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4-2006 Sex of the Soybean Aphid, Aphis glycines Matsumura, and Its Potential Use in -Based Control Junwei Zhu MSTRS Technologies, Inc.

Aijun Zhang United States Department of Agriculture

Kye-chung Park Iowa State University

Tom Baker MSTRS Tecnologies, Inc.

Brian J. Lang IFoowlalo Swta tthie Usn iaverndsit ay,dd bjlitaniong@ialas wtaorkte.edus at: http://lib.dr.iastate.edu/hort_pubs Part of the Agricultural Science Commons, Agriculture Commons, Entomology Commons, and See next page for additional authors the Horticulture Commons The ompc lete bibliographic information for this item can be found at http://lib.dr.iastate.edu/ hort_pubs/6. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html.

This Article is brought to you for free and open access by the Horticulture at Iowa State University Digital Repository. It has been accepted for inclusion in Horticulture Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Sex Pheromone of the Soybean Aphid, Aphis glycines Matsumura, and Its Potential Use in Semiochemical-Based Control

Abstract The newly invasive soybean aphid, Aphis glycines Matsumura, has seriously threatened soybean production in North America, after having spread to >20 states in the United States and several southern provinces of Canada. Control of A. glycineshas focused on applications of insecticides, which are not a long-term solution to soybean aphid pest management. In autumn, soybean aphids start producing alate females (gynoparae) that search for their overwintering host plants, the common buckthorn, Rhamnus cathartica. The gynoparae then produce pheromone-emitting wingless female offspring (oviparae) that attract male aphids. In this study, we report the chemical identification of the soybean aphid sex pheromone using gas chromatography–electroantennogram, gas chromatography–mass spectrometry, and nuclear magnetic resonance spectroscopy. Behavioral activities of males and gynoparous females in the field were also characterized. The potential applications using formulations containing specific os ybean aphid pheromone compositions for reducing overwintering populations are discussed.

Keywords Entomology, Extension and Outreach, sex pheromone, artificial induction of pheromone-emitting females, (1R, 4aS, 7S, 7aR)-nepetalactol, (4aS, 7S, 7aR)-nepetalactone, field trapping

Disciplines Agricultural Science | Agriculture | Entomology | Horticulture

Comments This article is from Environmental Entomology 35 (2006): 249–257, doi:10.1603/0046-225X-35.2.249. Posted with permission.

Rights This article is the copyright property of the Entomological Society of America and may not be used for any commercial or other private purpose without specific written permission of the Entomological Society of America.

Authors Junwei Zhu, Aijun Zhang, Kye-chung Park, Tom Baker, Brian J. Lang, Russell A. Jurenka, John J. Obrycki, William R. Graves, J. A. Pickett, D. Smiley, Kamlesh R. Chauhan, and Jerome A. Klun

This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/hort_pubs/6 Sex Pheromone of the Soybean Aphid, Aphis glycines Matsumura, and Its Potential Use in Semiochemical-Based Control Author(s): Junwei Zhu, Aijun Zhang, Kye-chung Park, Tom Baker, Brian Lang, Russell Jurenka, John J. Obrycki, William R. Graves, J. A. Pickett, D. Smiley, Kamlesh R. Chauhan, and Jerome A. Klun Source: Environmental Entomology, 35(2):249-257. 2006. Published By: Entomological Society of America DOI: http://dx.doi.org/10.1603/0046-225X-35.2.249 URL: http://www.bioone.org/doi/full/10.1603/0046-225X-35.2.249

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Sex Pheromone of the Soybean Aphid, Aphis glycines Matsumura, and Its Potential Use in Semiochemical-Based Control

JUNWEI ZHU,1, 2 AIJUN ZHANG,4 KYE-CHUNG PARK,3 TOM BAKER,1 BRIAN LANG,5 RUSSELL JURENKA,3 JOHN J. OBRYCKI,3, 6 WILLIAM R. GRAVES,7 J. A. PICKETT,8 D. SMILEY,8 4 4 KAMLESH R. CHAUHAN, AND JEROME A. KLUN

Environ. Entomol. 35(2): 249Ð257 (2006) ABSTRACT The newly invasive soybean aphid, Aphis glycines Matsumura, has seriously threatened soybean production in North America, after having spread to Ͼ20 states in the United States and several southern provinces of Canada. Control of A. glycines has focused on applications of insecticides, which are not a long-term solution to soybean aphid pest management. In autumn, soybean aphids start producing alate females (gynoparae) that search for their overwintering host plants, the common buckthorn, Rhamnus cathartica. The gynoparae then produce pheromone-emitting wingless female offspring (oviparae) that attract male aphids. In this study, we report the chemical identiÞcation of the soybean aphid sex pheromone using gas chromatographyÐelectroantennogram, gas chromatog- raphyÐmass spectrometry, and nuclear magnetic resonance spectroscopy. Behavioral activities of males and gynoparous females in the Þeld were also characterized. The potential applications using formulations containing speciÞc soybean aphid pheromone compositions for reducing overwintering populations are discussed.

KEY WORDS sex pheromone, artiÞcial induction of pheromone-emitting females, (1R,4aS,7S,7aR)- nepetalactol, (4aS,7S,7aR)-nepetalactone, Þeld trapping

THE SOYBEAN APHID, Aphis glycines Matsumura, is a ruses distort soybean growth and further reduce newly invasive species that seriously threatens yields. U.S. soybean production. It is the only aphid species Aphis glycines have a complex life cycle with Ͼ15 that develops large colonies on soybeans, Glycine max, generations per season that feed and reproduce on its in North America (Ragsdale et al. 2004). Since its Þrst secondary plant host, soybeans (Zhang and Zhong appearance in Wisconsin in 2000, it has spread to Ͼ20 1982). In the fall, A. glycines begin producing winged U.S. states and southern provinces of Canada (soybean female aphids (gynoparae) that ßy from soybean aphid watch 2005). Infestations of A. glycines reduce Þelds, searching for their overwintering host plant, plant growth so fewer pods and seeds develop, thereby buckthorns, Rhamnus spp. (primary host). In Asia, the lowering yields (data from Midwest Soybean Aphid most common overwintering hosts are R. davurica and Workshop, 5 February 2004). Soybean aphids also R. japonica (Zhang and Zhong 1983, Takahashi et al. transmit several plant viruses, including alfalfa mosaic, 1993). In North America, several Rhamnus species are soybean mosaic, soybean dwarf, soybean stunt, and used as a primary host (Ragsdale et al. 2004, Voegtlin bean yellow mosaic (van-den-Berg et al. 1997, Clark et al. 2004). Once on overwintering hosts, gynoparae and Perry 2002, Wang and Ghabrial 2002). These vi- produce pheromone-emitting wingless female off- spring (oviparae). Alate males are attracted to ovipa- rae through a speciÞc sex pheromone blend produced from glands on the hind legs of female aphids (Pet- 1 MSTRS Technologies, Inc., 2501 North Loop Dr., Ames, IA 50010. tersson 1970, 1971). The have been re- 2 Corresponding author. e-mail: [email protected]. 3 Department of Entomology, Iowa State University, Ames, IA ported from several other aphid species (Pickett et al. 50011. 1992). After mating, oviparae lay eggs that overwinter 4 USDAÐARS Plant Sciences Institute, Chemicals Affecting Insect on the buckthorn. The generation involving males and Behavior Laboratory, BARC-West, 10300 Baltimore Ave., Beltsville, oviparae occurred in the autumn is the only genera- MD 20705Ð2350. 5 Iowa State University Extension, Decorah, IA 52101. tion that uses sex pheromones for mate Þnding during 6 Department of Entomology, University of Kentucky, Lexington, the entire season. KY 40546. Thus far, pheromones have been identiÞed from 7 Department of Horticulture, Iowa State University, Ames, IA Ͻ15 aphid species worldwide (Dawson et al. 1990, 50011. 8 Biological Chemistry Division, Rothamsted Research, Harpenden, Pickett et al. 1992, Boo et al. 2000, Goldansaz et al. Hertfordshire AL5 2JQ, UK. 2004). All reported aphid pheromone structures are

0046-225X/06/0249Ð0257$04.00/0 ᭧ 2006 Entomological Society of America 250 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 2 comprised of either a mixture of the monoterpenoids with 3 ml of distilled hexane. The hexane eluent was (4aS,7S,7aR)-nepetalactone and (1R,4aS,7S,7aR)-nepe- concentrated to a volume of Ϸ50 ␮l. Pheromones talactol or one of these two compounds alone, except from Ͼ500 laboratory-induced oviparous soybean the damson-hop aphid, Phorodon humili, which uses aphids were collected for further analytical and elec- only (1S and R,4aR,7S,7aS)-nepetalactol as its phero- trophysiological analyses. mone (Birkett and Pickett 2003). The pheromone of To accumulate pure forms of the two soybean aphid two Aphis species, closely related to the soybean pheromone compounds, (4aS,7S,7aR)-nepetalactone aphid, is comprised of different blend ratios of and (1R,4aS,7S,7aR)-nepetalactol, we used a micro- (4aS,7S,7aR)-nepetalactone and (1R,4aS,7S,7aR)-nepe- preparative GC fractionation system previously de- talactol (Campbell et al. 1990, Dawson et al. 1990, scribed in Zhang et al. (2004). A Hewlett-Packard 6890 Pickett et al. 1992). The ratios of these two compounds gas chromatograph combined with a Gerstel prepar- in aphid sex pheromone communication seems ex- ative fraction collector (Gerstel, Baltimore, MD) was tremely important, because speciÞcity in male re- equipped with a 60 m by 0.53-mm ID, 0.50-␮m Þlm sponse relies on the species-speciÞc ratio that occurs thickness DB-1 capillary column (J&W ScientiÞc, in several sympatric species (Hardie et al. 1992, Boo Folsom, CA). Injector temperature was 32ЊC and in- et al. 2000). creased to 230ЊCat60ЊC/min to transfer solute onto This study reports on the chemical identiÞcation of the column; column temperature was held at 50ЊC for the sex pheromone of the soybean aphid and behav- 2 min and then programmed to 250ЊCat30ЊC/min and ioral responses of male and gynoparous soybean held for 10 min. Split ratio of column efßuent to ßame aphids to synthetic pheromones. We also discuss ion detection (FID) and fraction collector was set at their potential uses in pheromone mass trapping and 4:96. The collector was cooled to Ϫ20ЊC by circulating mating disruption for suppressing the overwintering MeOH from a benchtop refrigeration unit (Julabo population, therefore reducing the damage on soy- F25-MP; Julabo USA, Mertztown, PA). The collection beans in the next growing season. efÞciency was Ϸ70%. Electrophysiological and Chemical Analyses. For each collected pheromone sample, 2Ð3 ␮l was ana- Materials and Methods lyzed on both a DB-5 and DB-225 column (30 m by . Soybean aphids collected from soybean 0.25 mm ID; J & W ScientiÞc) in a Hewlett-Packard Þelds (Ames, IA) were raised on potted soybean 5890 Series II gas chromatograph interfaced to a plants. They were initially kept in a growth chamber Hewlett-Packard 5972 Mass Selective Detector (GC- maintained at 25 Ϯ 2ЊC and under L:D, 16:8 h photo- MS). The injector temperature was set at 200ЊC, and period as a stock colony producing only asexual, par- the split valve was opened 1 min after injection. The thenogenetic females. To induce sexual forms of soy- column temperature started at 40ЊC for 1 min after the bean aphids in the laboratory, 10 boxes of 20 winged injection and then linearly increased to 250ЊC at a rate or wingless asexual females were transferred and of 5ЊC/min. Mass spectra were recorded from 30 to reared on individual soybean plants under a short-day 450 a.m.u. after electronic impact ionization at 70 eV. photoperiod (L:D, 10:14 h) and temperature program The chemical structures of the putative components (L:D, 20:12ЊC) that mimics late autumn conditions in were tentatively identiÞed by comparison of retention the upper midwest of the United States. The progeny times and mass spectra with those of authenticated from these aphids was checked every week and seg- chemical standards and reference spectra in a mass regated by age throughout the next 4Ð5 wk until spectral library (Wiley 138K; John Wiley and Sons, winged aphids emerged. After the alate females (gy- New York, NY). noparae) were observed, buckthorn leaves (Rhamnus To ensure that possible additional components carthartica) were provided, and the alate females gave were not missed, we also performed combined GC/ birth to the sexually active oviparae that start produc- EAGs on the airborne-collected extract to determine ing sex pheromone. The later emerging male aphids if other compounds might be present to which male (winged) that were also produced under these con- antennal neurons can detect. For both GC-EAG and ditions were transferred into individual boxes and kept EAG experiments, we adopted an improved aphid for electroantennogram and gas chromatography- EAG technique (Park and Hardie 1998) that includes electroantennogram (EAG and GC-EAD) analyses. the whole intact body of the soybean aphid. This Pheromone Collection and Purification. Air en- technique resulted in a highly improved signal-to- trainment was used for collecting putative sex pher- noise ratio and greater sensitivity. The GC-EAG hard- omone components from oviparae. Thirty mature ware consisted of a Hewlett-Packard 5890 Series II oviparae, maintained on buckthorn leaves with the gas chromatograph equipped with the same columns ends of cut branches held in water-Þlled test tubes, described above. An efßuent split allowed simulta- were placed in a ventilated, 960-ml wide-mouth glass neous ßame ionization (FID) and EAG of pheromone bottle. Activated charcoalÐÞltered air was drawn from components. Helium was used as the carrier gas with the container at a rate of 0.5 liters/min. Volatiles were a ßow rate of Ϸ30 ml/min, and the efßuent split ratio entrained onto an adsorbent collector system com- was maintained at 1:1. Aphid pheromone extracts were prised of a glass tube containing 250 mg of Super Q, injected in splitless mode. The GC temperature pro- 80/100 mesh (Alltech, DeerÞeld, IL). The collection gram was the same as the GC-MS analysis described lasted for 6 h, and volatiles were desorbed by eluting above. The outlet for the EAG was continuously sup- April 2006 ZHU ET AL.: SEX PHEROMONE OF SOYBEAN APHIDS 251 plied with a puriÞed, humidiÞed air stream ßowing over the antennal preparation at a speed of 0.5 m/s. A restrained soybean aphid was mounted on a plastic base using thin copper wire restraints. A capillary recording Ag-AgCl electrode Þlled with saline (0.1 M KCl solution) was inserted into one of the aphidÕs compound eyes and used as the reference (ground) electrode, while the other electrode Þlled with the same solution was inserted into the intersegmental membrane between the third and the fourth antennal segments. The EAG setup consisted of a high-imped- ance DC ampliÞer with automatic baseline drift com- Fig. 1. GC-EAD analyses using a DB-5 column of extracts pensation, and a GC-EAG program (GC/EAG version of soybean aphid sex pheromone entrainment on antennae of 2.4; Syntech, Hilversum, The Netherlands) was used conspeciÞc males. to record and analyze the ampliÞed EAG and FID signals on a PC computer. In the EAG doseÐresponse tests, stimulus cartridges pheromone compounds was measured in soybean consisted of Pasteur pipettes containing a piece of Þelds of 6,000Ð12,000 m2. There were three to Þve Þlter paper (8 by 15 mm) on which a stimulus had replicates in each Þeld, with a total number of 10 been applied. Serial dilutions (0.001Ð10 mg in decadic replicates. Water traps were constructed using yellow steps) of the two aphid pheromone compounds dis- plastic bowls (250 ml; Solo Cup Co., Wheeling, IL) for solved in 10 ␮l of hexane were applied. A control puff collecting aphids. Each trap contained Ϸ100 ml of from a cartridge with just hexane was applied after water and two drops of odorless detergent. Traps each puff of a tested stimulus. The sequence of expo- were set 1 m above the ground. The two soybean sure to the stimulus compound on each antenna pro- aphid pheromone components were protected from ceeded from the lowest to the highest concentration. UV degradation by using brown borosilicate vials Chemicals. The two aphid pheromone compounds, (Chromacol, Trumbull, CT). Pheromones were emit- (4aS,7S,7aR)-nepetalactone and (1R,4aS,7S,7aR)-nepe- ted through a 1-mm-diameter hole drilled in the plas- talactol, were synthesized at Rothamsted Research tic cap of the vial. The lures were suspended centrally Laboratory, UK, and USDA-ARS Beltsville Labora- 3Ð4 cm above the water level. Traps were deployed on tory. The purity of these two compounds was Ϸ99 and the Þeld edges and inside soybean Þelds. Trap posi- 93% as analyzed by GC-MS, respectively. tions were spaced 10 m apart. Traps were checked Nuclear Magnetic Resonance Spectroscopy. The ex- three times a week, and within each replicate, trap tracts of several airborne collections were combined, position was randomized to minimize the effects of and the two pheromone candidate compounds were habitat heterogeneity. The captured aphids from each further puriÞed using a micro-preparative GC frac- trap were transferred to petri dishes and brought back tionation system for stereochemistry determination to the laboratory for counting and species identiÞca- before nuclear magnetic resonance (NMR) analyses. tion.

NMR spectra were recorded in a C6D6 solution on a Soybean aphids caught in the pheromone traps Bruker QE Plus spectrometer at 300 MHz for 1H and were identiÞed based on characters (caudal setae and 75 MHz for 13C. The chemical shifts are expressed in sensoria on the antennae) of specimens (preserved in ppm (␦ scale) relative to the residual solvent for 1H 90% alcohol) under a Olympus dissecting microscope ␦ 13 (C6H6 at 7.20) or to the central peak of solvent C (Olympus, Melville, NY), using keys published in signal (C6D6 at 128.5). (4aS,7S,7aR)-Nepetalactone: Zhang and Zhong (1983), Takahashi et al. (1993), and 1 ␦ 3 H NMR (C6D6): 0.69Ð0.80 (2H, m), 1.03 ( H, d, Voegtlin et al. (2004). Although other aphid species ϭ 3 J 6.44 Hz, CHCH3), 1.14 ( H, br s, CH3), 1.51Ð1.40 were caught in the pheromone traps, their identities (2H, m), 2.01Ð2.05 (2H, m), 2.08Ð2.21 (1H, m), 5.82 were not conÞrmed because of the complexity of ϭ 13 ␦ (1H, br m, CH); C NMR (C6D6): 169.45, 134.12, aphid species identiÞcation. 114.68, 49.39, 40.76, 39.70, 33.05, 30.95, 20.33, 15.30. Statistical Analysis. Unless otherwise mentioned, 1 ␦ (1R,4aS,7S,7aR)-nepetalactol: H NMR (C6D6): aphid captures (means) were compared by one-way 3 ϭ 0.75Ð0.92 (1H, m), 0.95 ( H, d, J 6.44 Hz, CHCH3), analysis of variance (ANOVA) followed by FisherÕs 3 1.05 (1H, m), 1.41 ( H, br s, CH3), 1.46Ð1.55 (1H, m), PLSD test (SPSS 10.0 for Windows) for signiÞcance at 1.60Ð1.75 (2H, m), 1.80Ð1.90 (1H, m), 2.09 (1H, OH, ␣ ϭ 0.05. d, J ϭ 5.91 Hz), 2.23 (1H, q, J ϭ 8.05 Hz), 4.61 (1H, t, J ϭ 5.91 Hz, O-CH-O), 6.07 (1H, br s, ϭ CH); 13C NMR ␦ Results (C6D6): 135.18, 113.21, 94.94, 50.73, 39.48, 36.15, 33.63, 31.24, 20.83, 16.42. Soybean Aphid Pheromone Identification and Elec- Field Trapping Test. In September and October of trophysiology. GC/EAG analyses of extracts from the the 2001 and 2002 seasons, Þeld trapping tests were airborne collection of calling oviparae on male anten- conducted in three soybean Þelds (Iowa State Uni- nae of soybean aphids revealed two EAG active peaks versity Farms, Ames, IA). The ability to trap males (Fig. 1). The GC-MS analyses of the same extract using either single or different ratio blends of aphid determined the mass spectra of peak A: 168 (81), 135 252 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 2

Table 1. Comparisons of pheromone titers and blend ratios between oviparae of laboratory-induced and field-collected soybean aphids (SBA)

Pheromone titers (ng/h/ovipara Ϯ SD) Pheromone ratios (% Ϯ SD) SBA sources Nepetalactone Nepetalactone Nepetalactol Nepetalactone Laboratory-induced 0.38 Ϯ 0.13 0.82 Ϯ 0.27 32 Ϯ 0.7 68 Ϯ 0.6 Field-collected 0.59 Ϯ 0.03 1.14 Ϯ 0.06 34 Ϯ 0.2 66 Ϯ 0.2

Data based on analyses of extracts of pheromone-producing oviparae from laboratory-induced colony or collected from their winter host plants, buckthorn (Rhamnus cathartica), in Ames, IA. For laboratory-induced colony, pheromones from a total of 106 calling females in three batches were analyzed. Approximately 60 Þeld-collected calling females in two batches were extracted. Student t-test, N ϭ 2Ð3, pheromone titers: nepetalactone, t ϭ 0.93, P Ͼ 0.05; nepetalactol, t ϭ 1.27, P Ͼ 0.05, pheromone ratios: nepetalactone and nepetalactol, t ϭ 1.99, P Ͼ 0.05.

(94), 97 (82), 84 (79), 81 (56), 71 (88), 67 (52), 58 Behavioral Responses of Soybean Aphids to Syn- (59), 55 (66), 43 (77), 41 (100). The GC-MS analyses thetic Pheromone Compounds. Field tests using of the same extract determined the mass spectra of traps baited with 10 mg of the two synthetic aphid peak B: 166 (78), 151 (8), 138 (17), 123 (83), 109 (51), pheromone compounds showed that a blend contain- 95 (81), 81 (100), 69 (95), 67 (66), 55 (39), 41 (72), 39 ing (1R,4aS,7S,7aR)-nepetalactol and (4aS,7S,7aR)- (65). Compound B had the same retention time and nepetalactone at a ratio of 35:65 caught the highest identical mass spectrum as one of the monoterpenes, numbers of males and gynoparous females compared (4aS,7S,7aR)-nepetalactone, identiÞed from the cat- with single compounds (Fig. 3). Traps with either nip plant, Nepata cataria (Peterson et al. 2002). The (1R,4aS,7S,7aR)-nepetalactol or (4aS,7S,7aR)-nepeta- mass spectrum of compound A with Mr 168 was similar lactone alone captured more males and gynoparaous to that of (1R,4aS,7S,7aR)-nepetalactol, which has females than those of the control. The order of attrac- been reported as a pheromone component used by tiveness for the three treatments for both males and several aphid species (Dawson et al. 1990). gynoparae was as follows: blend Ͼ (1R,4aS,7S,7aR)- The 1H NMR data for peak B were in good agree- nepetalactol Ͼ (4aS,7S,7aR)-nepetalactone. ment with those of the natural (4aS,7S,7aR)-nepeta- DoseÐresponse tests conducted in the same Þeld in lactone isolated from catnip oil. The structure of 2002 showed that traps with a dose of 30 mg soybean (1R,4aS,7S,7aR)-nepetalactol was also conÞrmed by aphid pheromone blend caught the highest numbers 1 H NMR (C6D6) analysis: characteristic peaks at 4.61 of both males and gynoparae (Fig. 4). We also tested ppm (1H, t, J ϭ 5.91 Hz, O-CH-O) and 2.09 (1H, OH, effects of different amounts of (1R,4aS,7S,7aR)- ϭ d, J 5.91 Hz). The 5.91-Hz coupling between H1 and nepetalactol added to the nepetalactone on their at- OH in the 1H NMR spectrum is characteristic of tractiveness to males and gynoparae. At least 10% (1R,4aS,7S,7aR)-nepetalactol based on an organic syn- (1R,4aS,7S,7aR)-nepetalactol in the two-component thetic product obtained from (2S,9S,)-5,9-dimethyl- blend was essential to attract males (Fig. 5). Gyno- 2(N-methylphenylamino)-3-oxabicyclo [4,3,0]-4-none parae responded equally well to traps baited with (Dawson et al. 1996). Based on the results from pheromone lures containing no (1R,4aS,7S,7aR)- GC-MS and NMR analyses, we concluded that the nepetalactol as they did to those with no (4aS,7S,7aR)- chemical structures of the soybean aphid pheromone nepetalactone. components are (1R,4aS,7S,7aR)-nepetalactol and EAG Responses of Male Soybean Aphids Pre-Ex- (4aS,7S,7aR)-nepetalactone. posed to Pheromone. EAG responses were measured We also compared pheromone titers and ratios from antennae of male soybean aphids after they were emitted between oviparae induced in the laboratory pre-exposed to a dispenser containing 5 g pheromone artiÞcially and those collected from buckthorn shrubs for 10 min. SigniÞcantly lower EAG responses were in the Þeld during the fall. The results showed that elicited from antennae of the pre-exposed soybean there were no differences in the amounts of the two aphids than to those naive males (Fig. 6). pheromone compounds produced by Þeld-collected individuals compared with those from our laboratory Discussion colony; nor were there any differences in the blend ratios emitted (Table 1). Oviparae of soybean aphids produce both (1R,4aS, The EAG analyses of both males and gynoparae 7S,7aR)-nepetalactol and (4aS,7S,7aR)-nepetalactone, revealed that olfactory receptors on their antennae which are the two most common aphid phero- were responding to the identiÞed pheromone com- mone compounds identiÞed from a number of other pounds (Fig. 2). Male antennae (Fig. 2, top) were aphid species (Dawson et al. 1990, Pickett et al. 1992, highly sensitive to the two pheromone compounds at Boo et al. 2000, Goldansaz et al. 2004). The ratio of relatively low doses, with the response decreasing these two pheromone compounds from A. glycines signiÞcantly when the dose exceeded 0.1 mg. In con- differs from those previously reported, which sup- trast, gynoparae (Fig. 2, bottom) showed a higher ports the hypothesis of species speciÞcity in aphid pher- EAG response when pheromone exceeded a 0.1-mg omone systems (Pickett et al. 1992, Guldemond et al. dose, particularly in response to (4aS,7S,7aR)-nepeta- 1993). The absolute stereochemistry of (1R,4aS,7S,7aR)- lactone. nepetalactol has been elucidated, but only with the use April 2006 ZHU ET AL.: SEX PHEROMONE OF SOYBEAN APHIDS 253

Fig. 2. EAG doseÐresponse curves of the two identiÞed soybean aphid pheromone compounds from both males and gynoparous females. Means with the same letter for a given compound are not signiÞcantly different (N ϭ 3Ð5; gynoparae to nepetalactol: F ϭ 0.95; df ϭ 4,20; P Ͼ 0.05; gynoparae to nepetalactone: F ϭ 6.42; df ϭ 5,24; P Ͻ 0.001; males to nepetalactol: F ϭ 8.99; df ϭ 5,12; P Ͻ 0.001; males to nepetalactone: F ϭ 2.19; df ϭ 5,12; P Ͼ 0.05).

of derivatives from synthetic preparations. This study One of the biggest obstacles for research on aphid reports the Þrst attempt to determine its stereochemistry pheromones is the difÞculties in locating pheromone- through NMR analyses on naturally collected phero- producing oviparae. The alternative to Þeld collection mone compounds. Although the stereochemistry of is to induce pheromone-producing females under au-

(4aS,7S,7aR)-nepetalactol at C1 position could not be tumnal conditions, such as lower temperatures and established based on the conÞguration of the natural shortened day lengths in the laboratory. In this study, pheromone, we propose that it is R, based on the positive we documented similarities in pheromone titers and Þeld-trapping results. ratios produced by Þeld-collected and laboratory-in- 254 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 2

Fig. 3. Total number of male and gynoparous soybean aphids caught in traps with different combinations of identiÞed sex pheromone compounds in 2001. Means with different letters on top of the bars indicate signiÞcant differences. (N ϭ 10; for gynoparae: F ϭ 75.42; df ϭ 3,20; P Ͻ 0.001; for males, F ϭ 99.04; df ϭ 3,36; P Ͻ 0.001). duced oviparae. Our results show the reliability of of this compound in mate Þnding. The signiÞcant EAG using artiÞcially induced oviparae for aphid phero- and behavioral responses of gynoparous females to mone characterization. Similarly, Goldansaz et al. (4aS,7S,7aR)-nepetalactone suggest that this com- (2004) used laboratory-induced oviparae to identify pound may be used as a chemical cue to locate over- pheromone components of the potato aphid. wintering host plants. This function has also been The review of the aphid sensory system by Ander- observed in several other aphid species (Campbell et son and Bromley (1987) suggests that the greater al. 1990, Hardie et al. 1992, 1996). Furthermore, we abundance of secondary rhinaria on the antennae of found that the volatile emission of buckthorn leaves alate morphs indicates their involvement in host lo- signiÞcantly decreased while oviparae were feeding cation and mate selection. Du et al. (1995) reported and producing pheromone compounds (J.Z., unpub- that the antennae of alate soybean aphids contain lished data). Further studies will determine if this is a placoid sensilla on their secondary rhinaria. Our EAG form of host plant defense by not emitting signature tests showed that the antennae of both male and gy- volatiles to prevent further damage caused by gyno- noparaous soybean aphids posses receptor neurons parae. responding to the two identiÞed pheromone com- The Þeld trapping of both male and gynoparous fe- pounds. The fact that male antennae are more speciÞc male soybean aphids with various ratios of (1R,4aS,7S, to (1R,4aS,7S,7aR)-nepetalactol indicates a function 7aR)-nepetalactol and (4aS,7S,7aR)-nepetalactone has

Fig. 4. Mean number of male and gynoparae soybean aphids caught in traps with different doses of the most attractive pheromone blend in 2002. Means with different letters on top of the bars indicate signiÞcant differences (N ϭ 10; for gynoparae: F ϭ 34.14; df ϭ 5,54; P Ͻ 0.001; for males: F ϭ 27.57; df ϭ 4,45; P Ͻ 0.001). April 2006 ZHU ET AL.: SEX PHEROMONE OF SOYBEAN APHIDS 255

Fig. 5. Mean number of male and gynoparae soybean aphids caught in traps baited with different ratios of the identiÞed sex pheromone compounds at a dose of 10 mg in 2002. Means with different letters on top of the bars indicate signiÞcant differences (N ϭ 10; for gynoparae: F ϭ 11.47; df ϭ 5,54; P Ͻ 0.001; for males: F ϭ 47.95; df ϭ 5,54; P Ͻ 0.001). shown the effectiveness of the nepetalactol to male at- shop, 5 February 2004). However, the pattern of trap traction. The attraction of both the alate male and gy- captures was consistent during the 2-yr Þeld-trapping noparous female forms of aphids has also been re- experiments. ported for the damson-hop aphid, Phorodon humuli The identiÞcation of sex pheromones of the soy- (Hardie et al. 1996, Lo¨sel et al. 1996a, b). The speciÞc bean aphid provides us with a unique opportunity to blend of a 35:65 ratio of these two components in this explore the possibility of suppressing soybean aphid study represents the actual emitted pheromone ratio populations using pheromone-based strategies. For from calling oviparae. The ratio was the most attractive aphids, possible immigration of gravid females into blend in 2 yr of Þeld trials. The relatively lower numbers the mating disruption area is negligible, because of A. glycines caught in pheromone traps in 2002 were oviparae are wingless. Mating disruption should be caused by the sparse occurrence of soybean aphids dur- particularly effective against such insects. The sup- ing that year (data from Midwest Soybean Aphid Work- pressive effects should be easily discerned in small

Fig. 6. Comparisons of absolute EAG responses of male soybean aphids pre-exposed with higher dosages of aphid pheromones to those without pre-exposure. Means with different letters on top of the bars indicate signiÞcant differences (Student t-test, N ϭ 12; for nepetalactone: t ϭ 5.08, P Ͻ 0.001; for nepetalactol: t ϭ 3.54, P Ͻ 0.005; for blank: t ϭ 1.18, P Ͼ 0.05). 256 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 2 plots. Furthermore, we also showed the sensory ad- Goldansaz, S. H., S. Dewhirst, M. A. Birkett, A. M. Hooper, aptation of soybean aphid antennal responses to the D.W.M. Smiley, J. A. Pickett, L. Wadhams, and two pheromone compounds. J. N. McNeil. 2004. IdentiÞcation of two sex pheromone Insect suppression by the use of mass trapping components of the potato aphid, Macrosiphum euphorbiae through combinations of sex pheromones and host (Thomas). J. Chem. Ecol. 30: 819Ð834. plant volatiles has shown increased success in con- Guldemond, J. A., A.F.G. Dixon, J. A. Pickett, L. J. Wadhams, and C. M. Woodcock. 1993. SpeciÞcity of sex phero- trolling major agricultural pests (Kobayashi et al. 1981, mones, the role of host plant odor in the olfactory at- Smit et al. 2001, Baker and Heath 2004). Mass trapping traction of males, and mate recognition in the aphid of the autumnal generation soybean aphids (including Cryptomyzus. Physiol. Entomol. 18: 137Ð143. gynoparae as they seek to Þnd winter host plants and Hardie, J., S. F. Nottingham, G. W. Dawson, R. Harrington, males ßying from soybean Þelds to locate oviparae) J. A. Pickett, and L. J. Wadhams. 1992. Attraction of may be a useful control strategy using this newly Þeld-ßying aphid males to synthetic sex pheromone. identiÞed sex pheromone. The subsequent yearÕs pop- Chemoecology 3: 113Ð17. ulation may be reduced by interfering with the pre- Hardie, J., J. R. Storer, F. J. Cook, C.A.M. Campbell, ceding fall aphid generationÕs ability to locate mates or L. J. Wadhams, R. Lilley, and L. Peace. 1996. Sex pher- buckthorn plants. Although the mass-trapping tech- omone and visual trap interactions in mate location strat- egies and aggregation by host-alternating aphids in the nique against the soybean aphid may be more labo- Þeld. Physiol. Entomol. 21: 97Ð106. rious than mating disruption, further research may Kobayashi, M., T. Wada, and H. Inoue. 1981. A comparison prove its effectiveness. of communication disruption technique and mass-trap- ping technique for controlling moths using sex phero- mone of Spodoptera litura (F.) (Lepidoptera: Noctu- Acknowledgments idae). Proc. Japan/USA Symp. On IPM., 32Ð40. Lo¨sel, P. M., M. Lindemann, J. Scherkenbeck, J. Maier, This project was funded by National Science Founda- B. Engelhard, C.A.M. Campbell, J. Hardie, J. A. Pickett, tion-SBIR Program 0450032 to J.Z. Technical support from L. J. Wadhams, A. Elbert, and G. Thielking. 1996a. The J. Jones, J. Heath, and B. Ellingson is appreciated. We also potential of for control of Phorodon hu- appreciate the support from the ISU-IPRT. muli (Homoptera: Aphididae). Pest. Sci. 48: 293Ð303. Lo¨sel, P. M., M. Lindemann, J. Scherkenbeck, C.A.M. Camp- bell, J. Hardie, J. A. Pickett, and L. J. Wadhams. 1996b. References Cited Effect of primary-host kairomones on the attractiveness Anderson, M., and A. K. Bromley. 1987. Sensory system, of the hop-aphid sex pheromone to Phorodon humuli pp. 153Ð162. In A. K. Minks and P. Harrewijn (eds.), males and gynoparae. Entomol. Exp. Appl. 80: 79Ð82. Aphids: their biology, natural enemies, and control. Midwest Soybean aphid Workshop, February 5, 2004. http:// _ Elsevier, Amsterdam, The Netherlands. www.ipm.uiuc.edu/Þeldcrops/insects/soybean aphids/ Baker, T. C., and J. J. Heath. 2004. PheromonesÑfunction workshop/index.html. and use in insect control, pp. 407Ð460. In L. I. Gilbert, Park, K. C., and J. Hardie. 1998. An improved aphid elec- K. Iatro, and S. Gill (eds.), Comprehensive insect phys- troantennogram. J. Insect Physiol. 44: 919Ð928. iology, biochemistry, pharmacology and molecular biol- Peterson, C. J., L. T. Nemetz, L. M. Jones, J. R. Coats. 2002. ogy. Elsevier, Amsterdam, The Netherlands. Behavioral activity of catnip (Lamiaceae) essential oil Birkett, M. A., and J. A. Pickett. 2003. Aphid sex phero- components to the German cockroach (Blattodea: Blat- mones: from discovery to commercial production. Phy- tellidae). J. Econ. Entomol. 95: 377Ð380. tochemistry 62: 651Ð656. Pettersson, J. 1970. An aphid sex attractant. I. Biological Boo, K. S., M. Y. Choi, I. B. Chung, V. E. Eastop, J. A. Pickett, studies. Entomol. Scand. 1: 63Ð73. L. J. Wadhams, and C. M. Woodcock. 2000. Sex phero- Pettersson, J. 1971. An aphid sex attractant. II. Histological, mone of the peach aphid, Tuberocephalus momonis, and ethological and comparative studies. Entomol. Scand. 2: optimal blends for trapping males and females in the Þeld. 81Ð93. J. Chem. Ecol. 26: 601Ð609. Pickett, J. A., L. J. Wadhams, C. M. Woodcock, and J. Hardie. Campbell, C.A.M., G. W. Dawson, D. C. Griffiths, J. Petters- 1992. The chemical ecology of aphids. Annu. Rev. son, J. A. Pickett, L. J. Wadhams, and C. M. Woodcock. Entomol. 37: 67Ð90. 1990. Sex attractant pheromone of damson-hop aphid Ragsdale, D. W., D. J. Voegtlin, and R. J. O’Neil.. 2004. Phorodon humuli (Homoptera, Aphididae). J. Chem. Soybean aphid biology in North America. Ann. Entomol. Ecol. 16: 455Ð465. Soc. Am. 97: 204Ð208. Clark, A. J., and K. L. Perry. 2002. Transmissibility of Þeld Smit, N.E.J.M., M.C.A. Downham, P. O. Laboke, D. R. Hall, isolates of soybean viruses by Aphis glycines. Plant Dis. 86: and B. Odongo. 2001. Mass-trapping male Cylas spp. 1219Ð1222. with sex pheromones: a potential IPM component in Dawson, G. W., D. C. Griffiths, L. A. Merritt, A. Mudd, sweetpotato production in Uganda. Crop Protect. 20: 643Ð J. A. Pickett, L. J. Wadhams, and C. M. Woodcock. 1990. 651. Aphid semiochemicals - a review, and recent advances on Soybean aphid watch 2005. http://www.ncpmc.org/alerts/ the sex pheromone. J. Chem. Ecol. 16: 3019Ð3030. soybeanaphid.cfm. Dawson, G. W., J. A. Pickett, and D.W.M. Smiley. 1996. The Takahashi, S., M., Inaizumi, and K. Kawakami. 1993. Life aphid sex pheromone cyclopentanoids: synthesis in the cycle of the soybean aphid Aphis glycines Matsumura, in elucidation of structure and biosynthetic pathway. Japan. Jap. J. Appl. Entomol. Zool. 37: 207Ð212. Bioorg. Med. Chem. 4: 351Ð361. van-den-Berg, H., D. Ankasah, and A. Muhammad. 1997. Du, Y.-J., F.-S. Yan, X.-L. Han, and G.-X. Zhang. 1995. Ol- Evaluating the role of predation in population ßuctua- faction in host selection of the soybean aphid Aphis tions of the soybean aphid Aphis glycines in farmersÕ Þelds Glycine. Acta Entomol. Sinica. 37: 385Ð391. in Indonesia. J. Appl. Ecol. 34: 971Ð984. April 2006 ZHU ET AL.: SEX PHEROMONE OF SOYBEAN APHIDS 257

Voegtlin, D. J., S. E. Halbert, and G. Qiao. 2004. A guide to mealybug, Maconellicoccus hirsutus, contains an unusual separating Aphis glycines Matsmura and morphologically cyclobutanoid monoterpene. Proc. Natl. Acad. Sci. U.S.A. similar species that share its hosts. Ann. Entomol. Soc. 101: 9601Ð9606. Am. 97: 227Ð232. Zhang, G. X., and T. S. Zhong. 1982. Experimental studies on Wang, R. Y., and S. A. Ghabrial. 2002. Effect of aphid be- some aphid-cycle patterns. Sinozoology 2: 7Ð17. havior on efÞciency of transmission of Soybean mosaic Zhang, G. X., and T. S. Zhong. 1983. Economic Insect Fauna virus by the soybean-colonizing aphid, Aphis glycines. of China, Fasc. 25: Homoptera: Aphidinea, part 1. Science Plant Dis. 86: 1260Ð1264. Press, Beijing, China. Zhang, A., D. Amalin, S. Shirali, M. S. Serrano, R. A. Franqui, J. E. Oliver, J. A. Klun, J. R. Aldrich, D. E. Meyerdirk, and Received for publication 23 February 2005; accepted 3 Jan- S. L. Lapointe. 2004. Sex pheromone of the pink hibiscus uary 2006.