeles County Museum of Natural History (A.M.R. and I.P.S., unpublished data). Larvae of A. inflaticollis bore within and eventually girdle branches of box thorn (Lycium cooperi Gray, Solanaceae) and black greasewood [Sarcobatus vermiculatus (Hook) Torrey, Chenopodiaceae; Hovore et al. 1978, Cope 1984]. In the process, they produce a linear series of small holes through which they expel frass (Hovore and Giesbert 1976, Solomon 1995). Larvae may require 1Ð2 yr to complete development (Solomon 1995). The crepuscular adults are cryptically colored and have been collected in May (Linsley 1963). There is no published information on the behaviors of adult A. inflaticollis (see Solomon 1995). We provide evidence that (R)-3-hydroxyhexan-2one, the most abundant sex-speciÞc compound produced by male A. inflaticollis, attracted both sexes in Þeld trials. The addition of a blend of the minor components [(S)-2-hydroxyhexan-3-one, 2,3-hexanedione, and (2R,3R)- and (2R,3S)-2,3-hexanediol] to (R)-3-hydroxyhexan-2-one further increased attraction of beetles.
0046-225X/09/1462Ð1466$04.00/0 䉷 2009 Entomological Society of America
October 2009
RAY ET AL.: AGGREGATION PHEROMONE OF A. inflaticollis Materials and Methods
We reared adult A. inflaticollis from branches of box thorn that were collected on 16 March 2007 from a site in the Mojave Desert (⬇56 km northeast of Mojave, Kern Co., CA, State Hwy. 14; 35⬚28⬘12⬙ N, 117⬚57⬘43⬙ W, 964-m elevation). Other dominant perennial plants at the site were creosote bush [Larrea tridentata (de Candolle) Coville, Zygophyllaceae], Joshua tree (Yucca brevifolia Engelman, Agavaceae), and cholla cactus (Cylindropuntia sp., Cactaceae). Branches of box thorn were placed in a 40-liter plastic storage container on a laboratory bench (12:12-h L:D, 20⬚C, ⬇50% RH). Adult beetles that emerged were caged individually in the laboratory in 0.1-m3 cylindrical cages of aluminum screen with glass petri dishes at top and bottom. Beetles were provided 10% sucrose solution in 8-ml vials plugged with cotton rolls. Males in cages were observed to adopt a body posture analogous to the pushup stance of calling males of other cerambycine species (see Lacey et al. 2007a, b). Headspace volatiles were collected from three female and three male A. inflaticollis held individually in ⬇0.3-liter glass vacuum traps lined with aluminum screen to provide a perch. One nipple of each chamber was connected with Teßon tubing to a collector consisting of a glass tube (6 cm by 4 mm ID) that contained 100 mg of 80/100 mesh SuperQ (Alltech Associates, DeerÞeld, IL) held between plugs of silanized glass wool. Charcoal-puriÞed air was pulled through the apparatus with a water aspirator (0.7 liter/min). One male and one female were aerated simultaneously on a laboratory windowsill for 24 h, with different beetles aerated on 3 consecutive d. Collectors were eluted with three 0.5-ml aliquots of methylene choride (CH2Cl2). The resulting extracts were analyzed on a Hewlett-Packard (HP; Sunnyvale, CA) 5973 mass selective detector interfaced to an HP 6890 gas chromatograph, Þtted with a DB5-MS column (30 m by 0.25 mm ID, 0.25-m Þlm thickness; J&W ScientiÞc, Folsom, CA); temperature was programmed from 40 (1-min hold) to 250⬚C at 10⬚C/min. Injector temperature was 250⬚C, and injections were made in splitless mode. Absolute conÞguration of the insectproduced compounds was determined by analysis of an extract on a Cyclodex-B GC column (30 m by 0.25 mm ID, 0.25-m Þlm thickness; J&W ScientiÞc) programmed from 50 (1-min hold) to 200⬚C at 5⬚C/min; injector and detector temperatures were 100 and 200⬚C, respectively. IdentiÞcations of peaks were conÞrmed by coinjections of extracts with authentic standards. 2,3-Hexanedione was purchased from Aldrich (Milwaukee, WI). (R)-3-hydroxyhexan-2-one was prepared by kinetic resolution of the racemate as described by Lacey et al. (2007a). (S)-2-Hydroxyhexan-3-one and (2R,3R)- and (2R,3S)-2,3-hexanediols were prepared as described by Lacey et al. (2008). Specimens of both sexes of A. inflaticollis were prepared for scanning electron microscopy using methods described by Ray et al. (2006). We used the same beetles that had been used to collect headspace volatiles (see above), coating the excised prothoraces with
1463
6 m of gold-palladium, and imaging them with an environmental scanning electron microscope equipped with a Þeld-emission electron gun (Philips model XL30; FEI Company, Hillsboro, OR) operated at 5.0 kV. Remnants of specimens have been retained by A.M.R. at the University of California, Riverside. We tested the response of adult A. inflaticollis to the speciÞc blend of compounds released by males (see Results) and to (R)-3-hydroxyhexan-2-one alone in Þeld bioassays from 26 April to 17 June 2008 (daily air temperatures: maxima, 16 Ð39⬚C; minima, 4 Ð21⬚C; maximum daily sustained wind speeds 19 Ð71 km/h). We included the treatment with only (R)-3-hydroxyhexan-2-one because that compound commonly is the most active component in pheromone blends of cerambycine species (Hanks et al. 2007). The prolonged trapping period was necessary because literature reports of the ßight period of A. inflaticollis and collection dates of museum specimens range from April through June (Linsley 1963; A.M.R., unpublished data). We conducted bioassays at the desert study site where infested host material previously had been collected (see above). We initially used ßight-intercept panel traps (black, 1.2 by 0.30 m, model PT Intercept; APTIV, Portland, OR) suspended from branches of Joshua trees, but they often were knocked down by wind. We therefore used bucket traps after 13 May, partially burying the traps to prevent their being dislodged by wind. Bucket traps were constructed from 19-liter plastic buckets Þtted with 30-cm-diameter aluminum funnels (Catalog 2851B; BioQuip Products, Rancho Dominguez, CA). Each trap was buried such that the lip of the bucket was ⬇8 cm above ground level to exclude vertebrates. Traps were placed ⬇10 m apart in three northeasterly transects that were ⬇0.4 Ð 0.8 km apart. Lures consisted of clear self-seal polyethylene bags (Bagette model 14770, 5.1 by 7.6 cm, 0.05-mm wall thickness; Cousin, Largo, FL) that were sealed and suspended with wire from the handle of the bucket over the center of the funnel. Lures were loaded with (1) 1 ml of an ethanol solution of the reconstructed blend of pheromone components found in aeration extracts from male A. inflaticollis (see Results), including (per ml of solution) 2,3-hexanedione (19 l), (R)-3-hydroxyhexan-2-one (100 l), (S)-2-hydroxyhexan-3-one (6 l), (2R,3R)-2,3-hexanediol (11 l), and (2R,3S)-2,3-hexanediol (6 l); (2) 1 ml of a solution of 100 l of (R)-3-hydroxyhexan-2-one in ethanol; or (3) 1 ml of ethanol (solvent control). Each transect included one trap for each treatment. We checked traps for beetles every 3Ð 4 d, at which time lures were replaced and treatments were rotated one position along the transect to control for positional effects. Differences between treatments in numbers of beetles captured were tested with the two-way nonparametric FriedmanÕs test (blocked by day; PROC FREQ with CMH option; SAS Institute 2001) because assumptions of analysis of variance (ANOVA) were violated by heteroscedasticity (Sokal and Rohlf 1995). Differences between pairs of means were tested with the REGWQ means-separation test
1464
ENVIRONMENTAL ENTOMOLOGY
Table 1. Retention times/elution order of hydroxyhexanones and 2,3-hexanediols on a Cyclodex-B GC column, programmed from 50°C/1 min, 5°C/min, to 200°C Compound
Retention time (min)
(R)-2-hydroxyhexan-3-one (R)-3-hydroxyhexan-2-one (S)-2-hydroxyhexan-3-one (S)-3-hydroxyhexan-2-one (2S,3S)-2,3-hexanediol (2R,3R)-2,3-hexanediol (2R,3S)-2,3-hexanediol (2S,3R)-2,3-hexanediol
10.49 10.56 10.67 11.10 15.12 15.31 15.77 15.87
to control maximum experimentwise error rates (SAS Institute 2001). We include in the analysis data from only two trapping periods during which we captured adult A. inflaticollis (see Results). Voucher specimens of A. inflaticollis have been deposited in the Entomology Research Museum at the University of California, Riverside, and the Smithsonian National Museum of Natural History, Washington DC. Results Comparisons of extracts of headspace volatiles collected from male and female A. inflaticollis showed that there were Þve male-speciÞc compounds. These compounds were tentatively identiÞed (in order of elution) as 2,3-hexanedione, 3-hydroxyhexan-2-one, 2-hydroxyhexan-3-one, (2R*,3R*)-2,3-hexanediol, and (2R*,3S*)-2,3-hexanediol by matches of their mass spectra with spectra from our library of known cerambycid pheromone components. The identiÞcations were conÞrmed by matching the mass spectra and retention times with those of authentic standards on DB-5 and Cyclodex B columns (see below). Having determined the gross structures of the two hydroxyhexanones and the two 2,3-hexanediols, the absolute conÞgurations of the insect-produced compounds were determined by analysis of the extracts on a chiral stationary phase Cyclodex-B column (Table 1). IdentiÞcations were conÞrmed by coinjection of aliquots
Vol. 38, no. 5
of extract with the racemic hydroxyhexanones or diols, to show conclusively that one of the two peaks in a racemate was enhanced by coinjection of the extract. In total, these analyses unambiguously identiÞed the Þve male-speciÞc compounds as (R)-3-hydroxyhexan-2-one (⬇70%), (S)-2-hydroxyhexan-3-one (4%), 2,3-hexanedione (14%), (2R,3R)-2,3-hexanediol (8%), and (2R,3S)-2,3-hexanediol (4%). Extracts of headspace aerations of female A. inflaticollis did not contain any of these compounds in detectable quantities. The prothoraces of male and female A. inflaticollis are strongly dimorphic in punctation and shape of the pronotum (Hovore and Giesbert 1976). Pronota and pleura of males had groups of pores within indentations, whereas females had indentations but lacked the pores (Fig. 1). We captured adult A. inflaticollis during only two 2-d trapping periods (17Ð19 and 19 Ð21 May 2008) of the 34 d that bucket traps were in place in the Mojave Desert (with the exception of one female that was captured on 1 June). A total of 27 beetles were captured (13 females, 13 males; one specimen was damaged and could not be sexed), with a maximum of seven adults captured in one trap that was baited with the blend of pheromone components. Treatment means were signiÞcantly different from one another (Fig. 2; sexes combined, FriedmanÕs Q2,17 ⫽ 11.7, P ⫽ 0.003). Traps baited with the A. inflaticollis–speciÞc blend and those baited with (R)-3-hydroxyhexan-2one as a single component captured signiÞcantly more beetles than did controls, and the blend attracted more than twice as many beetles as (R)-3-hydroxyhexan-2-one alone (Fig. 2). Traps baited with synthetic pheromone did not capture any other insect species in consistent or signiÞcant numbers. Discussion (R)-3-hydroxyhexan-2-one is known to be an important component or the sole component of maleproduced pheromones of 14 species of cerambycine 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
Fig. 2. Mean (⫾ SE, N ⫽ 6 replicates) number of adult A. inflaticollis (sexes combined) that were captured on 17Ð19 and 19Ð21 May 2008 in the Mojave desert with bucket traps baited with ethanol solutions of the blend of male-speciÞc components [(R)-3-hydroxyhexan-2-one, (S)-2-hydroxyhexan-3-one, 2,3hexanedione, (2R,3R)- and (2R,3S)-2,3-hexanediols], (R)-3hydroxyhexan-2-one alone (R-ketone), or ethanol (control). Bars with different letters are signiÞcantly different (REGWQ test P ⬍ 0.05).
cerambycine species respond as strongly to (R)-3hydroxyhexan-2-one alone as they do to more complete reconstructions of blends of headspace volatiles collected from live males (Hanks et al. 2007). In this context, the markedly stronger behavioral response of adult A. inflaticollis to the blend compared with their responses to (R)-3-hydroxyhexan-2-one alone is noteworthy and indicates that one or more of the minor components also forms part of the pheromone blend for A. inflaticollis. The 3-hydroxyhexan-2-one, 2-hydroxyhexan-3-one, and 2,3-hexanediol structures of the major and minor components produced by male A. inflaticollis (tribe Elaphidiini) match the structural motif shared by the pheromones identiÞed for cerambycine species in the tribes Anaglyptini, Callidiini, and Clytini (Lacey et al. 2004, 2007a, 2008, 2009; Ray et al. 2006; Hanks et al. 2007). We have not attempted to further optimize the blend and narrow down the speciÞc minor component(s) that contribute to the increased attraction because of the scarcity of the species and the very short time window that adult beetles apparently are active every year (e.g., we captured beetles during only two trapping periods). Thus, Þeld trials to determine the role of each of the minor components and the exact nature of the full pheromone blend could require many Þeld seasons. It also is important to note that the minor component (S)-2-hydroxyhexan-3-one was not simply an artifact of isomerization of the rather thermolabile (R)-3-hydroxyhexan-2-one (Leal et al. 1995), because isomerization would have resulted in approximately equivalent amounts of (R)and (S)-2-hydroxyhexan-3-one, as well as lesser amounts of (S)-3-hydroxyhexan-2-one from isomerization in the reverse direction. The pores on the prothorax of male A. inflaticollis are similar in structure to the pheromone gland pores
1465
in males of other cerambycine species that are also known to produce hydroxyketone and/or 2,3-alkanediol 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. inflaticollis. Male A. inflaticollis displayed the characteristic “pushup” stance that has been associated with calling behaviors of other cerambycines (Lacey et al. 2007a, b). The small numbers of A. inflaticollis that were captured 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 sexes of A. inflaticollis in Þeld bioassays to lures baited with the species-speciÞc blend, or (R)-3-hydroxyhexan-2-one alone, suggests that males produce an aggregation rather than a sex pheromone according to the accepted deÞnitions of these terms (Wyatt 2003), as seems to be true for many other cerambycine species (Hanks et al. 2007). However, it should be pointed 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 of Þnding females. This “satellite male” strategy, whereby noncalling males intercept females responding to calling males, has been reported for other types of insects [e.g., the cockroach Nauphoeta cinerea (Olivier); Moore et al. 1995]. Alternatively, the pheromone signal may be increased when calling males aggregate, thereby improving the chances of attracting receptive females and allowing females to choose among a number of males. Analogous types of mating aggregations are known for insects as diverse as arctiid moths (Wunderer et al. 1986) and true fruit ßies (Aluja and Norrbom 2000). In summary, we identiÞed and conducted bioassays of a male-produced aggregation pheromone blend for a rare desert-dwelling cerambycid beetle. The most abundant component of the pheromone, (R)-3-hydroxyhexan-2-one, conforms to the structural motif characteristic of pheromones for related species in several tribes of the Cerambycinae, lending further support to our working hypothesis that male-produced pheromones are common and may be conserved across this large and diverse subfamily, which is represented by nearly 5,000 species and 65 tribes in the New World alone (Monne´ and Hovore 2006). Acknowledgments We thank D. Erskine-Hellrigel for assistance with Þeld work, S. Lingafelter for helpful comments and assistance with museum specimens, and two anonymous reviewers whose constructive comments helped to improve the manuscript. Scanning electron microscopy was made possible through collaboration between A.M.R. and Bugscope, The Imaging Technology Group, Beckman Institute for Advanced Science and Technology (http://bugscope.beckman.uiuc.edu/), University of Illinois at Urbana-Champaign (UIUC). This project was supported by a Francis M. and Harlie M. Clark Research Support Grant (UIUC, to A.M.R.), the Alphawood
1466
ENVIRONMENTAL ENTOMOLOGY
Foundation (to L.M.H.), and the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, Grant 2006-35302-17457 (to J.G.M. and L.M.H.). This research was in partial fulÞllment of a PhD degree for A.M.R. from UIUC.
References Cited Aluja, M., and A. L. Norrbom (eds). 2000. Fruit ßies (Tephritidae): phylogeny and evolution of behavior. CRC, Boca Raton, FL. Cope, J. 1984. Notes on the ecology of western Cerambycidae. Coleop. Bull. 38: 27Ð36. Hanks, L. M., J. G. Millar, J. A. Moreira, J. D. Barbour, E. S. Lacey, J. S. McElfresh, F. R. Reuter, and A. M. Ray. 2007. Using generic pheromone lures to expedite identiÞcation of aggregation pheromones for the cerambycid beetles Xylotrechus nauticus, Phymatodes lecontei, and Neoclytus modestus modestus. J. Chem. Ecol. 33: 889 Ð907. Hovore, F. T., and E. F. Giesbert. 1976. Notes on the ecology and distribution of Western Cerambycidae (Coleoptera). Coleop. Bull. 30: 349 Ð360. Hovore, F. T., R. L. Penrose, and E. F. Giesbert. 1978. Notes on North American Cerambycidae (Coleoptera). Entomol. News 89: 95Ð100. Lacey, E. S., M. D. Ginzel, J. G. Millar, and L. M. Hanks. 2004. Male-produced aggregation pheromone of the cerambycid beetle Neoclytus acuminatus acuminatus. J. Chem. Ecol. 30: 1493Ð1507. Lacey, E. S., J. A. Moreira, J. G. Millar, A. M. Ray, and L. M. Hanks. 2007a. Male-produced aggregation pheromone of the cerambycid beetle Neoclytus mucronatus mucronatus. Entomol. Exp. Appl. 122: 171Ð179. Lacey, E. S., A. M. Ray, and L. M. Hanks. 2007b. Calling behavior of the cerambycid beetle Neoclytus acuminatus acuminatus (F.). J. Insect Behav. 20: 117Ð128. Lacey, E. S., J. A. Moreira, J. G. Millar, and L. M. Hanks. 2008. A male-produced aggregation pheromone blend consisting of alkanediols, terpenoids, and an aromatic alcohol from the cerambycid beetle Megacyllene caryae. J. Chem. Ecol. 34: 408 Ð 417. Lacey, E. S., J. G. Millar, J. A. Moreira, and L. M. Hanks. 2009. Male-produced aggregation pheromones of the ceram-
Vol. 38, no. 5
bycid beetles Xylotrechus colonus and Sarosesthes fulminans. J. Chem. Ecol. 35: 733Ð740. Leal, W. S., X. Shi, K. Nakamuta, M. Ono, and J. Meinwald. 1995. Structure, stereochemistry, and thermal isomerization of the male sex pheromone of the longhorn beetle Anaglyptus subfasciatus. Proc. Natl. Acad. Sci. U.S.A. 92: 1038 Ð1042. Linsley, E. G. 1963. The Cerambycidae of North America, part IV. Taxonomy and classiÞcation of the subfamily Cerambycinae, tribes Elaphidionini through Rhinotragini. Univ. Calif. Publ. Entomol. 21: 105. Monne´, M. A., and F. T. Hovore. 2006. Checklist of the Cerambycidae (Coleoptera) of the Western Hemisphere. BioQuip Publications, Rancho Dominguez, CA. Moore, A. J., N. L. Reagan, and K. F. Haynes. 1995. Conditional signaling strategiesÑ effects of ontogeny, social experience, and social status on the pheromonal signal of male cockroaches. Anim. Behav. 50: 191Ð202. Ray, A. M., E. S. Lacey, and L. M. Hanks. 2006. Predicted taxonomic patterns in pheromone production by longhorned beetles. Naturwissenschaften 93: 543Ð550. Ray, A. M., J. G. Millar, J. S. McElfresh, I. P. Swift, J. D. Barbour, and L. M. Hanks. 2009. Male-produced aggregation pheromone of the cerambycid beetle Rosalia funebris. J. Chem. Ecol. 35: 96 Ð103. SAS Institute. 2001. SAS/STAT userÕs guide for personal computers, release 8.01. SAS Institute, Cary, NC. Sokal, R. R., and F. J. Rohlf. 1995. Biometry, 3rd ed. Freeman and Co., New York. Solomon, J. D. 1995. Guide to insect borers in North American trees and shrubs. U.S. Dep. Agric. Forest Service Agriculture Handbook 706. USDA, Washington, DC. Wunderer, H., K. Hansen, T. W. Bell, D. Schneider, and J. Meinwald. 1986. Sex pheromones of two Asian moths (Creatonotos transiens, C. gangis; Lepidoptera-Arctiidae): behavior, morphology, chemistry and electrophysiology. Exp. Biol. 46: 11Ð27. Wyatt, T. D. 2003. Pheromones and animal behaviour: communication by smell and taste. Cambridge University Press, Cambridge, United Kingdom. Received 24 November 2008; accepted 2 July 2009.
