The female-produced sex pheromone of the scarab beetle Phyllophaga lanceolata was identified as the methyl ester of an essential amino acid, l-leucine. During field testing, 239 male P. lanceolata were caught in traps baited with l-leucine methyl ester. l-Isoleucine and l-valine methyl esters, similar in structure to l-leucine methyl ester and previously identified as female-produced sex pheromone compounds employed by other Phyllophaga species, were also tested. Addition of l-valine or l-isoleucine methyl esters to the l-leucine methyl ester in 1:1 ratios completely inhibited attraction of P. lanceolata males. Males of P. squamipilosa were also captured using l-leucine methyl ester. This is the first record of P. squamipilosa from Kansa

Morris, Bruce

Nojima, Satoshi

Robbins, Paul

Roelofs, Wendell

Salsbury, Glenn

Villani, Michael

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P1: JQXJournal of Chemical Ecology [joec] pp997-joec-473733 October 17, 2003 19:55 Style file version June 28th, 2002Journal of Chemical Ecology, Vol. 29, No. 11, November 2003 (°C 2003)L-LEUCINE METHYL ESTER: THE FEMALE-PRODUCEDSEX PHEROMONE OF THE SCARAB BEETLE,Phyllophaga lanceolataSATOSHI NOJIMA,1 PAUL S. ROBBINS,1;⁄ GLENN A. SALSBURY,2BRUCE D. MORRIS,1;3 WENDELL L. ROELOFS,1and MICHAEL G. VILLANI1;41Department of EntomologyCornell UniversityNew York State Agricultural Experiment StationGeneva, New York 14456, USA2Kansas Department of Agriculture222 East IowaGreensburg, Kansas 67054, USA(Received February 19, 2003; accepted July 18, 2003)Abstract—The female-produced sex pheromone of the scarab beetle Phyl-lophaga lanceolata was identified as the methyl ester of an essential aminoacid, L-leucine. During field testing, 239 male P. lanceolata were caught intraps baited with L-leucine methyl ester. L-Isoleucine and L-valine methyl es-ters, similar in structure to L-leucine methyl ester and previously identified asfemale-produced sex pheromone compounds employed by other Phyllophagaspecies, were also tested. Addition of L-valine or L-isoleucine methylesters to the L-leucine methyl ester in 1:1 ratios completely inhibited attrac-tion of P. lanceolata males. Males of P. squamipilosa were also captured us-ing L-leucine methyl ester. This is the first record of P. squamipilosa fromKansas.Key Words—L-Valine, L-isoleucine, L-leucine methyl ester, sex pheromone,electroantennogram, Tostegoptera, squamipilosa, Scarabaeidae, Melolonthinae.3Current address: Hultz Hall, North Dakota State University, P.O. Box 5346, Fargo, North Dakota58105, USA.4Deceased May 15, 2001.⁄To whom correspondence should be addressed. E-mail: psr1@cornell.edu24390098-0331/03/1100-2439/0 C° 2003 Plenum Publishing CorporationP1: JQXJournal of Chemical Ecology [joec] pp997-joec-473733 October 17, 2003 19:55 Style file version June 28th, 20022440 NOJIMA, ROBBINS, SALSBURY, MORRIS, ROELOFS, AND VILLANIINTRODUCTIONThe genus Phyllophaga (sensu lato) (Coleoptera: Scarabaeidae: Melolonthinae),encompasses about 200 species in the United States north of Mexico (Woodruffand Beck, 1989). Only two species are found in the subgenus Tostegoptera, P .(Tostegoptera) lanceolata and P: (Tostegoptera) squamipilosa Saylor. Whereas P.lanceolata was described in 1824, P. squamipilosa was not described until 1936by L.W. Saylor, who discovered the species in a series of P. lanceolata from Texas(Saylor, 1936). Nothing is known of the life history or biology of P. squamipilosa.P. lanceolata is unusual among the Phyllophaga in that it is diurnal, and thefemales are flightless (Travis, 1939; Reinhard, 1940). Travis (1939) reported thaton days that are “hot and sunny,” the greatest number of beetles “are visible between8 and 10 o’clock in the morning.” He further reports that “On many occasions,males were noted feeding, apparently unconcerned, on the same leaf or plant witha female until a female extruded her genitalia. Immediately, males within a radiusof 15 to 20 yards flew toward her.”P. lanceolata is distributed throughout the Great Plains including Colorado,Iowa, Kansas, Missouri, Nebraska, Nevada, New Mexico, Oklahoma, SouthDakota, Texas, and Wyoming. It has also been reported from the province ofOntario, Canada (Luginbill and Painter, 1953). It has been an important pest speciesin both the larval and adult stages on wheat, cotton, corn (Hayes, 1919), and sun-flowers (Rogers, 1978). Reinhard (1940) listed a series of truck crops on whichP. lanceolata fed including spinach, lettuce, carrot, cowpea, rape, turnip, mustard,onion, beans, and Irish potato. We present here the identification of the female-produced sex pheromone of P. lanceolata and the results of field trapping tests.METHODS AND MATERIALSPheromone Collections. Third-instar larvae of P. lanceolata were dug frominfested cattle range on a ranch in Kiowa County, Kansas, in late April 2000. Larvaewere individually housed in »30-ml plastic cups in a 3:1 mix of greenhouse sandand screened peat moss raised to about 12% moisture. The cups were housed ina controlled environment room maintained at 25–C during the 16-hr photophaseand 20–C during the 8-hr scotophase. After pupation and adult emergence, femaleswere placed in observation cages. When females were observed calling (abdominalpheromone gland everted, see Figure 1) (Leal et al., 1993) during the photophase,they were removed from the cages, and the glands were excised and soaked in200„l of dichloromethane. After 20 min, the glands were removed and the extractconcentrated under a nitrogen stream to a volume of about 20 „l.GC-EAD Analysis. GC-EAD instrumentation used in this study was the sameas described previously (Nojima et al., 2003), except for using an acrylic holderP1: JQXJournal of Chemical Ecology [joec] pp997-joec-473733 October 17, 2003 19:55 Style file version June 28th, 2002THE FEMALE-PRODUCED SEX PHEROMONE OF THE SCARAB BEETLE 2441FIG. 1. Female P. lanceolata in calling posture with sex pheromone gland everted.designed for a beetle’s antenna. A nonpolar SPB-1 capillary column (30 m £0.25 mm ID, 0.25-„m film thickness; Supelco, Bellefonte, PA) or a polar EC-WAX Econo-Cap capillary column (30 m£ 0.25 mm ID, 0.25-„m film thickness;Alltech, Deerfield, IL) was used for GC-EAD analyses. Nitrogen was used ascarrier gas (flow 2.0 ml/min). The GC was programmed for 40–C for 5 min, then15–C/min to 250–C and held for 10 min. Injector and detector temperatures were260–C. A fl-DEX 120 chiral capillary column (30 m £ 0.25 mm ID, 0.25-„mfilm thickness; Supelco, Bellefonte, PA) was used for resolution of enantiomers,with N2 carrier gas (flow 2.0 ml/min). The GC program was 40–C for 5 min,4–C/min to 130–C, then 10–C/min to 220–C and held for 20 min. Injector anddetector temperatures were 260–C. Six individual virgin female gland extractswere subjected to GC-EAD analysis, and antennae from four different males wereused for EAD recordings.Chemical Analysis. GC-MS analysis was performed on a Shimadzu QP5050A equipped with a nonpolar DB-1ms capillary column (30 m £ 0.25 mmID, 0.25-„m film thickness; J&W Scientific, Folsom, CA) or a polar EC-WAXEcono-Cap capillary column (30 m £ 0.25 mm ID, 0.25-„m film thickness;P1: JQXJournal of Chemical Ecology [joec] pp997-joec-473733 October 17, 2003 19:55 Style file version June 28th, 20022442 NOJIMA, ROBBINS, SALSBURY, MORRIS, ROELOFS, AND VILLANIAlltech, Deerfield, IL) in EI mode (70 eV) and the same conditions as those ofGC-EAD analyses, but with helium as the carrier gas at constant flow (1.0 ml/min).The EAD active compound was tentatively identified by mass spectral matchesto library spectra. The identity was confirmed by comparing GC retention times,mass spectra, and EAD activity with those of an authentic sample.Chemicals. L-Leucine, L-valine, and L-isoleucine methyl esters were ob-tained as hydrochloric acid salts (Aldrich, Milwaukee, WI). Free amino acid methylesters were regenerated by dissolving the salts in an aqueous alkali solution andextracting the mixture with three portions of ether. Ether layers were combined,washed with brine, dried over sodium sulfate, and concentrated in vacuo. Theresulting methyl esters were used without further purification. D-Leucine methylester was prepared by methyl esterification of D-Leucine (Aldrich, Milwaukee,WI) according to Anand and Vimal (1998).Preparation and Field Evaluation of Synthetic Lures. Lures were formulatedby dissolving the neat compounds in hexane (20 „g/„l), dispensing appropriateamounts into 5-mm rubber septa (Thomas Scientific, Swedesboro, NJ), and allow-ing the hexane to evaporate in a fume hood. Lures were deployed in the field inlab-constructed cross-vane traps.The six treatments tested included the methyl esters of L-leucine, L-isoleucine,and L-valine, each tested alone at 4 mg/septum, L-leucine C L-isoleucine (1:1) at4 mg each/septum, L-leucine C L-valine (1:1) at 4 mg each/septum, and a solventonly control.L-Isoleucine and L-valine methyl esters have been reported as sex pheromonecomponents for other Phyllophaga species (Zhang et al., 1997; Leal et al., 2003).They were included in the field test because when antennae of P. lanceolata maleswere challenged with these two compounds in GC-EAD assays, they elicited anten-nal responses similar to those produced by the L-leucine methyl ester (Figure 2B).Three replicates of the six treatments were deployed June 1, 2001, on cattlerange at the ranch from which the larvae had been obtained and where adultactivity had been observed in previous years. Each replicate of the six treatmentswas deployed in a line with traps placed about 15 m apart. Traps were hung on metalstakes such that the bottom of the trap touched the ground. Trap positions within aline were randomized at initial placement and rerandomized each time they werechecked. Traps were checked nine times between June 2 and June 16, when thetraps were removed from the field. The pooled data were log transformed (x C 1)to insure homogeneity of variance before calculating one sample t confidenceintervals (0.95) for those treatments that had catches greater than 0.RESULTS AND DISCUSSIONPheromone Identification. GC-EAD analyses of pheromone gland extracts offemale P. lanceolata using both nonpolar and polar columns consistently revealedP1: JQXJournal of Chemical Ecology [joec] pp997-joec-473733 October 17, 2003 19:55 Style file version June 28th, 2002THE FEMALE-PRODUCED SEX PHEROMONE OF THE SCARAB BEETLE 2443FIG. 2. GC-EAD traces using the antennae of male P. lanceolata stimulated with the femalepheromone gland extracts (A) and synthetic amino acid methyl esters (B) on a nonpolarcapillary column.one EAD active compound (Figure 2A). The mass spectrum of the compoundmatched that of leucine methyl ester. The identity was confirmed by comparingGC retention times and MS spectra with those of authentic samples. The chiralityof the leucine methyl ester was confirmed by means of a chiral GC-EAD analysis.When run on a chiral column, authentic L-leucine methyl ester had a retention time(32.43 min) and EAD activity similar to that of the natural compound (32.48 min),whereas the D-leucine methyl ester did not (31.98 min).Field Evaluation of Synthetic Lures. Of the 240 male P. lanceolata capturedin field testing of the compounds, 239 were captured in the L-leucine methyl esterbaited traps (Table 1). No female beetles were taken in the traps. We conclude thatL-leucine methyl ester is the sex pheromone of P. lanceolata. The four treatmentsthat compared the L-isoleucine and L-valine methyl esters, each alone and each incombination with the L-leucine methyl ester, yielded only a single male P. lance-olata that was captured in a trap baited with L-valine methyl ester. The confidenceintervals calculated for the two treatments that caught beetles indicated the catchP1: JQXJournal of Chemical Ecology [joec] pp997-joec-473733 October 17, 2003 19:55 Style file version June 28th, 20022444 NOJIMA, ROBBINS, SALSBURY, MORRIS, ROELOFS, AND VILLANITABLE 1. AVERAGE NUMBER OF MALES CAPTURES/TREATMENT (§SE)L-Leucine L-Leucine SolventL-Leucine L-Isoleucine L-Valine C L-Isoleucine C L-Valine control79.7 § 18.6 0a 0.33 § 0.33a 0a 0a 0aa Not significantly different, one sample t confidence interval, 95%.from the traps baited with the L-valine methyl ester (a single beetle) were not sig-nificantly different from those traps that captured no beetles (Table 1), whereas thecatches from the L-leucine methyl ester baited traps were different. L-Valine andL-isoleucine methyl esters are clearly nonattractive to the males when presentedalone and demonstrated antagonistic effects when presented in combination withL-leucine methyl ester, the sex pheromone of P. lanceolata.In light of the identification of L-leucine methyl ester as the sex pheromoneof P. lanceolata, the work of Travis (1939) is of particular note. In that publication,Travis reported results of field tests of a series of 24 volatile compounds assessingwhether they would attract P. lanceolata adults. One of the compounds tested wasisoamylamine. Travis states.The last named (isoamylamine) was the only one of these compounds that proved attractiveto the males, and none of them seemed to stimulate the females. Isoamylamine produced amale response similar to that made to crushed or to sexually active females. The crushed andreceptive females appeared to produce an emanation or odor that was rapidly disseminatedby air movement, as male activity was always greater on the leeward side of the female.The same type of response was secured when isoamylamine was released.A comparison of the structures of L-leucine methyl ester, the authentic pheromone,and isoamylamine (3-methyl-butylamine), (Figure 3) demonstrates that the work ofTravis presaged the identification of L-leucine methyl ester as the sex pheromoneof P. lanceolata. Future field tests will compare the relative attractancy of themethyl ester of L-leucine and isoamylamine to P. lanceolata males.FIG. 3. Structures of L-leucine methyl ester and isoamylamine.P1: JQXJournal of Chemical Ecology [joec] pp997-joec-473733 October 17, 2003 19:55 Style file version June 28th, 2002THE FEMALE-PRODUCED SEX PHEROMONE OF THE SCARAB BEETLE 2445Of additional interest is the capture of P. squamipilosa using L-leucine methylester. In an unreplicated test in Stevens County in southwestern Kansas in mid-Juneof 2001 (G.A.S., personal observation), a septum loaded with 4 mg of L-leucinemethyl ester was hung (using a piece of wire) in low brush in grassy rangelandhabitat. No trap was used. Beetles were captured both crawling on the septumas well as while flying upwind in the pheromone plume. The 16 male beetlescaptured were subsequently identified as P. squamipilosa. This is the first recordof P. squamipilosa from the state of Kansas. Very little is known regarding thedegree of overlap in the ranges of P. lanceolata and P. squamipilosa, and nothingis known about their interspecific relationships where they occur sympatrically.Methyl esters of essential amino acids are employed as sex pheromones inthe genus Phyllophaga (Zhang et al., 1997; Leal et al., 2003) as well as the closelyrelated genus Holotrichia (Leal et al., 1992). They are termed essential amino acidsbecause they are generally required for growth and development by insects andother animals and cannot be synthesized de novo (McFarlane, 1985). Therefore,prior to utilization of these compounds as sex pheromones (subsequent to methylesterification) they must be sequestered from plants or produced by endosymbionts.Virgin female P. anxia (LeConte) individuals are capable of emitting L-valine andL-isoleucine methyl esters before any adult feeding has taken place (P.S.R., unpub-lished data). Furthermore, the sex pheromone of Costelytra zealandica (White),another melolonthine scarab beetle, identified as phenol (Henzell and Lowe, 1970),was later found to be produced by bacterial endosymbionts (Hoyt and Osborne,1971). Further work is planned to identify the source of the essential amino acidsused by these Phyllophaga species to make sex pheromone compounds.Acknowledgments—Financial support for this project was provided by the Rawlins GraduateStudent Endowment Fund, administered by the Department of Entomology at Cornell University. Wethank the owners of the Greenleaf Ranch, Kiowa County, Kansas, for allowing access to dig larvae anddeploy traps, and Charles E. Linn Jr. of Cornell University for careful review of this manuscript whilein preparation.REFERENCESANAND, R. C. and VIMAL 1998. A mild and convenient procedure for the esterification of amino acids.Synth. Commun. 28:1963–1965.HAYES, W. P. 1919. The life cycle of Lachnosterna lanceolata Say. J. Econ. Entomol. 12:109–117.HENZELL, R. F. and LOWE, M. D. 1970. Sex attractant of the grass grub beetle. Science 168:1005–1006.HOYT, C. P. and OSBORNE, G. O. 1971. Production of an insect sex attractant by symbiotic bacteria.Nature 230:472–473.LEAL, W. S., MATSUYAMA, S., KUWAHARA, Y., WAKAMURA, S., and HASEGAWA, M. 1992. An aminoacid derivative as the sex pheromone of a scarab beetle. Naturwissenschaften 79:184–185.LEAL, W. S., OEHLSCHLAGER, A. C., ZARBIN, P. H. G., HIDALGO, E., SHANNON, P. J., MURATA, Y.,GONZALEZ, L., ANDRADE, R., and ONO, M. 2003. Sex pheromone of the scarab beetle Phyllophagaelenans and some intriguing minor components. J. Chem. Ecol. 29:15–25.P1: JQXJournal of Chemical Ecology [joec] pp997-joec-473733 October 17, 2003 19:55 Style file version June 28th, 20022446 NOJIMA, ROBBINS, SALSBURY, MORRIS, ROELOFS, AND VILLANILEAL, W. S., SAWADA, M., MATSUYAMA, S., KUWAHARA, Y., and HASEGAWA, M. 1993. Unusualperiodicity of sex pheromone production in the large black chafer Holotrichia parallela. J. Chem.Ecol. 19:1381–1391.LUGINBILL, P. and PAINTER, H. R. 1953. May Beetles of the United States and Canada, United StatesDepartment of Agriculture. Tech. Bull. 1060:1–102, (78 plates).MCFARLANE, J. E. 1985. Nutrition and digestive organs, pp. 59–89, in M. S. Blum (ed.). Fundamentalsof Insect Physiology. Wiley, New York.NOJIMA, S., LINN, C. E., JR., MORRIS, B. D., ZHANG, A., and ROELOFS, W. L. 2003. Identificationof host fruit volatiles from hawthorn (Crataegus spp.) attractive to hawthorn-origin Rhagoletispomonella flies. J. Chem. Ecol. 29:319–334.REINHARD, H. J. 1940. The life history of Phyllophaga lanceolata (Say) and Phyllophaga crinita(Burmeister). J. Econ. Entomol. 33:572–578.ROGERS, C. E. 1978. Phyllophaga lanceolata: A new pest of sunflower in west Texas. Southwest.Entomol. 3:85–87.SAYLOR, L. W. 1936. A new Texas scarab (Coleoptera). Can. Entomol. 68:280.TRAVIS, B. V. 1939. Habits of the June beetle, Phyllophaga lanceolata (Say) in Iowa. J. Econ. Entomol.32:690–693.WOODRUFF, R. E. and BECK, B. E. 1989. The scarab beetles of Florida (Coleoptera: Scarabaeidae)Part II. The May or June beetles (genus Phyllophaga), pp. 226. In Arthropods of Florida andNeighboring Land Areas, Vol. 13.ZHANG, A., ROBBINS, P. S., LEAL, W. S., LINN, C. E., JR., VILLANI, M. G., and ROELOFS, W. L. 1997.Essential amino acid methyl esters: Major sex pheromone components of the cranberry whitegrub, Phyllophaga anxia (Coleoptera: Scarabaeidae). J. Chem. Ecol. 23:231–245.

l-Leucine Methyl Ester: The Female-Produced Sex Pheromone of the Scarab Beetle, Phyllophaga lanceolata

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l-Leucine Methyl Ester: The Female-Produced Sex Pheromone of the Scarab Beetle, Phyllophaga lanceolata

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