Journal alChemical Ecology. Vol. 13, No.4. 1987 6174 *

SEMIOCHEMICAL ATTRACTANTS OF Diabrorica undecimpuncrara hOlVardi BARBER, SOUTHERN CORN ROOTWORM, AND Diabrorica virgijera virgijera LECONTE, THE WESTERN CORN ROOTWORM (COLEOPTERA: CHRYSOMELIDAE)

R.L. LAMPMAN,' R.L. METCALF,' and J.F. ANDERSEN1

I D~partmem 01 Entomology Unh'ersiry ofIllinois Urbana-Champaign. Illinois 61801 2USDA-ARS North Regional Research Center Pt!oria. Illinois. 61604

(Received April 22, 1986; accepted June 9. 1986)

Abstract-DUring the summers of 1984 and {98S. a variety of structurally related benzenoid compounds was evaJuated in sweet com plms as anractams for adult southern com rootworms (SCR), western com rootworms (WCR), and nonhem com rootwonns (NCR). Field response to the volatiles was mea· sured by beetle COUntS on baited cylindrical sticky traps placed inside the corn plots al a height of 1 m above ground level. SCR adults were auracled late in the season (last week of August through September. 1984 and 1985) to numerous aromatic compounds. inclUding phenylacetaldehyde. benzyl ace~ tone. phenethyJ alcohol. phenyl acetate. indole. veratrole. methyl eugenol. methyl isoeugenol. eugenol. and isoeugenol. Although many compounds at­ tracted SCR adults late in the season. only verauule. phenylacetaldehyde. and chavicol were significantly active in early and middle August 1985. WCR adul[S were attracted to a different group of compounds. namely estragole. trans-anethole. and indole. Estragole (4-methoxy-l-ailylbenzene) was an ef­ fective WCR attractant from com tasseling in earJy AugUSt 1985. until the end of the trapping period in late September and early October 1985. Indole and trans-anethole (4-methox;y-/-propenylbenzene) were less effective anrac~ tams than estragole and were most active at the beginning and/or end of the com season. Traps baited with 100 mg of estragole caught an average of 20 times more WCR adul[S [han unbaited control traps. and the females outnum­ bered the maJes in the baited lraps. Estragole dosage tests were conducted in three sweel com plots on different dates in 1985 and Ihe minimum effective dose ranged between 5 and 30 mgltrap. Field tests with stroctural anaJogs reveaJed the impormnce of the site of unsaturation in the ally lie side chain of

959

(l()q1l·onI/1l7'0J11O·oq~qSO~oom .c. 1987 Plenum Publlshln8 COrpl,lr.lUon 960 LAMPMAN ET AL.

esuagole and the effect of different ring substimcnts on WCR response. The phenylpropanoids. eugenol and isoeugenol. significantly attracted NCR adults. even though these beetles were in low abundance in the test com plots. Field tests indicate there IS no cross-species response by WCR and NCR adults to their related phenylpropanoid attractants. However. in late August. SCR adults do respond to some WCR and NCR attractants (indole and several eugenol analogs). Electroantennographic analysis of SeR males reve:lIed they can perceive peripherally a wide range of benzcnoid com­ pounds.

Key Words-Diabrorica. Coleoptera. ChrysomeJidat:. western com root­ wann, southern com roorwonn. nonhem com rootwonn. attractants. semi­ ochemicals. vcrturcle. esrragole. frans-anethole. indole. eugenol. phenyl~ acetaldehyde.

INTRODUCTION

Larvae and adults of the northern com rootworm (NCR). Diabrorica barben Smith and Lawrence, and the western com rootWorm (WCR). D. virgifera vir­ gifera LeConte, are serious root and silk pests of field and sweet com in the com belt. The univoltine NCR and WCR overwinter as diapausing eggs in North America and the oligophagous larvae develop only on the roots of grasses (Po­ aceae), primarily com (Branson and Krysan, 1981: Branson and Ortman. 1967, 1971; Smith. 1966). In contrast to these two Diabrotica species. the multivol­ tine southern com rootworm (SCR) = spotted cucumber beetle. D. undecim­ punctata howardi Barber. overwinters in southern areas as an adult. annually reinvades the Midwest. and both larvae and adults are polyphagous (Smith. 1966: Branson et al., 1978). SCR bettles are pests of cucurbits. peanuts. com. and a variery of vegetable and field crops (Campbell and Emery. (967). Although the New World diabroticites (Chrysomelidae: Galerucinae: Lu­ perini: Diabroticina) have greatly expanded their host distribution and range from their origins. they apparently retain an ancestral compulsive feeding re­ sponse to the bitter-tasting cucurbiracins found in the cotyledons. foliage. fruit. and flowers of many species of the Cucurbiraceae (Chambliss and Jones. 1966: Sharma and Hall. 1973: Howe and Rhodes. 1976: Metcalf et al.. 1982: Fer­ guson et aJ.• 1983; Andersen. 1984). Cucurbiracins are undoubtedly potent kai­ romones for the diabroticites. as well as potent allomones for many other her­ bivores and for some diabroticite predators (Metcalf, 1979; Tallamy. 1985: Ferguson and Metcalf. 1985). yet their high molecular weights and low vapor pressures make them ineffective as volatile semiochemicals (Branson and Guss. 1983). Considerable progress has been made recently in understanding Diabrorica response to volatile attractants. The sex pheromone of WCR virgin females. 8­ methyl-2-decyl propanoate. attracts both WCR and NCR males; however, the ROOTWORM AlTRACTANTS 961 two species demonstrate temporal differences in response and high doses of the racemic synthetic mixture inhibits NCR response (Guss et al., 1982. (985). SCR females release lO-methyl-2-uidecanone and the R enantiomer is the most active anractant for male SCR and D. u. undecimpunctata. the western sponed cucumber beetle (Guss et a1., 1983). Furthermore. the nonpheromonal phen­ ylpropanoids eugenol. isoeugenol. and 2-methoxy-4-propylphenol strongly at­ tract NCR adults, whereas WCR adults do not respond to these compounds (Ladd et al.• 1983; Ladd. 1984). Andersen and Metcalf (1986) isolated the shikimic acid derivative indole from the male blossoms of Cucurbita maxima cultivars. where Diabrotlca and Acalymma adults often congregate in large numbers. and found adult WCR and striped cucumber beetles. Acalymma vlt­ tarum (Fabr.), are anracted early and late in the season to as linle as 5 mg of indole. Although indole is clearly a kairomonal attractant of WCR adults. a host-plant relationship has not been established in the literature for the attrac- tants of NCR adults (Ladd. 1984). . During the summers of 1984 and 1985. we investigated the response of WCR. SCR, and NCR adults to a wide variety of benzenoid compounds using baited. one-quart (0.95-liter) white cylindrical sticky traps in sweet com plots. Chemical analogs of anractive compounds were used to determine the specific­ ity of response by the com rootworm species. This report summarizes the data obtained with more than 20 aromatic compounds. many of which have been detected in com orcucurbits (Bunety et al.. 1978. 1980. 1982; Andersen. 1984). A preliminaty electroantennographic (EAG) study with SCR males evaluates their response to several benzenoid compounds.

METHODS AND MATERIALS

Field Tests. During the summers of 1984 and 1985. various volatile com­ pounds were evaluated for com lOotworm attraction on the South Farms of the University of Illinois in Urbana. Illinois. In 1984. tests were conducted in a single sweet com plot which measured 52 x 22 m with eight lOWs each of cultivars. "Florida Stay Sweet" and "Gold Cup." The com plot was bordered on one side by a grass lane and on the other side by a cucurbit plot. The adjacent cucurbit plot included several common squash varieties (predominantly Cucur­ blta maxima cv. "Blue Hubbard"). as well as high cucurbitacin-containing spe­ cies and hybrids (Cucurbita andreana, C. texana. and C. andereana x C. max­ Ima hybrids) (Rhodes et al .. 1980). Large numbers of SCR adults (> 20,000 beetles) aggregated on the leaves. blossoms. and fruits of the bitter cucurbits and "Blue Hubbard" cultivar in 1984 and 1985. In both years. SCR adults were ca. 10-20 times more abundant than WCR and A. vltratum adults during the test period. The experimental design for the main plot used in 1985 was essentially the same as in 1984. except the com was a monoculture of 20 rows 962 LAMPMAN ET AL. of cultivar "lllini Xtra Sweet." Tests were also conducted in 1985 in two non­ adjacent sweet com plots located approximately 150 m and 250 m from the main plot. These auxilliary plots were not adjacent to any cucurbits and WCR adults were the predominant rootworm species present. The 1984 test plot was planted on June 12 and initial tests were conducted in late August after tasseling. Afterthe first frost (last week ofSeptember 1984). field tests were discontinued. In 1985. trapping began in mid-July. before tas­ seling. and continued until the com foliage was totally dry in late September. The two auxiliary sweet com plots were used to test WCR attractants and were planted approximately tWO and four weeks. respectively. after the main plot (June 6. 1985) and were used until the first week of October. Pretest whole­ plant counts were taken every other week in the main plot and once in the two auxiliary plots by counting beetles in the rows used for testing treatments. All compounds tested. except two. were from commercial sources and ver­ ified as being :,,95% pure by gas chromatographic analysis. Chavicol (4-hy­ droxy-I-allylbenzene) was prepared (R.L.M.) by the method of Buu-Hoi et al. (1954) and 4-methoxy-l-propylbenzene by reacting the sodium salt of 4-pro­ pylphenol with dimethyl sulfate. The products were glass distilled and purity and identiry confirmed by boiling points. infrared analysis. and gas chromatog­ raphy-mass spectrometry. Compounds were field tested for attraction of roOt­ worms by treating dental cotton wicks with 200 mg or less of each compound (see Tables). In order to prolong volatilization of the treatment compound. the wicks (ca. 15 mm long x 6 mm diameter) were soaked in polyethylene glycol and the excess squeezed out prior to treatment with each test compound. The treated wicks were attached to the tops of one-quart (0.95-liter) white paper caftons. similar to those described by Hein and Tollefson (1984). The paper cartons were 15.2 cm high and had a circumference of 27.9 cm and were evenly coated with clear Tangletrap'" in the field. The baited sticky traps were placed in the com plots in the early afternoon (1-4 PM) and beetle counts were taken periodically for one to three days. In 1984, the traps were placed ca. I m high by breaking off the com stalk at ear level and placing the hollow carton trap on top of the stalk. In 1985. the traps were placed in com rows on I m-high wood stakes. In both years. the baited traps were arranged in a randomized complete block design with treat­ ments 3 to 5 m apart and blocks 2 to 3 m apart. depending on the number of compounds and replicates in a test. Results were analyzed by an ANOVA pro­ gram and. in the case of multiple comparisons. means were separated by Dun­ can's mUltiple-range test with significance levels set at P = 0.05 (Nie et aJ.. 1975). Electroantennographic Analysis. Several SCR attractants with variable field activity were selected for EAG analysis (see Table 4 for a list of compounds). Antennae of male SCR adults were excised at the head following removal of the distal segment. The antennae were placed between twO glass capillaries ROOTWORM AlIRACTANTS 963 filled with 0.9% NaC! solution. Silver-silver chloride wires were used as elec­ trodes and connected by shielded leads to a Grass P-18 preamplifier. Sample compounds were dissolved in paraffin oil (Fisher) at 100- and 1000-fold dilu­ tions. and filter paper triangles were impregnated with 5 iLl of each solution. The treated triangles were then inserted into Pasteur pipets and a glass syringe used to pass 0.4 ml of air through the pipet into a charcoal-filtered airstream (100 ml of air per minute) which flowed over the beetle antennae. Since EAG response diminishes through time. the response of the antenna to each treatment is expressed as a percent relative response in comparison to a standard com­ pound (cis-3-hexen-I-ol) at a I : 100 dilution in paraffin oil (similar to that used by Visser. 1979). Six replicates were conducted for each compound at both dilutions. and the laboratory-reared beetles ranged in age from 2 to 4 weeks postemergence. Each treatment was compared to the control by a single-cIa:;­ sification ANOVA (Sokal and Rohlf. (969).

RESULTS

SCR Attractants. [n the first field test. August 28. 1984. the mean 24-hr trap catches (± standard error) for phenylacetaldehyde (PAA). indole. eugenol, and control traps were 238.0 ± 14.5.95.8 ± 3.3. 55.5 ± 5.9. and 16.0 ± 2.6 SCR adults per trap. respectively (N = 6: dosage = 200 mg/trap). All three treatments were significant attractants: however. PAA traps significantly at­ tracted 2.5 and 4.3 times more SCR adults than indole- and eugenol-baited traps. The downwind surface of all baited traps had the greatest concentration of beetles. suggesting an anemOlactic response by the insects. Since a pretest whole-plant count in the com plot yielded a mean (± standard error) of 3.0 ± 0.4 SCR adults per com stalk (N = 50). the ~a. 1500 beetles captured on the six PAA traps were more than could have been present on the surrounding com stalks. In fact. SCR adults were often observed. in both years. flying from the adjacent cucurbit plot into the com plot and orienting to the treated traps from several meters downwind. Since PAA was such an active semiochemical for SCR adults. structurally related compounds were evaluated in the same com plot over a 4-hr period for potential activity (September 9. 1984) (Table I). Although PAA-baited traps had the highest mean trap catch. ca. 8 times greater than the control traps. they were not significantly more attractive to SCR adults than benzyl acetone-baited traps. Phenethyl alcohol and benzyl acetate were significantly less active than PAA-baited traps: however. both compounds were still 5.6 and 3.9 times more attractive. respectively. than controls. The structural similarity of these active compounds suggests that they may interact with the same sensory receptors. A substantial decrease in SCR attraction was observed between compounds which differed only by one carbon and two hydrogens. For example. PAA traps were 964 LAMPMAN ET AL.

TABLE I. MEAN TRAP CATCH OF SOUTHERN CORN ROOTWORM ADULTS ON STICKY TRAPS BAITED WITH 200 mg PHENYLACETALDEHYDE AND RELATED COMPOUNDS IN CORN PLOT ON SEPTEMBER 9, 1984 (SOUTH FARMS, URBANA, lLUNorsl

Treatment Chemical formula Xltrap ± SEa RA(%)'"

Phenylacetaldehyde C,H,CH,C(OlH 93.0 ± 15.0 ar 100 Benzyl acetone C,H,CH,CH,C(OlCH, 11.0 ± 4.2ab 76 Phenethyf alcohol C,H,CH,CH,OH 63.3 ± 2.7 be 68 Phenyl acetate C,H,OClOlCH, 43.7 ± 16.3 cd 47 Benzyl alcohol C,H,CH,OH 36.7 ± 9.0 ue 39 Methyl benzoate C,H,ClOlOCH, 28.0 ± 8.5 def 30 Benzyl acetate C,H,CH,OC(O)CH, 26.7 ± 6.2 def 29 p·Anisaldehyde ....CH,O.C,H,C(OlH 26.3 == 1.5 def 28 Acetophenone C,H,ClO)CH, 25.3 ± 5.8 def 27 p.Anisyl alcohol "-CH,O.C,H,CH,OH 24.0 ± ;.t.1 uef 26 Benzaldehyde C,H,C(OlH 14.0 ± 3.2 ef 15 Control 11.3 ± 1.0 ef 12 Anisole Cf)H.!OCH, 9.3 ± i.5 f 10

a Mean number of beetles per lrap 3.fter a 4·h period ± standard error of the mean (SE); three replicates per treatment, & RA (%) = percent relative amactancy (mean of treatment/mean of PAA) x tOO. r Means followed by the same letter are not significan!ly different Jt the 5% level by Duncan's multiple-range tCSt.

6.6 times more attractive than benzaldehyde traps; phenethyl alcohol traps. 1.7 times more than benzyl alcohol traps; and phenyl acetate traps. 1.6 times more than benzyl acetate-baited traps. Eugenol-related compounds were also investigated (Table 2) in 1984. This evaluation was conducted in mid-September and SCR activity was reduced due to lower day and night temperatures (compare controls in Tables I and 2). The 24-hr mean beetle catches of veratrole-. isoeugenol-. methyl isoeugenol-. and methyl eugenol-baited traps were significantly greater than the control beetle catch. Veratrole (orthodimethoxybenzene) consistently allracted more beetles than the other compounds in this series; however, the metadimethoxybenzene and paradimethoxybenzene isomers of veratrole were not significantly different. from the control trap catch. This indicates the ortho ring position of the methoxy· groups is important for activity. Eugenol and indole had higher mean trap catches than controls in this test but were not significantly different from con­ trols. Apparently. the propenylbenzenoid compounds are slightly more attrac­ tive than the allylbenzenoid compounds (Table 2). Field tests with SCR adults were discontinued in late September 1984 after the first frost, [n 1985, veratrole- and PAA-baited traps were reevaluated in the main com plot from July 18 to September 13. Veratrole-baited traps attracted ROOTWORM ATIRACTANTS 965

TABLE 2. MEAN TRAP CATCH OF SOUTHERN CORN ROOTWORM ADULTS ON STICKY TRAPS BAiTED WITH 200 mg VERATROLE AND RELATED COMPOUNDS IN CORN PLOT ON SEP1"EMBER IS. 1984 (SOUTH FARMS. URBANA. ILLINOIS)

Treatment ChemicaJ fonnula X/trap + SE" RA l%y'''

Veratrole 1.2~(CH~OhC6H" 45.0 ± 6.7.' 100 [sOt:ugenol 3-CH,OA-HO.C"H,CH=CHCH, 27.3 :t 7.1 b 60 Methyl isoeugenol 3.·l-(CH,Ol,C,H,CH=CHCH, D.J ± J.8 be 5Z Methyl eugenol 3A-(CH,Ol,C,H,CH,CH=CH, Z1.7 ± 1.7 bed 48 . Eugenol 3-CH,O.4- 17.7 ± 3.7 bcde 39 HO.C,H,CH,CH=CH, Indole CoH" . C!H'!NH 16.J ± 5.5 bede 36 m-Dimethoxybenzene I.J-(CH,Ol,C,H. 15.J ± 0.3 ede 34 Anethole '-CH,0.C,H.CH=CHCH, 12.7 ± 1.7 cde 48 Safrole 3.4- lO.O ± 2.9 de 21 (OCH,O)-C,H,CH,CH=CH, 3.4-Dimethoxypropylbenzene 3A-(CH,OhC,H,CH,CH,CH, 9.3 ± 3.5 e 41 Control 1.3 ± (.2 e 16 p-Oimethoxybenzene IA-(CH ,OhC,H. 7.3±1.ge 16 Estragole ';"CH,O.C,H.CH,CH=CH, 1.3 ±: 1.ge 16

"Mean number of beetles per trap after a 24-hr period ± standard error of the mean (SE); three replicates per treatment. . /;IRA (%) = percent relauve attractancy = (mean of treatmentlmean of vemtrole) x 100. .. Means followed by Ihe same letter are not significantly different :n the 5% level by Duncan· s multiple~range tCSt.

an average of twO times more SCR adults than PAA traps and eight times more than control traps (Table 3). Initial baited trap catches of beetles in 1985 were much lower than the late season beetle catches in 1984; however. trap catches dramatically increased at the end of AugUSt and early September for both treat­ ments and controls (in Table 3 compare mean. tmp catches from July 18 to August 13 to those from August 25 to September 13). Analysis of the 1985 data by two-way ANOV A (blocks versus treatments) yielded significantly different block mean squares for SCR trap catches in all late-August and September tests; therefore. there was a substantial field effecl. The beetle trap catches in the main plot were 3-10 times greater in the block closest to the cucurbit plot than the trap catches in the other blocks. The whole­ plant count in the com plot on August 20 revealed there were 4.9 ± 0.54 SCR adults per stalk (mean ± standard error. N = 20) on the cucurbit side of the plot and 1.5 ± 0.36 SCR per stalk (N = 20) on the opposite. open field side of the plot. Within a four-day period from August 16 to August 20. the whole­ plant count doubled. Mean (± standard error) whole-plant counts in 1985 were 0.1 ± 0.05 (July 23. N = 79). 0.7 ± 0.12 (August 7. N = 70). 1.5 ± 0.16 966 LAMPMAN ET AL.

TABLE 3. MEAN TRAP CATCH OF SOUTHERN CORN ROOTWORM ADULTS ON STICKY TRAPS BAITED WITH VERATROLE AND PHENYLACETALDEHYDE (PAA)

Treatment

Dale (1985) Dose (mgJ/trnp Vernuole PAA Control

July 18 100 5.00 ± 0.71a" 1.00 ± OAlb O±O c August 2 100 6.75 ± 3.28a 2.50 ± 0.96b 0.75 ± O.25c August 13 20 8.25 ± 3.77. ·'.15 ± 1.19. 0.75 ± OA8b August 18 100 23.25 ± 6.75a 3.50 ± L19b 1.50 ± 0.50b August 25 30 26.75 ± 6.34a 6.75 ± U5b August 27 200 92.50 ± 9.9Oa 72.20 ±,7.86b 4.50 ± 2.JOc September 1.3 100 33.00 ± 9.70. - 13.00 ± 5.24b a Mean trap catch per 24 hr ± 5 Error: N = 4, Means in the same row followed by the same letter are not significantly different at the 5% level by Duncan's muhiple-range test. I>Compound not tested on that date.

(August 16. N = 54). and 3.2 ± 0.28 (August 20. N = 40) SCR adults per stalk. The field effect and late season increase in SCR trap catches was appar­ ently the result of an increase in SCR dispersal from the cucurbit plot into the com test plot. Other field tests conducted in August 1985 showed chavicol (4-hydroxy-l­ allylbenzene) also significantly attracted SCR adults (Table 5). Related com­ pounds like estragole (4-methoxy-I-allylbenzene). trans-anethole (4-methoxy­ l-propenylbenzene). and 4-methoxy-l-propylbenzene. as well as several eu­ genol-related compounds. were not significant attractants of SCR adults in early and mid-August. 1985. However. the late August and September results in the main plot were similar to those obtained in 1984. with eugenol. isoeugenol. PAA. veratrole. and indole having significantly greater trap catches than con­ trols. SCR trap catch increases late in the season and previously unattractive compounds become significant attractants. This suggests SCR adults in central OIinois exhibit an enhanced response to volatiles in late August and early Sep- tember. . Elecrraanrennagrams of SCR Males. The relative EAG response (com­ pared to cis-3-hexen-I-01) for male SCR was determined for several aromatic compounds (Table 4). Males were used for the EAG analysis since they often outnumber females in high cucurbitacin-comaining cucurbit plots (Andersen. 1981). and our plot was adjacent to such a cucurbit plot. The EAG response to indole. phenethyl alcohol. PAA. benzyl acetate. benzyl acetone. eugenol. and veratrole. at 100- and 1000-fold dilutions in mineral oil. was seven or more times greater than that of control air puffs. Indole. phenethyl alcohol. and PAA had EAG responses 20-40 times greater than that of the control. This indicates ROOTWORM ATTRACTANTS 967

TABLE 4. MEAN RELATIVE ELECTROANTENNOGRAPHIC RESPONSea OF MALE SOUTHERN CORN ROOTWORMS TO SEVERAL AROMATIC COMPOUNDS

Dilution in mineral oil

Tn:::ument I: 1000 I: 100 cis·)·Hexenol 100 Indole 80.3 ± 6.l b 154.5 :!: 8.2 Phenethy I alcohol 30.3 ± ~.5 I~. 7 ;t 3.0 Phenyillcetaidehyde 3U ± ".5 106.5 ± 3~ Benzyl acetate ~6.6 ± 9.3 74.5 :!: 2.3 Benzyl acetone 33.1 ± ~.5 55.5 ± ).1 Eugenol "6.. ± ~.3 58.. ± ~.O Ver:ltrole 19.1 ± 5.1 .7.3 " 6.5 Control ~.O ± 2.5 ~.O !: 2.5

"EAG response is relative to a I: 100 dilution of cis-3-hexcnol. /OMean response:!: SO: N = 6.

SCR males are able to perceive peripherally a wide variety of aromatic com­ pounds. Although all the compounds evaluated yielded significant responses.. veratrole, one of the most active field attractants. had the lowest EAG response. and indole. a moderately active field attractant, had the highest EAG response. WCR Attractants. In a late season experiment. October 7, 1984, trans­ anethole- and indole-baited traps significantly attracted WCR adults; therefore. these and related compounds were intensively evaluated in 1985. Estragole (4­ methoxy-I-a1lylbenzene). a structural analog of trans-anethole (4-methoxy-l­ propenylbenzene), was the most active WCR attractant throughout the 1985 field evaluation of volatiles. Estragole was initially tested in the main plot on August 9, 1985, approximately one week after the sweet com tassels had started to shed pollen. The treated (100 mg) mean trap catch (± standard error) was 34.25 ± 6,86 and the control. 1.5 ± 0.65 WCR adults per trap per 24 hr (N = 4). The test was replicated on August II, and estragole traps had ca. -1-0 times more WCR adults than control traps (estragole. 32.5 ± 6.60 WCR per trap and control. 0.8 ± 0.96 WCR per trap per 24 ht; N = 4). Fifty WCR adults per trap were removed 48 hr later (August 13) and sexed in the labora­ tory. The estragole traps had sex ratios (female-male) of 4.3: I, 1.4: 1. 2.1: 1. and 4.1: I; a mean sex ratio of 2,98 females to 1.0 male per trap. Whether the predominance of females reflected field. chemical, or trap bias was not determined. Estragole (loo mg/trap) and control traps were also tested in the main plot on August 16 and 28 and September 3, 1985, with mean trap catches (± stan­ dard error) 01'23.4 ± 2.93, 31.0 ± 6.94, and 7.25 ± 2.53 WCR per trap per 968 LAMPMAN ET ..\L.

24 hr for estragole-baited traps and 3.5 ± 0.65. 2.25 ± 0.75. and 1.5 ± 0.50 WCR per trap per 24 hr for control traps (N = 4 for each trap date). On all dates. estragole was significantly different from controls (P < 0.05) by single classification ANaVA. Whole-plant COUnts in the main com plot yielded means (± standard errors) of 2.1 ± 0.22 (August 7. N = 54). 0.8 ± 0.15 (August 16. N = 37). and 0.5 ± 0.07 (August 28. N = 45) WCR per stalk. Although the WCR population decreased throughout August (unlike the SCR population), estragole traps consistently attracted WCR adults. The attraction of WCR adults to estragole and several phenylpropanoid compounds was investigated in the main plot on August 9 and August 16. 1985 (Table 5). Estragole trap catches were 3.64 ± 0.90 (mean ± standard error) times greater than that of the propenyl isomer trans-anethole and 4.6 ± 1.50 times greater than the propyl analog. 4-methoxy-l-propyl-benzene (MPB) (N = 28 for the estragole-anethole comparison). Fulthermore. estragole with a para­ methoxy ring substituent was eight times more attractive to WCR adults than chavicol. an estragole analog with a parahydroxy ring substiutent (see Table 5). Other allyl and propenyl compounds. such as eugenol. methyl eugenol, isoeu­ genol, and methyl isoeugenol. as well as veratrole and PAA. were not signifi­ cantly different from control WCR trap catches. WCR attraction to estragole is characterized by a highly specific structure-activity relationship; the position of the double bond and the methoxy ring substituent are critical for activity. A nitrogen containing phenylpropanoid derivative. indole. was attractive to WCR adults early and late in the test period. On July 18. 1985. in the main plot prior to com tasseling. indole (l00 mg/trap) attracted a mean (± standard error) of 10.8 ± 5.57 WCR per trap per 48 hr compared to a control trap catch of 4.0 ± 0.82 WCR per trap per 48 hr. (N = 4). After this test (August 2. 13. and 16. 1985). indole waS not significantly different from controls until August 23. 1985 (indole traps. 8.8 ± 2.87 WCR per trap per 24 hr and control traps. 1.5 ± 1.00 WCR per trap per 24 hours; N = 4). When indole (20 mg/trap) was tested on September 28. 1985 in the auxiliaty plot 250 m from the main plot. 22.0 ± 8.29 WCR were caught per trap per 24 hr versus a control of 2.8 ± 2.36 WCR per trap per 24 hr. The early and late season attraction of WCR adults by indole agrees with the results of Andersen and Metcalf (l986). Dosage-activity tests with estragole were conducted in all three experi­ mental plots on different dates (Table 6). In the auxiliary field 150 m from the main plot. the minimum dose per trap which significantly attracted WCR adults was 5 mg. and the highest dose. 100 mg/trap. attracted ca. 31 times more bee­ tles than the control (tested from August 20 to 22. 1985). [n the main plot the minimum effective dose was 30 mg and the highest dose. 100 mg/trap. attracted 18 times more beetles than the control (tested from August 28 to 30. 1985). In the plot 250 m from the main experimental plot. the minimum effective dose was to mg and the highest dose. 30 mg/trap. attracted four times more WCR adults than the control (tested from September 19 to 20. 1985). [n the last dos- ! '"3: TAUl.E 5. MEAN TRAI' CATCIl 01' WESTERN (WCRl. SOUTHERN (SCRl. AND NORTIIERN (NCR) CORN Rom'WORM ADUl.TS ON BAlTl.O (100 mgITRAP) STICKY TRAPS ~ Augusl 9. J985 August 16, 1985 ~

8ail WCR scn NCR WCR seR NCR

CUillrol 1.50 ± 0.65." 3.00 ± 1.0H. 0.25 ± 0.25. 3.50 ± O.tH.c 1.50 ± 0.50. 0.00 ± 0.00. E:>Iragoh: 3~.25 ± 6.H6b 4.25 ± 0.H5.b 0.00 ± 0.00. 23.50 ± 5.H6b 6.50 ± 3.33.b 0.25 ± 0.25. Im/u-Anethole 6.25 ± 1.61k 2.75 ± 0.H5. 0.25 ± 0.25. 7.75 ± 1.79, 2.00 ± O.82u 1.00 ± 0041. Chuvicul 3.75 ± ,0.H5.c Hoo ± 1.20bc 1.00 ±0.71. 3.50 ± 0.96., 10.25 ±4.l3b 0.75 ± 0.25. 4-McdlOxy·l-propylbcfllcnc 2.75 ± 1.03. 2.53 ± 1.71a 0.50 ± 0.50. Eugenol 2.25 ± O.95u 4.75 ± I.II.b 6.25 ± 1.60b Methyl eugenol 2.25 ± 1.03a 4.00 ± 1.73,b 0.75 ± 0.25. IsllcugcJlul 3.25 ± O.~H.c 5.25 ± 2.25ub 3.75 ± 0.75b Melhyl bocugcnul 3.25 ± 0.H5., 3.00 ± 0.H2, 0.50 ± 0.50. 5alrulc 1.25 ± 1.25. 2.25 ± UAH. 025 ± 0.25. PhcnyJul:clitluchyl.Jc 4.00 ± l.bMut: 9.25 ± 1.97c 0.00 ± 0.00. -"

"Meitn ± SE; N :: ... MCUlls in the sallie column Jultuwcd by the salile teller arc nut significantly ..hlrercnt at the 5% level by Dum:uu's multiple-runge IC:l1- "Nul tested un dust dale.

$ 970 LAMPMAN ET Al.

TABLE 6. DOSAGE TESTS WITH ESTRAGOLE FOR ATTRACTION OF WESTERN CORN ROOTWORM ADULTS IN THREE NONAOJACENT FIELDs" (SOUTH FARMS. URBANA. IL)

Field 1 Field 2 Field} Dose (mg)/lrap (Aug. 20-22. 1985) lAug. 28-30. 1985) (Sept. 19-20. 1985)

h 0 1.27 ± ,O.75 ;{ 1.75 ± GASa 17.0 ± 2.51a 1 - 13.25 ± 1.56. 3 22.5 ± 3.37ab 5 7.25 ± l.44b 6 2.50 ± O.87ab 10 11.50 ± !.73bc 50.0 ± 3.05b 20 28.00 ± 6.81kd 30 6.0 ± 1.J6b 71.75 ± 16.56b 50 ~7.50 ± 13.86d 60 14.0 ± 7.15ab 100 39.43 ± 7.30d 3\.0 ± 6.9

.. Field .2 was the main test plot; field J. 150 m from field 2: field 3. 250 m from field 2. IJ Mean WCR trap catch ± SE; N =: 4. for all treatments. "Means in a column followed by the same letters are not significantly different from each other at the 5% level by Duncan's muhiplcMrJnge lest. oJ Not tested on that date. , Mean number WCR adults per smJk ± SE.

age test, the 24-hr control trap catch was ca. 10 times greater than the "8-hr control trap catchesin the two previous tests. Since this field was planted one month after the main plot. it had the only fresh host available in the test area on that date. The high pretest whole-plant count (Table 6) suggests beetles were attracted to this field. NCR Attractants. Northern corn rootworms were significantly attracted to eugenol- and isoeugenol-baited traps in mid-August (Table 5). with eugenol­ baited traps having a higher mean trap catch than 'the isoeugenol baited traps. A pretest Whole-plant COUnt found only one NCR per 45 stalks. and the control traps caught no NCR adults; therefore. both compounds must be very active attractants. In the auxiliary plot 250 m from the main plot, 10 mg eugenol­ baited traps had a mean (± standard error) of3.8 ± 2.06 NCR per trap per· 24 hr and 30 mg eugenol-baited traps, 8.0 ± 0.82 NCR per trap per 24 hr. with control trap catch of 0.8 ± 0.96 NCR per trap per 24 hr (N = 4, tested on September 19. 1985). None of the related compounds. such as estragole. aneth­ ole. chavicol. 4-methoxy-I-propylbenzene. methyl eugenol. methyl isoeugenol. or safrole significantly attracted NCR adults. Veratfole and PAA, the two major SCR attractants. also did not attract NCR adults. The results of this study in- ROOTWORM ATTRACTANTS 971 dicate both the methoxy and hydroxy ring substituents and the propanoid side chain in eugenol and isoeugenol are critical for NCR attraction. which agrees with the results of Ladd (1984).

DISCUSSION

Southern. western. and northern corn rootworm adults exhibited a species­ specific pattern of response to sticky traps baited with various benzenoid com­ pounds. Southern com rOotWorm (SCR) adults were attracted in ~arly and mid­ AugUSt to traps baited with veratrol~ (l.2-dimethoxybenzenel. phenylacetal­ dehyde. and chavicol (4-hydroxy-l-allylbenzene). In late August and Septem­ ber. 1984 and 1985. SCR trap catches dramatically increased for veratrole and phenyiacetaldehyde. as well as for some previously unattractive compounds. such as indole. several eugenol-related compounds. benzyl acetone. and phen­ ethyl alcohol. The rapid change in trap catch was at least partially due to an increase in SCR beeties dispersing from an adjacent cucurbit plot into the sweet corn test plol. Environmental conditions andior host quality in mid- to late Au­ gUSt apparently enhanced SCR response to volatiles. Regardless of the seasonal variability in beetle response. SCR adults demonstrated a relatively specific structure-activity relationship for veratrole and phenylacetaldehyde. since closely related compounds were always less attractive to beetles. An electroan­ tennographic analysis (EAG) of SCR males revealed the beeties can perceive peripherally a variety of benzenoid compounds; however. the level of EAG response did not reflect the magnitude of field response. Subsequent field tests indicate that indole. which yielded a high EAG response but low field response. imeracts synergistically with veratrole which had a low EAG response. but a high field attraction of beeties (unpublished data). A more detailed EAG anal­ ysis of all three Diabrotica species with single compounds and mixtures is in progress to determine whether species differences in field attraction to volatiles can be related to different characteristics of the EAG response. Western corn rootworm (WCR) adults were significantly attracted to the phenylpropanoid estragole in three nonadjacent corn plots from early August to the end of the trapping period in late September 1985. In the main experimental plot WCR trap catches with 100 mg of estragole per trap were ca. 20 times greater than that of blank controls. and the minimum amount of estragole re­ SUlting in a significant trap catch was 5 mg/trap. Furthermore. estragole at­ tracted more females than males. comparable to the results recorded by Ladd et aI. (1985) for eugenol and NCR adults. The paramethoxy ring substituent and the position of the double bond in the propanoid side chain are critical for maximum WCR response; compounds differing in either aspect were less at­ tractive. For example. estragole caught an average of 3.6 times more beeties .than its structural analog trans-anethole. Despite the specificity of estragole for 972 LAMPMAN ET AL.

WCR attraction. another shikimic acid derivitive. indole. was active early and late in the season. Andersen and Metcalf (1986) had previously noted that in­ dole showed a decrease in activiry during com tasseling and silking. which may rellect multiple sensory cues from the com planES outcompeting the attraction to single-component traps or it may indicate a seasonal change in beetle re­ sponse to volatiles. A greatly enhanced response to volatiles. comparable to that of the SCR adulES under the late season conditions. was not recorded for WCR adults. Northern com roolWorm (NCR) adulES were relatively rare in this test; control trap catches never exceeded one to two beetles per trap. and the mean number of NCR per plant was typically less than 0.1 adult throughout the season in all pIOES. Despite the low bettie population. eugenol and isoeugenol were significant attractants and 10 mg of eugenol per trap caught a significant number of beetles. None of the other test compounds. such as estragole. rrans­ anethole. chavicol. and 4-methoxy-l-propylbenzene. attracted NCR adulES. Ladd (1984) found NCR response is optimal for phenylpropanoids with a metamethoxy and parahydroxy substituent. In our study conducted in mid-Au­ gust using 100 mg/trap. more NCR adults were attracted to eugenol. an allyl­ benzenoid. then isoeugenol. a propenylbenzenoid. analogous to the greater at­ traction of estragole to WCR adults than rrans-anethole. Ladd (1984). using 1500 mg/trap. found NCR attraction to isoeugenol was 1.6 times greater than eugenol in early August. roughly equivalent in mid-August. and the results reversed in late August. NCR adulES apparently display a seasonal change in response to attractanES similar to that previously discussed for SCR and WCR adults in this study. Whether this seasonal variability in beetle response to vol­ atiles is due to changes in the environment. host plant. and/or beetle biology is undetermined. A comparison between the rootworm species indicates the ecologically similar NCR and WCR adults are attracted to structurally related phenylpro­ panoids and both species respond to changes in the ring substituents of the major attractants. However. WCR and NCR adults are not attracted to the same phen­ ylpropanoids. although SCR adults are occasionally attracted to indole. a WCR attractant, and eugenol and isoeugenol. NCR attractants. An increase in SCR trap catch has also been recorded for either eugenol or veratrole added to in­ secticide-impregnated cucurbitacin baiES (unpublished data). Apparently. SCR adults cross-respond (especially late in the season) to some WCR and NCR attractants, although the main attractants for each species is highly specific. Perhaps one of the most intriguing aspects about the newly described Dia­ bratica semiochemicals is the establishment of an a1lelochemical basis for their activiry. Indole has been isolated from natural aggregation sites. cucurbit blos­ soms, as well as com whorls (Andersen and Metcalf. 1986; Thompson et a1 .. 1974). The major SCR attractants. veratrole and phenylacetaldehyde. are pres­ ent in com husk and silk volatiles (Cantelo and Jacobson. 1979b; Buttery et al.. (978), and phenylacetaldehyde and several di- and trimethoxybenzenes have ROOTWORM ATIRACTANTS 973 been detected in Cueurbita maxima blossoms (Andersen. 1984). The presence of veratrole. phenylacetaldehyde. and indole in larval or adult host plants sug­ gests they act as kairomonal attractants. Other NCR, WCR, and SCR attractants (such as eugenol. isoeugenol, estragole, and trans-anethole) have not been de­ scribed from the essential oils of the primary larval host, Zea mays. or cucurbits (Thompson et a!., 1974; Flath et al .. 1978: Buttery et al.. 1980: Buttery and Ling. 1984; Andersen. 1984). However. vinyl gUiacol, which is structurally similar to eugenol and isoeugenol. has been isolated from com husks (Buttery et al.. 1978). Ladd et al. (1983) originally described eugenol as a "food-type" attractant present in a number of alternative NCR adult hosts. SCR and NCR adults are often found on composites, grasses, legumes. and cucurbits not adjacent to com. especially late in the season. whereas WCR adults are considered to have a closer association with com and weeds in the immediate area (Cinerski and Chang. 1968; Ludwig and Hill. 1975; Branson and Krysan. 1981; Rhodeset al.• 1980; Messina and Root. 1980; Sholes. 1984). In Illinois. NCR. SCR. and D. eristata are often found feeding on the pollen in flower heads of Canada thistle (Carduus arversis). white heath aster (Aster pilosis). goldenrod (Soli­ dago spp.). and sunflowers (Helianthus spp.) in late August and September (personal observation). Since eugenol and phenylacetaldehyde are volatile con­ stitutents of sunflowers (Etievant et al .. 1984). these phenylpropanoids may be the cues SCR and NCR adults utilize to locate pollen sources. This would sug­ gest that eugenol and/or phenylacetaldehyde may also attract D. cristata. as it is occasionally found on the same plant species. It also suggests the other pollen hosts previously mentioned may release volatile compounds that attract Diabro­ ticites. Phenylacetaldehyde. which attracts numerous Lepidoptera. Diptera, Co­ leoptera. and Hymenoptera and which is widely distributed among the plant families, may act as a general insect attractant to numerous pollen or nectar sources (Cantelo and Jacobson. 1979a.b. references therein. and unpublished data). The major WCR attractant. estragole. has not been detected in cucurbits, com, or any of the aforementioned wildflowers: therefore. a host-plant rela­ tionship remains to be established.

Acknowledgmems-This research was supported in part by a gram from the SEA. USDA Competitive Research Grnnt Office. 81:CRCR·OI·0659; ··Kairomont:~ in I.;om .loti .;ucurbits In monitoring and controlling com rootworms:' Any opinion5. Iindings. conclusions. or recommen· dations are !hose of the authors and do not necessarily rerlect the view of the USDA. We would like (0 !hank Phil Lewis lor his valuable field assistance and Drs. Berenbaum and Ferguson for their criticisms of this manuscript. We wnuld like to thank Lavella Whited for typing numerous drafts of this manuscript.

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