The female-produced sex pheromone of Phyllophaga crinita (Burmeister) (Coleoptera: Scarabaeidae: Melolonthinae; the adult has no common name) is identified as methyl 2-(methylthio)benzoate. This is the first identification of a sulfur-containing, long-distance, female-produced sex attractant from any insect taxa. The root-feeding larvae of this species are serious pests in many crops in Texas and Mexico. In field tests, many P. crinita males were captured in traps baited with the authentic compound. Interestingly, a heteroatom analog, methyl 2-methoxybenzoate, also captured P. crinita males, but only at a dose 10,000 times higher than the lowest tested dose of the authentic pheromon

Crocker, R.

Morris, B.

Nojima, S.

Robbins, P.

Roelofs, W.

Villani, M.

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Naturwissenschaften (2003) 90:517–520DOI 10.1007/s00114-003-0469-5S HORT COMMUN I CA T I ONP. S. Robbins · R. L. Crocker · S. Nojima ·B. D. Morris · W. L. Roelofs · M. G. VillaniMethyl 2-(methylthio)benzoate:the unique sulfur-containing sex pheromone of Phyllophaga crinitaReceived: 11 April 2003 / Accepted: 28 August 2003 / Published online: 11 October 2003 Springer-Verlag 2003Abstract The female-produced sex pheromone of Phyl-lophaga crinita (Burmeister) (Coleoptera: Scarabaeidae:Melolonthinae; the adult has no common name) isidentified as methyl 2-(methylthio)benzoate. This is thefirst identification of a sulfur-containing, long-distance,female-produced sex attractant from any insect taxa. Theroot-feeding larvae of this species are serious pests inmany crops in Texas and Mexico. In field tests, many P.crinita males were captured in traps baited with theauthentic compound. Interestingly, a heteroatom analog,methyl 2-methoxybenzoate, also captured P. crinitamales, but only at a dose 10,000 times higher than thelowest tested dose of the authentic pheromone.IntroductionThe larvae of Phyllophaga crinita (Burmeister) (Coleop-tera: Scarabaeidae: Melolonthinae) are root-feeding pestsof some economic consequence. There is no commonname for this species. In Texas, the larvae have beenreported feeding in rangeland pasture, ornamental turf,sugarcane, grain sorghum, cotton, and wheat, as well ascorn, parsley, and cabbage (Reinhard 1940; Teetes 1973,1976; Huffman et al. 1976; Huffman and Harding 1980).P. crinita larvae are also serious pests in Mexico(Rodriguez-del-Bosque 1984, 1995). In the USA, P.crinita is reported from Texas, Louisiana, Mississippi,and Alabama (Luginbill and Painter 1953). Until recently,the most southerly known distribution of P. crinita wasSan Fernando, Tamaulipas, Mexico, 137 km south of theMexico–USA border (Rodriguez-del-Bosque et al. 1995).However, Rodriguez-del-Bosque (2003) recently reportedP. crinita larvae feeding on grain sorghum in Tampico,southern Tamaulipas, 275 km south of San Fernando, andindicates that the larvae of this insect have the potential tobecome a pest of some significance there.P. crinita has long been regarded as a member of thegenus Phyllophaga (sensu stricto), and as such, is one ofabout 200 species of Phyllophaga (sensu lato) found inNorth America (Woodruff and Beck 1989). However, arecent publication (Coca-Abia 2002) hypothesized themonophyly and subsequent re-establishment of the genusTrichesthes and removed P. crinita from the genusPhyllophaga to Trichesthes. We report here the identifi-cation of the unique sulfur-containing female sex pher-omone of P. crinita as methyl 2-(methylthio)benzoate(CAS number 3704-28-7).Materials and methodsPheromone collectionsThird instar larvae of P. crinita were collected by digging in Dallas,Texas, in April 2000 and were shipped to Geneva, New York, viaovernight mail. Larvae were individually housed in ~30-ml plasticcups in a 3:1 mixture of greenhouse sand and screened peat mossraised to about 12% moisture. The cups were kept in a controlledenvironment room maintained at 25C during the 16-h photophaseand 20C during the 8-h scotophase. After pupation and adultemergence, females were placed in observation cages. When thefemales were observed calling (abdominal pheromone glandeverted) (Leal et al. 1993) during the scotophase, they wereremoved from the cages and their glands excised and soaked in200 l of dichloromethane. After 20 min, the glands were removedand the extract concentrated under a nitrogen stream to a volume ofabout 20 l.In memory of M.G. Villani, who died on 15 May 2001. Hisenthusiasm for soil-dwelling scarab beetles and contributions totheir research are greatly missed.P. S. Robbins ()) · S. Nojima · B. D. Morris · W. L. Roelofs ·M. G. VillaniDepartment of Entomology,New York State Agricultural Experiment Station,Cornell University, Geneva, NY 14456, USAe-mail: psr1@cornell.eduR. L. CrockerDepartment of Entomology, Texas A & M University,Dallas, TX 75252, USAB. D. MorrisHultz Hall, North Dakota State University,PO Box 5346, Fargo, ND 58105, USACoupled gas chromatographic-electroantennographic detection(GC-EAD) analysisThe GC instrumentation used in this study was the same as thatdescribed by Nojima et al. (2003a). A Hewlett Packard 5890 seriesII gas chromatograph equipped with either a nonpolar SPB-1capillary column (30 m  0.25 mm i.d., 0.25 m film thickness;Supelco, Bellefonte, Pa.) or a polar EC-WAX econo-cap capillarycolumn (30 m  0.25 mm i.d., 0.25 m film thickness; Alltech,Deerfield, Ill.) was used for analysis in splitless mode. Nitrogenwas used as the carrier gas at a head pressure of 138 Pa (flow rate,2.0 ml/min). The oven temperature was programmed 40C for2 min, increased at 15C/min to 250C and held for 10 min. Injectorand detector temperatures were set at 250 and 280C, respectively.The column effluent was combined with nitrogen make-up gas(30 ml/min) and then split 1:1 to the flame ionization detector (FID)and EAD. The EAD outlet was secured in a charcoal-filtered andhumidified airstream, refrigerated by a modified condenser flushedwith 0C water, flowing at 500 ml/min over the antennalpreparation.EAD recordingsAn antenna plucked from the head of a beetle was bridged betweentwo slits in a custom-made acrylic holder in such a position that thelamellae were held open (Fig. 1). Each slit was filled with 0.9%NaCl saline, to which pure gold indifferent and recordingelectrodes were connected, respectively. The holder was placedinside of the cooling condenser and maintained at about 5C. Goodrecordings could often be made for several hours on these antennalpreparations.The output signal from the antenna was amplified 100 by acustomized single-step high-output impedance DC amplifier (No-jima et al. 2003a). The signal was filtered by a simple resistance/capacitor high-pass filter with a cutoff frequency of about 0.5 Hzand recorded on an HP 3390A integrator synchronized with the GCintegrator. A total of seven antennae from different males (2–8runs/antenna) were used for the GC-EAD analysis.Chemical analysisGC-MS analyses were performed on a Shimadzu GC-17A equippedwith a nonpolar DB-1 ms capillary column (30 m  0.25 mm i.d.,0.25 m film thickness; J&W Scientific, Folsom, Calif.) or a polarEC-WAX econo-cap capillary column (30 m  0.25 mm i.d.,0.25 m film thickness; Alltech). The GC was coupled to aShimadzu QP-5050A quadrupole mass spectrometer running in theEI (electron ionization at 70 eV) scan mode. Helium was used asthe carrier gas at an initial head pressure of 53.8 Pa at constant flowrate (1.0 ml/min). The time for splitless injection was 1 min. Oventemperature was programmed 40C for 2 min, increased at 15C/min to 250C and held for 10 min. Injector and interfacetemperatures were set at 280 and 250C, respectively. The EADactive component was tentatively identified by matches to libraryspectra followed by comparisons of retention time and mass spectrawith that of an authentic sample. The antennal activity of thesynthetic compound was confirmed by GC-EAD.ChemicalsMethyl 2-(methylthio)benzoate and methyl 4-(methylthio)benzoatewere purchased from Lancaster Synthesis (Pelham, N.H.) andMaybride Chemical Company. (UK), respectively. Methyl 3-(methylthio)benzoate was prepared from 3-mercaptobenzoic acid(Toronto Research Chemical, Canada) by methylation withtrimethylsilyl diazomethane (Aldrich, Milwaukee, Wis.). Methyl2-methoxybenzoate was also obtained from Aldrich.Field evaluation of compoundsA total of six treatments were field-tested in 2001. These treatmentsincluded methyl 2-(methylthio)benzoate at 100, 300, and 1,000 g,and methyl 2-methoxybenzoate at 1,000 and 1,000,000 g, as wellas a solvent-only treatment. The 100, 300, and 1,000 g treatmentswere made by dissolving each chemical in hexane (10 g/l) andapplying the appropriate amount into 5 mm rubber stopper septa(Thomas Scientific, Swedesboro, N.J.) and allowing the hexane toevaporate in a fume hood. The 1,000,000 g lure was made byapplying 1.0 g of neat methyl 2-methoxybenzoate into an evapo-rative dispenser (GenPore, Reading, Pa.).Four sets of the six treatments of cross-vane traps and lureswere deployed at four locations in and around Dallas, Texas, inMay 2001. Traps were placed about 20 m apart in a line. Trapswere hung on stakes such that the trap bottom was about 60 cmfrom the ground. Treatment positions were randomized at deploy-ment and once each week thereafter. Traps were checked andbeetles counted a total of 24 times between 31 May and 19 August.Lures were replaced every 4 weeks.Fig. 1 Simultaneous responsesof GC-FID and GC-EAD of themale antenna of P. crinita to thefemale pheromone gland extracton a nonpolar capillary column.Photograph of antenna in holderapparatus518StatisticsThe total numbers of beetles caught in each trap at each locationover the 24 count dates were combined. Data were log transformed(x+1) before analysis to establish homogeneity of variance.Treatments were compared with the Student-Newman-Keuls mul-tiple range test (at P<0.05).ResultsIdentification of the sex pheromone componentGC-EAD analyses of female P. crinita gland extracts onnonpolar and polar columns consistently revealed a singleEAD-active component (Fig. 1). Mass spectral matches tolibrary spectra tentatively identified the compound asmethyl 2-(methylthio)benzoate. The identity was con-firmed by comparison of mass spectra, GC retention timesand EAD activity on both polar and nonpolar columnswith the authentic standard. The retention times and MSfragment patterns of the other isomers, methyl 3-(methylthio)benzoate or 4-(methylthio)benzoate, weredifferent from the natural compound.Field testsResults from the field test can be seen in Fig. 2. A total of8,664 male beetles were captured. No females werecaptured. The 1,000 g dose of the synthetic pheromone(methylthio) captured 337 times more beetles than theoxygen analog (methoxy) at the same dose and wassignificantly different from it (F5,15=88.04 ;P>0.0001).The oxygen analog, when presented to the beetles at1,000,000 g (1.0 g), caught only slightly more than halfthe beetles than the pheromone did at 100 g, a rate10,000 times smaller. No significant differences werefound between those two treatments.DiscussionThis is the only sulfurous, long-distance, female-producedsex attractant that has been identified from any insecttaxa. Field data indicate that the presence of the sulfuratom is important for pheromonal activity. Interestingly,this compound is crystalline at room temperature. Ashort-range, sulfur-containing, male-produced sex attrac-tant has been described from the cockroach, Nauphoetacinerea (Sreng 1990; Sirugue et al. 1992). Leal recentlysummarized the current knowledge regarding scarabbeetle sex pheromones (Leal 1998, 1999). The rutelinebeetles generally employ sex pheromones that are fattyacid derivatives reminiscent of moth sex pheromonebiosynthetic pathways. Melolonthine beetles, however,have been found to utilize a more diverse suite ofcompounds than the rutelines. Although melolonthinepheromones do include fatty acid derivatives such as(R,Z)-7,15-hexadecadien-4-olide (Leal et al. 1996a) and2-tetradecanone (Zhang et al. 2003) as sex attractants,they also make use of compounds such as methyl esters ofl-valine and l-isoleucine (Leal et al. 1992, 2003; Zhanget al. 1997), l-leucine methyl ester (Nojima et al. 2003b),phenol (Henzell and Lowe 1970), anisole (Leal et al.1996b; Ward et al. 2002), linalool (Leal et al. 1993), andthe alkaloid 1,3-dimethyl-2,4-(1H,3H)-quinazolinedione(Leal et al. 1997). To this group of compounds is nowadded methyl 2-(methylthio)benzoate. There is a paucityof information on the biosynthetic pathways by whichmost melolonthine sex pheromone compounds are pro-duced. The sex pheromone of the grass grub, Costelytrazealandica, identified as phenol (Henzell and Lowe1970), was ultimately found to be produced by endosym-biotic bacteria (Hoyt and Osborne 1971). Whether othermelolonthines such as P. crinita also employ symbioticbacteria to produce semiochemicals will be the subject ofour future research. Methyl 2-(methylthio)benzoate isinexpensive and commercially available off the shelf. It ishoped that the identification of this compound as the sexattractant of P. crinita will find some use in detection and/or management systems for this pest.Acknowledgement The authors thank Dr. Richard Cowles of theConnecticut Agricultural Experiment Station for statistical consul-tation.ReferencesCoca-Abia MM (2002) Reestablishment of the genus TrichesthesErichson, 1847 (Coleoptera: Scarabaeidae, Melolonthinae)based on phylogeny. J N Y Entomol Soc 110(1):95–114Henzell RF, Lowe MD (1970) Sex attractant of the grass grubbeetle. Science 168:1005–1006Hoyt CP, Osborne GO (1971) Production of an insect sex attractantby symbiotic bacteria. Nature 230:472–473Huffman FR, Harding JA (1980) Biology of Phyllophaga crinita inLower Rio Grande Valley sugarcane. 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Methyl 2-(methylthio)benzoate: the unique sulfur-containing sex pheromone of Phyllophaga crinita

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Methyl 2-(methylthio)benzoate: the unique sulfur-containing sex pheromone of Phyllophaga crinita

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