CA2024085C - Volatile attractants for diabrotica species - Google Patents

Abstract

The compounds 4-methylphenylethanol, 4-chlorophenylethanol, 4-fluorophenylethanol, phenylethanol, phenylpropanol, 4-methoxyphenylethanol, 3-methoxyphenylethanol, 2-methoxyphenylethanol, 4-methoxyphenylpropanol, phenylethylamine, and phenylpropylamine may be used as attractants for northern corn rootworm or in combination with diabroticides and Diabrotica species non-volatile compulsive feeding stimulants as toxic bait for northern corn rootworm.

Description

202~085 BACRGROUND OF THE INVENTION
The present invention relates generally to lures for attraeting and eontrolling Diabrotiea species. In partieular, the invention relates to lures eomprising one or more compounds found in the volatile fraction of Cueurbita blossoms, or analog~ thereof, alone or in eombination with other lures, in~ectieides, and/or eompulsive feeding stimulants.
The ehrysomelid genera Diabrotica and Acalymma contain numerous pest species, ineluding the western eorn rootworm (WCR), Diabrotica virgifera virgifera LeConte; the southern eorn rootworm (SCR) or, the spotted cueumber beetle, D. undecimpunctata howardi lD.
duodecimpunctata Fab.]; the northern eorn rootworm (NCR), D. barberi Smith and Lawrenee; and the striped cucumber beetle (SCB), Acalymma vittatum (Fabr.).
The western, northern, and southern corn rootworms are the most expensive insect pests of North America and A~n~ ly cost U.S. farmers approximately one billion dollars in costs associated with lost crop yield and with preventative treatments with soil insecticides. The era of relatively inexpensive crop protection againQt these pests has ended because of - generalized ~ootworm resistance to organochlorine insecticides and the withdrawal by the U.S. EPA of registrations for these insecticides due to widespread environmental contamination. The newer organophos-phorous and earbamate insecticides are more expensive and subject to accelerated microbial degradation in soils and a rapid loss of activity. Furthermore, due to the persistence of many of these soil inseeticides, 2a2~085 groundwater and surfaee run-off pollution is of mueh eoneern to state and federal agencies. Because of the uneertain performanee and safety of the major products eurrently used for larval rootworm eontrol, such as earbofuran (Furadan~), isofenphos (Amaze~), phorate (Thimet~), terbufos (Counter~), a teehnologieal void exists for eontrolling these pests. Even standard eultural methods of pest management sueh as crop rotations of eorn-soybean-eorn and eorn are endangered as evidenee exists that the northern eorn rootworm ean undergo an extended diapause for two seasons. Henee, the benefits of yearly erop rotation are threatened.
Present soil inseetieide teehnology for eorn rootworm eontrol is rapidly beeoming unworkable. The use of volatile attraetants, singularly and in eombi-nations with other eontrol methods, ean become the basis for a new integrated pest management (IPM) technology for rootworm control that is economically favorable for the farmer and eertainly mueh less environmentally objeetionable. In this regard, Diabrotiea and Aealymma are known to show a elose assoeiation with host plants of the family Cueurbitaeeae, partieularly with the genus Cueurbita. Adult beetles are most eommonly found in the blossoms of Cueurbita speeies where they feed on pollen (in staminate flowers) and on neetar. In most instances, adult beetles showed a preference for the blossoms of C. maxima Duehesne eultivars over those of C. pepo L. and C. mosehata Poir.
The blossom eharaeteristies, i.e., eolor, size, shape, and/or fragranee, responsible for this preference are not fully understood, although Diabrotiea attraetion to eertain eompounds, termed semioehemieals, has been reported. By way of background, semioehemicals are plant-produced compounds which act by diffusion through air to produce behavorial responses in asso-ciated insect species. Kairomones are those semio-- 2(~24G~5 chemicals which act to benefit the receiving species;
allomones are those which benefit the sending species;
and synomones, e.g., floral volatiles involved in pollination, benefit both the emitting plant, through pollination, and the perceiving insect by rewards of nectar and pollen or through more intangible ecological rewards of aggregation or lek formations which lead to mating.
One of the earlier reports relating to Diabrotica attraction to compounds appeared in Morgan, et al., J. Econ. Entomol., 21:913 (1928). This collection of preliminary results on the chemotropic response of certain insects included the observation that the spotted cucumber beetle, D. undecimpunctata howardi (as D. duodecimpuntata Fab.), was attracted to cinnamaldehyde and cinnamyl alcohol. These results were obtained using white granite-ware pans of about 2-quart capacity set in rows on stakes 2 feet high, and filled with 1 quart of water to which was added 1 cc. of the chemical to be tested. The areas in which experiments were conducted included a field white with the blossoms of the field daisy (Erigeron annuus) oatfields, margins of woods, hedge rose, a cane brake, a slaughter-house lot, and a livery-stable yard. There is no indication in the reference as to the degree to which, or the conditions under which, cinnamic aldehyde and cinnamic alcohol acted as attractants. Snapp, et al., J. Econ.
Entomol., 22:98 (1929) disclosed a preliminary report indicating that the spotted cucumber beetle was attracted by oil of thyme (white) and benzyl alcohol.
However, in tests by Lampman, et al, J. Chem. Ecol., 13:959 (1987) benzyl alcohol was shown to have negligible attractant value.
Ladd, et al., J. Econ. Entomol., 76:1049 (1983), studying a mixture of phenethyl proprionate, eugenol, geraniol, a food-type lure for Japanese 2~2~08~

beetles, reported that eugenol was attraetive to adults of D. barberi Smith & Lawrenee (NCR), but not attractive to D. virgifera virgifera LeConte (WCR). Ladd, T.L., J.
Econ. Entomol., 77:339 (1984) tested nine compounds elosely related to eugenol for attractancy to NCR
ineluding four groups: eugenol and its elose relatives with isomerie or saturated hydroearbon side ehains;
eugenol aeetate and its isomer; anethole and its analogs with isomerie or saturated side ehains; and a miseellaneous group eonsisting of methyleugenol and 2-allyl-6-methoxyphenol (orthoeugenol). The author reported two new attraetants for NCR, isoeugenol and 2-methoxy-4-propylphenol and eoneluded that while neither the position of the double bond in the side ehain nor the degree of saturation appeared eritieal in attracting NCR, the presence of the methoxy and hydroxyl groups at their respective positions (adjacent), particularly the latter, seemed to be important. Yaro, et al., Environ.
Entomol., 16:126 (1987) reported that eugenol and 2-methoxy-4-propyl phenol were highly attractive to D.
cristata Harris (a non-pest species) and D. barberi Smith & Lawrenee, whereas isoeugenol aeetate was not attraetive. None of the eompounds was highly attraetive to D. virgifera vigifera LeConte. Ladd, et al., J.
Eeon. Entomol., 77:652 (1984) also eondueted tests to study the influenee of eolor and height of eugenol-baited sticky traps and reported that the traps were most effective when painted yellow and placed 0 to 0.25 m above ground.
Another recent report of investigations concerning a ehemical basis for Diabrotica orientation to the ~lossoms or foilage of Cucurbita species was in Andersen, et al., J. Chem. Ecol., 12:687 (1986). The authors screened C. maxima "Blue Hubbard" blossom volatiles for eleetroantennogram ~EAG) activity, a laboratory test for inseet electrophysiologieal response 202~08~ - -to volatile compounds, and found two fractions had significant activity for the southern corn rootworm.
The first peak consisted of indole and the second, smaller peak, was not characterized. Indole was then field tested for insect responsiveness and found to be a - potent attractant of the western corn rootworm and of the striped cucumber beetle. However, the southern corn rootworm was not attracted at any dosage level despite the strong EAG response.
Andersen, et al., J. Chem. Ecol., 13:681 (1987) subsequently characterized the blossom constituents of attractive Cucurbita floral volatiles from a number of cultivars representing C. moschata, C.
E~E~ and C. maxima, and examined other factors, including nutritional and secondary chemical characteristics, that might influence beetle field distribution in blossoms. They found para-dimethoxybenzene to be a major constituent of headspace samples from cultivated Cucurbita cultivars. The Diabrotica species showed a clear preference for certain cultivars. SCR preferred the blossoms of C. maxima cultivars, while WCR preferred the cultivars of C.
maxima as well as a single cultivar of C. pepo. The authors suggested that NCR and SCR are similar in their host preferences, each strongly favoring C. maxima, whereas WCR appeared to find a broader range of cultivars acceptable. No firm conclusions were reached with respect to whether the gustatory cues, i.e., the levels of cucurbitacins, and olfactory cues, i.e., floral odors and level of release, acted individually or in concert, along with visual qualities to produce the patterns of preferences exhibited by the various species of Diabrotica.
Seasonal variations in responses to attractants have also been observed. As noted earlier, Andersen, et al., J. Chem. Ecol., 12:687 (1986) found 2~2~085 .

that indole elicited a strong EAG response from SCR, but was wholly ineffective as an attractant in field tests. However, Lampman, et al., J. Chem. Ecol., 13:959 (1987) subsequently found that indole was in fact a moderately active attractant for SCR late in the season. In that study, the authors field tested various blossom volatiles and related compounds and found that southern, western, and northern corn rootworm adults exhibited not only a species specific response but also a seasonal pattern of response to sticky traps baited with various benzenoid compounds. SCR adults were attracted in early to mid-August to traps baited with veratrole (1,2-dimethoxybenzene), phenylacetaldehyde, and chavicol (4-hydroxy-1-allylbenzene). In late August and September, SCR trap catches dramatically increased for veratrole and phenylacetaldehyde, as well as for some compounds previously unattractive compounds, such as indole, several eugenol-related compounds, benzyl acetone, and phenethyl alcohol. WCR adults were significantly attracted to a different group of compounds, namely estragole, trans-anethole, and indole. Estragole (4-methoxy-1-allylbenzene) was an effective WCR attractant from early August until the end of the trapping period in late September and early October. Indole and trans-anethole (4-methoxy-1-propenylbenzene) were less effective attractants than estragole and were most active at the beginning and/or end of the corn season. The para-methoxy ring substituent and the position of the double bond in the propanoid side chain were noted to be critical for maximum WCR response; compounds differing in either aspect were less attractive. The phenyl propanoids, eugenol and isoeugenol, significantly attracted NCR
adults. Ladd, T.L., J. Econ. Entomol., 77:339 (1984) previously found that NCR response is optimal for phenylpropanoids with a meta-methoxy and para-hydroxy ~24085 substituent. Both Ladd, supra, and Lampman, et al., su~ra, noted a seasonal variability in response to eugenol and isoeugenol.
A comparison between the rootworm species indicated that the ecologically similar NCR and WCR
adults are attracted to structurally related phenylpropanoids 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 phenylpropanoids, although SCR adults are occasionally attracted to indole (a WCR attractant) and to eugenol and isoeugenol (NCR attractants). The authors in Lampman, et al., supra, also noted (and subsequently reported in Metcalf, et al., J. Econ.
Entomol., 80:870 (1987)) an increase in SCR trap catch for either eugenal or veratrole added to insecticide-impregnated cucurbitacin baits. Apparently, SCR adults cross-responded (especially late in the season) to some WCR and NCR attractants, although the main attractants for each species was highly specific.
The foregoing results suggested that Diabrotica are attracted to certain single component attractants; however, none of these attractants demonstrated the requisite degree of attraction required to effectively control and manage populations of Diabrotica. Thus, there exists a need in the art for an alternative safe and effective method for management of Diabrotica species which is effective in controlling the more visible stage of the insect pests, i.e., the adult, yet which is economical and which abolishes the need for the use of soil insecticides.

BRIEF SUMMARY OF T~E INVEWTION

The present invention provides novel attractants for the control of Diabrotic species 2~40~5 inseets, novel mixtures of attractants and novel compositions ineluding, in eombination, Diabrotiea species attractants, diabroticitieidal insecticides and eompounds and eompositions funetional as eompulsive stimulants of Diabrotieite feeding behavior.
Novel attraetants provided aeeording to the invention inelude: ~-ionone; 4-methoxyeinnamaldehyde;
4-methoxyeinnamonitrile; 4-methoxy-1-vinyl-benzene; 4-methoxy-l-propyl-benzene; 4-methoxy phenyl ethyl ether;
4-methoxy phenyl aeetonitrile; allyl benzene;
cinnamonitrile; 2-methoxy cinnamaldehyde einnamyl acetate; cinnamie aeid methyl ester; dihydroeinnamyl aldehyde; and phenyl propionitrile.
Also provided are the following novel attraetants: 4-methylphenylethanol, 4-chlorophenylethanol, 4-fluorophenylethanol, phenylethanol, phenylpropanol, 4-methoxyphenylethanol, 3-methoxyphenylethanol, 2-methoxyphenylethanol, 4-methoxyphenylpropanol, phenylethylamine, and phenylpropylamine.
These novel attractants are suitable for use individually or in eombination with eaeh other or known Diabrotiea speeies attraetants.
Novel mixtures of attraetants according to the invention are seen to include a compound selected from the group eonsisting of dimethoxybenzene, trimethoxybenzene, and guaiacol admixed with one or more compounds selected from the group eonsisting of indole, phenylaeetaldehyde, anethole, eugenol, einnamaldehyde and cinnamonitrile. Presently preferred dimethoxybenzene eompounds include ortho-dimethoxybenzene ("vératrole") as well as the meta- and para-forms. Of the isomeric forms of trimethoxybenzene, the 1,2,4-trimethoxy form is preferred. Presently 35 preferred attractant mixtures include the following: ~
veratrole and indole (VI); veratrole and phenylaeet-2~24085 g aldehyde (VP); veratrole, indole and phenylacetaldehyde (VIP); meta-dimethoxybenzene, indole and phenylacet-aldehyde (mDMBIP); para-dimethoxybenzene, indole and phenylacetaldehyde (pDM8IP); veratrole, indole, phenylacetaldehyde, anethole and eugenol (VIPAE);
trimethoxybenzene, indole, and cinnamaldehyde (TIC);
trimethoxybenzene and indole (TI); trimethoxybenzene and cinnamaldehyde (TC); and guaiacol, indole and phenylacetaldehyde (GIP).
Alternately, constituted attractants for Diabrotica species according to the present invention are selected from the group consisting of the following mixtures: cinnamaldehyde indole (CI); 3-ionone and indole (BI); and B-ionone and cinnamaldehyde (BC).
According to another aspect of the invention, compounds effective as attractants for southern corn rootworm include; meta-dimethoxybenzene, para-dimethoxybenzene, indole, phenylacetaldehyde, trimethoxybenzene, 1,2,4-trimethoxy benzene, trans-cinnamaldehyde, allyl benzene, cinnamonitrile, 4-methoxy cinnamaldehyde, cinnamyl acetate, phenyl propionitrile, and phenyl ethanol. Particularly useful are unsubstituted phenylpropanoids having an unsaturated side chain, and having an aldehydic carbonyl group or a nitrile group at the free terminus of said side chain.
Compounds herein shown to be effective as attractants for western corn rootworm include:
trimethoxybenzene, 1,2,4,-trimethoxybenzene, indole, cinnamaldehyde, trans-cinnamaldehyde, ~-ionone, estragole, trans-anethole, 4-methoxy-1-vinyl benzene, 4-methoxy-l-propyl benzene, 4-methoxybenzyl methyl ether, 4-methoxyphenyl ethyl ether, 4-methoxyphenyl acetonitrile, 4-methoxycinnamonitrile, cinnamonitrile, 4-methoxy cinnamaldehyde, 2-methoxy cinnamaldehyde, cinnamyl acetate, and cinnamyl alcohol. Particularly useful are phenylpropanoids having a para-methoxy group, - 202~08~

having an unsaturated side chain, and having an aldehydic carbonyl group or nitrile group at the free terminus of the side chain.
Compounds herein shown to be effective as attractants for northern corn rootworm include:
- estragole, trimethoxybenzene, 1,2,4-trimethoxybenzene, cinnamaldehyde, trans-cinnamaldehyde, cinnamonitrile, cinnamyl alcohol, cinnamyl acetate as well as 4-methylphenylethanol, 4-chlorophenylethanol, 4-fluorophenylethanol, phenylethanol, phenylpropanol, 4-methoxyphenylethanol, 3-methoxyphenylethanol, 2-methoxyphenylethanol, 4-methoxyphenylpropanol, phenylethylamine, and phenylpropylamine. Particularly useful NCR attractants are phenyl-propanoids such as (1) phenyl alkanols having a para-methoxy group and a 2- or 3-carbon alkanol; (2) phenyl alkanols with 2- or 3-carbon side chains including the unsaturated cinnamyl alcohol; and (3) phenyl alkylamines with 2- or 3-carbon side-chains.
Compounds shown herein to have particularly long term (persistent) effectiveness as NCR attractants include: phenyl propanol, phenylethyl amine, and 4-methoxyphenylethanol.
Compounds herein shown to be effective as attractants for Diabrotica cristata include: guaiacol, estragole, trimethoxybenzene, 1,2,4-trimethoxybenzene, indole, cinnamaldehyde, trans-cinnamaldehyde, s-ionone, 4-methoxycinnamaldehydej cinnamyl alcohol, and cinnamonitrile.
Attractant compounds and compositions of the invention attract both male and female rootworms and can be used with sticky traps and other types of insect traps at suitable attractive dosages, preferably ranging from approximately 1 mg to 100 mg per trap, although doses of 0.01 mg to 200 mg have been found to be effective, to attract hundreds of beetles per day. The 202408~

VIP mixture attraets SCR adults at rates of 10 to 100 times greater than eontrols. The TIC mixture attraets WCR and SCR adults at rates of 10 to 100 times greater than eontrols. The CI mixture attraets WCR and SCR
adults at rates of 10 to 100 times greater than eontrols. 4-Methoxyeinnamaldehyde, 4-methoxyeinnamonitrile, and B-ionone attraet WCR at rates 10 to 100 times eontrols; einnamyl aleohol attraets NCR
at rates 5-20 times eontrols; and einnamaldehyde and einnamonitrile attraet SCR at rates 5-20 times eontrols.
4-Methylphenylethanol, 4-ehlorophenylethanol, 4-fluorophenylethanol, phenylethanol, phenylpropanol, 4-methoxyphenylethanol, 3-methoxyphenylethanol, 2-methoxyphenylethanol, 4-methoxyphenylpropanol, phenylethylamine, and phenylpropylamine attraet NCR at rates approximately 5-50 times eontrols.
Attraetants aeeording to the invention may be used to manipulate adult eorn rootworm behavior for monitoring and eontrolling eorn rootworm populations.
Through the use of these lures, a totally new teehnology for eorn rootworm eontrol is possible. By using these attraetants, individually or in eombination, the presenee, speeies distribution, and reproduetive state of adult eorn rootworms ean be monitored and pest management decisions be made for the next season infestation. Moreover, these attractants are so potent that they can mobilize adult corn rootworm populations in large fields and attraet them to speeifie sites where they ean be destroyed by spraying limited areas with eonventional insecticides.
Alternatively, these lures can be used as eomponents of a toxie bait for adult beetles. The toxic bait eomprises: i) a Diabrotiea speeies attraetant, ii) a eompulsive feeding stimulant, and iii) a diabrotieitieidal inseetieide. These baits ean 202~085 .

combine: i) as attractant, either the multi-component mixtures, e.g., VIP or TIC mixtures and/or the single component lures, e.g., eugenol, indole, veratrole, cinnamaldehyde, cinnamonitrile, cinnamyl alcohol, cinnamyl acetate, 4-methoxycinnamaldehyde, 4-methoxycinnamonitrile, 4-methoxy-1-vinylbenzene, B-ionone, trimethoxybenzene, phenylethanol, phenylpropanol, 4-methoxyphenyl ethanol, 4-methoxyphenyl propanol, phenylethylamine, phenylpropylamine and/or other compounds found in volatile fractions of blossoms of Cucurbita plants (the exact choice of lure depending on the relative economic importance of the three Diabrotica species); ii) as compulsive feeding stimulant, cucurbitacins obtained from, for example, bitter squash, and iii) as the diabroticiticidal insecticide, a relatively small dosage of a wide variety of conventional insecticides including organophosphorous, carbamate and pyrethroid insecticides, such as carbaryl, methomyl, isofenphos, malathion, and dimethoate. As used herein the term diabroticiticidal includes a Diabrotica species insecticide and includes any and all compounds capable of killing Diabrotica species. (See, e.g., Canadian Patent 1,195,222 to R. L. Metcalf and A.M. Rhodes for the cucurbitacin-insecticide bait). This toxic bait combination of ingredients provides a long range and persistent (long term) attraction of beetles to a poison bait that acts as a contact feeding stimulant.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description thereof which includes numerous illustrative examples of the practice of the invention.

2~40~5 nl;!'rATT.~ n~sc~TpTIoN

The following examples illustrate practice of the invention in the production of lures for attracting and controlling Diabrotica species. Specifically, the invention relates to lures comprising one or more compounds found in the volatile fraction of Cucurbita blossoms, or analogs thereof, alone or in combination with other lures, insecticides, and/or compulsive feeding stimulants.
Example 1 relates to the attraction of Diabrotica species to single-component and multi-component lures in corn fields.
Example 2 relates to the response of Diabrotica species (D. cristata and D. barberi) in a prairie habitat to eugenol analogs and to attractants for D. v. virgifera and D. barberi.
Example 3 relates to the evaluation of twenty-four compounds, including estragole and estragole analogues, as attractants.
Example 4 relates to the preparation and use of poison (toxic) baits containing cucurbitacins, volatile attractants and insecticides.
Example 5 relates to Cucurbita blossom aroma and-Diabrotica rootworm beetle attraction.
Example 6 relates to the evaluation of seven compounds, including phenylethanol analogues, as attractants.
The examples which follow are for illustrative purposes only and are not intended in any way to limit the scope of the invention.

202~0~5 Single and Multicomponent Kairomonal Lures for Southern and Western Corn Rootworms:
The attraction of Diabrotica species to single-component and multicomponent lures was evaluated in corn fields. The field experiments on attraction of SCR and WCR adults were conducted in 0.405-ha (l-acreJ
plots of "Illini Xtra Sweet" corn grown on the University of Illinois South Farms at Urbana, Illinois, over a two year period. Most of the tests were conducted in August to correspond with the peak silk-feeding and oviposition periods of corn rootworm adults in central Illinois. In both years, the sweet corn plot was adjacent to a 0.405-ha trap crop of C. maxima cultivar "~lue Hubbard", to provide high densities of SCR and WCR adults in the corn plo`ts (~owe, et al., Environ. Entomol., 5:745-751 (1976), t~mpm~n, et al., J.
Chem. Ecol., 13:959-975 (1987).
Attraction of Diabrotica species to volatile compounds was measured by the average number of beetles caught during a 24-h period on l.0-liter cylindrical paper cartons (15.2 cm high, 27.9 cm circumference) which were evenly coated with clear insect adhesive, TangleTrap~ (Tanglefoot Co., Grand Rapids, Mich.J. The candidate attractants were applied by capillary micropipettes to cotton dental wicks (13 mm long by 6 mm diameter) that were previously impregnated with-mineral oil to prolong volatilization. The chemicals were purchased from Aldrich Chemical Co. (Milwaukee, Wis.)-and assayed by gas chromatography as >97% pure. Indole, a solid at ambient temperatures (m.p. 52-54~), was dissolved in glass-distilled acetone before dosing the cotton wicks. For dosage-activity tests, the various components were mixed in equal quantities by weight and diluted to the appropriate dosage with acetone. Treated 2û24~5 and untreated (eontrol) wieks were attached to the top of the paper eartons with TangleTrap~ and the inverted traps were plaeed on l-m-high posts between the sweet eorn rows. Four replicates of each treatment and a eontrol were eondueted in a randomized complete block design. The sticky traps within a block (ea. 50-m row ~ of eorn) were 5-10 m apart (depending on the number of treatments in a partieular test) and the bloeks were 3 m apart. The traps were baited and plaeed in the field between 1000 and 1300 hours and beetle counts were taken after 24 h. The significance of treatments was determined by analysis of variance and the individual means were separated by Duncan's multiple range test (Statistical Paekage for Soeial Seiences (SPSS) (Nie, et al., SPSS: statistical package for the social seienees, 2nd ed. McGraw-Hill, New York (1975)). Student's t test was used for comparisons of two means (Sokal, et al., Biometry, Freeman, San Francisco (1969). Significance levels were set at P=0.05 at all statistical analyses.
The initial field test in August of the first year evaluated the response of SCR and WCR adults of a mixture of veratrole (V), indole (I), phenylacetaldehyde IP), trans-anethole (A), and eugenol (E) (=VIPAE
mixture), as well as to the individual components. Each component in the VIPAE mixture was present at a dosage of 20 mg per trap and was compared with the individual eomponents at 100 mg per trap. The response of SCR and WCR adults to a range of VIPAE doses, 1-300 mg, was eonducted on the following day. Fifty SCR adults were removed from each 100-mg VIPAE trap, and returned to the laboratory for sex determination. Additional analyses in August evaluated the reduction in trap catches produced by the sequential removal of the individual components from the VIPAE lure. After the primary components of the initial lure were determined, a modified multicomponent lure consisting of veratrole, 2~240~
.

indole, and phenylacetaldehyde (=VIP mixture) was compared with its individual components to determine whether the attraction of SCR adults was the result of interactive or additive factors. The attraction and synergistic response of SCR adults of the VIP mixture was verified by tests conducted in August of the following year.
The importance of the ortho-dimethoxy configuration of veratrole to the overall activity of the VIP mixture was demonstrated in early September of the first year and August of the second year using mixtures containing compounds chemically related ~o veratrole. In late August of the second year, the VIP
mixture was compared with a mixture containing compounds structurally related to veratrole and phenylacetaldehyde. Two mixtures, VIP (veratrole, indole, and phenylacetaldehyde) and TIC (1,2,4-trimethoxybenzene, indole, and trans-cinnamaldehyde), were evaluated at doses ranging from l to 30 mg.
The VIPAE mixture caught 26 times more SCR
adults than the control traps and was 3 times more active than the only other significant attractant, veratrole (Table l).

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- 2~240~5 The SCR response to VIPAE exceeded the additive response that was expected, based on the stimulation of a single receptor type by each of the individual components. In contrast to the SCR response, fewer WCR adults were caught on the VIPAE-baited traps than on those baited with 100 mg of trans-anethole.
Furthermore, SCR adults responded to the mixture in a concentration-dependent manner. WCR adults did not respond to the VIPAE mixture at any concentration;
therefore, the activity of the lure was species-- specific. Although both male and female SCR were present on the baited traps, the overall sex ratio, as taken from the four 100-mg VIPAE-baited traps, was 4.6 males to 1.0 female (n=200). It was not determined whether the skewed sex ratio reflected a field bias or a distinct sexual preference. Single-component lures for NCR and WCR adults, such as eugenol, indole, and estragole, attract primarily females (Ladd, et al., J.
Econ. Entomol., 78:844-847 (1985), Andersen, et al., J.
Chem. Ec~l., 13:681-699 (1986), rAmr~ n, et al., J.
Chem. Ecol., 13:959-975 (1987)).
Components of the VIPAE mixture were sequentially removed to evaluate their contribution to the overall activity of the multicomponent lure. Traps baited with VIPAE caught 27-30 times as many SCR adults as untreated controls, and all of the four-component mixtures caught significantly more beetles than the controls. However, the absence of veratrole from the VIPAE mixture resulted in a dramatic 77% reduction of SCR trap catch relative to the complete mixture.
Although the mean trap catches were not significantly different, the removal of indole or phenylacetaldehyde from the VIPAE mixture produced 58 and 60% reductions in trap catches, respectively, whereas mixtures without trans-anethole or eugenol resulted in only 22 and 24%
fewer beetles being caught, respectively. These results 2024a~
.

suggested the primary attractants in the VIPAE mixture to be veratrole, indole, and phenylacetaldehyde, and a comparison of the three-component VIP mixture with the five-component VIPAE mixture showed no significant difference in SCR trap catches between the two lures.
Two-component mixtures without either phenylacetaldehyde, indole, or veratrole (VI, VP and IP
mixtures) caught significantly fewer SCR adults than traps baited with VIPAE. A comparison of the VIP, VI, IP, and VP trap ca^tches with their four- and five-component counterparts containing either anethole, eugenol, or both compounds (i.e., VIPAE, VIPA, and VIPE
compared with VIP; IPAE with IP; VIAE with VI; and VPAE
with VP) further supports the conclusion that anethole and eugenol were less important components of the original VIPAE mixture. WCR trap catches did not differ from the untreated controls in any of the tests conducted in the first year.
The VIP mixture was also compared with its individual components and several two-component mixtures. The VIP-baited traps caught ca. 8 times as many SCR adults as the mean additive response that would be expected based on the stimulation of different receptors by the individual components therefore, SCR
adults respond to the multicomponent lure in an interactive and synergistic manner. SCR adults also exhibited synergistic responses to the two-component mixtures, VP and VI, although less than half as many beetles were attracted to these traps as compared with the VIP-baited traps.
The importance of veratrole to the overall activity of the VIP mixture was demonstrated by the reduction in trap catch with mixtures containing indole, phenylacetaldehyde, and either the meta- or para-isomer of veratrole (ortho-dimethoxybenzene). These mixtures caught 36 and 84~ fewer SCR adults, respectively, than 202~0~5 traps baited with the VIP mixture. The substitution of guaiacol ~2-methoxyphenol) for veratrole in the VIP
mixture also reduced trap catch-ca. 80% (Table 2).
These data imply that the ortho-dimethoxy configuration i5 critical for beetle response to the mixture. The higher mean SCR trap catches with VIP-baited traps in September, as compared with those in August of the first year, probably reflect a seasonal increase in trap catch which has also been documented for NCR and WCR adults (Ladd, J. Econ. Entomol., 77:339-341 (1984), Andersen, et al., J. Chem. Ecol., 13:681-699 ~1986), Lampman, et al., J. Chem. Ecol., 13:959-975 (1987)).
In the second year the attraction and synergistic response of SCR adults to the VIP mixture was verified. There were 14-50 times more SCR adults caught on VIP-baited traps than on untreated controls.
Furthermore, the number of beetles captured with the multicomponent lure was 3-4 times greater than the expected mean additive trap catch based on the performance of the individual components (Table 2).

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202~G85 Although ehemical analyses of the floral eomponents of Cueurbita blossoms and plant parts of Zea mays have revealed an extensive array of "green leaf volatiles", aromatie eompounds, hydroearbons, and terpenoids, the eomponents of the VIP mixture apparently do not eo-oeeur in any one plant part (Buttery, et al., J. Agrie. Food Chem., 26:866-869 (1978), Buttery, et al., J. Agrie. Food Chem., 26:771-774 (1980), Itokawa, _ al., Phytoehemistry, 21:1935-1937 (1982), Buttery, et al., J. Agrie. Food Chem., 32:1104-1106 (1984), and Andersen, _ al., J. Chem. Eeol., 13:681-699 (1987).
However, two related eompounds, 1,2,4-trimethoxybenzene and trans-einnamaldehyde, are present, along with indole, in the blossom extraets of several C. maxima eultivars (Andersen, et al., J. Chem. Ecol., 13:681-699 (1987); unpublished data). Two mixtures, VI~ and TIC
(1,2,4-trimethoxybenzene, indole, and trans-einnamaldehyde), were eompared for attraetion of SCR
adults and appeared similar for this species at any dosage (Table 3).

- 2(~2~85 c e _____ __~,p .~
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The two multicomponent lures varied significantly in their attraction of WCR adults. The TIC-baited traps caught 5.7 times more WCR adults than controls at 1 mg per trap and 28.6 times more at 30 mg per trap, whereas the VIP-baited traps were not significantly different from controls at 1 mg per trap and were only 3.7 times greater than control WCR trap catch at 30 mg per trap (Table 3). The attraction of WCR adults to the VIP-baited traps at the higher dosages was probably due to the increased amount of indole per trap (Andersen, et al., J. Chem. Ecol., 12:687-699 (1986)) because the WCR trap catches with indole and the VIP mixture were not significantly different on two dates in the second year (Table 2).
The VIP and TIC lures were active during the peak oviposition and silk-feeding periods of WCR and SCR
adults; therefore, they can be useful to monitor or to attract beetles to a toxicant. Ongoing field studies have shown that the TIC lure can concentrate WCR and SCR
at the edges of 2-4-ha field plots, thereby redefining the spatial distribution of the beetle within the agroecosystem and that the TIC lure attracts significantly more female WCR than males.

The Response of Diabrotica species (D. cristata and D. barberi) in a Prairie Habitat to Eugenol Analogs and Other Attractants for D. v. virgifera and D. barberi:
The candidate lures in all experiments were purchased commercially and exceeded 98~ purity. The test chemicals were applied by capillary micropipettes to cotton dental wicks which were attached to the tops of 1.0 liter cylindrical paper cartons covered with TangleTrap~ (Tanglefoot Co., Grand Rapids, Mich.). The 202~0~5 sticky trap techniques used were as described in Example 1. Cinnamyl alcohol and E~-methoxycinnamaldehyde, which are solids, were dissolved in glass-distilled acetone prior to dosing the cotton wicks. All treatments were applied at a dosage of 100 mg per trap. 1,2,4-trimethoxybenzene, indole, and trans-cinnamaldehyde were mixed in a ratio of 1:1:1, by weight, immediately prior to dosing the cotton wicks.
Control traps had untreated cotton wicks. Four replicates of treated and untreated traps were randomly - positioned 10 meters apart on top of 1 m high posts along the border of a eight foot wide strip cut through the middle of a 4.05 ha relict prairie at Trelease Woods (ca. two miles northeast of Urbana, Illinois). The field consisted primarily of Andropogon gerardii Vitman (big bluestem) and a variety of composites. The tests were conducted in mid-August of two years and were timed to coincide with the presence of D. cristata and D.
barberi adults in the flower heads of Canada thistle (Carduus arvensis Robson). In the second year, several new attractants of D. v. virgifera and D. barberi were tested in Trelea~e Woods and along the edge of a 0.8 ha field of hybrid corn located at the Pomology South Farms, Urbana, ~llinois. Attraction of Diabrotica species to the various lures was measured by the mean number of beetles caught after 24-h. On one date in late August of the first year, D. cristata and D.
barberi were removed from the traps and sexed in the laboratory. The significance of treatments in all tests was determined by an analysis of variance and the individual means were separated by Duncan's multiple range test (Statistical Package for Social Science, Nie, et al., SPSS: statistical package for the social sciences, 2nd ed. McGraw-~ill, New York (1975). Data were logarithmically transformed for statistical analysis and the means and standard deviations are presented for the untransformed data.

2 ~ 2~

Counts in the flower heads of Canada thistle indicated both D. cristata and D. barberi adults were present in the prairie test site. D. cristata adults were not present in the corn field and D. v. virgifera adults displayed no affinity for Canada thistle. In the initial experiment with the candidate lures, traps baited with eugenol and isoeugenol caught significantly more adult D. cristata and D. barberi than methyl eugenol baited traps or the unbaited traps in the prairie habitat (Table 4).

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2024~8~

A subsequent comparison of eugenol with several analogs showed guaiacol also significantly attracted both Diabrotica species, albeit to a lesser S extent than either eugenol or isoeugenol (Table 4). As previously found for D. barberi in corn agroecosystems (Ladd, J. Econ. Entomol., 77:339-341 (1984)), compounds lacking the hydroxyl and methoxy groups, present on the eugenol-type analogs, were inactive for both species (Table 4). The sex ratios of the beetles on the eugenol traps were 16.6:1 (female:male) for D. barberi adults and 6.9:1 for D. cristata (n=60 for both species). The beetle catches on the control traps were so low a reliable estimate of field sex ratios was not lS determined. However, a comparison of control catches with treatment catches in (Yaro, et al., Environ.
Entomol., 16:126-128 (1987)) also indicates that eugenol and 2-methoxy-4-propylphenol are more active for D.
barberi females than D. cristata females.
In the final test, estragole and a 100 mg mixture (1:1:1) of 1,2,4-trimethoxybenzene, indole, and trans-cinnamaldehyde (TIC mixture) were as active as eugenol or isoeugenol for attracting D. barberi and D.
cristata (Table 4). The TIC mixture also attracted a significant number of D. u. howardi. These lures when tested in or along side corn fields are primarily attractants for D. v. virgifera adults (Lampman, et al., J. Econ. Entomol., 80:1137-1142 (1987)).
In the second year, estragole, B-ionone, the TIC mixture, 4-methoxycinnamaldehyde, cinnamyl alcohol and eugenol attracted more D. cristata than control traps in the prairie habitat (Table 5).
.

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202~85 Eugenol and einnamyl alcohol consistently had higher trap eatches of D. cristata than the other attractants. In addition to the attraction of D.
cristata, B-ionone and para-methoxycinnamaldehyde caught significantly more D. v. virgifera than controls and the TIC mixture was an active lure for D. u. howardi (Table 5). Eugenol, cinnamyl alcohol, and the TIC mixture also attracted D. barberi; however, cinnamyl alcohol was ca.
3.5 times more active than the TIC mixture (Table 5).
Estragole was a significant attractant for D. barberi in one test, but not in the other. Eugenol and cinnamyl alcohol were 2 and 5 times more active for D. barberi than estragole, respectively (Table 5). Although D.
barberi and D. cristata respond in a similar fashion to eugenol analogs, both species are also attracted to compounds that are structurally distinct from eugenol, such as cinnamyl alcohol, B-ionone~ and 4-methoxycinnamaldehyde. Methyl eugenol and ~-ionone were inactive as attractants for any of the Diabrotica species in these tests.
A comparative study was conducted with several attractants for D. barberi and D. v. virgifera along the outside row of hybrid field corn. Cinnamyl alcohol and eugenol exhibited roughly equivalent activity for D.
barberi (Table 6) as previously observed in the prairie tests (Table 5).

~2408~ ~

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2~2g~

Cinnamyl alcohol was also active, albeit at a low level, for D. u. howardi. Estragole, ~-ionone, the TIC
mixture, and 4-methoxycinnamaldehyde were highly active attractants for D. v. virgifera, but not D. barberi, when-tested along the corn field. The TIC mixture also caught significantly more D. u. howardi than control traps (Table 6). D. cristata adults were never found in any of the treatment or control traps in the corn tests. Although the TIC mixture and estragole exhibited activity for D. barberi in the prairie tests, they were not active for this species in the corn tests which agrees with previously published data for this species (Ladd, J. Econ. Entomol., 77:339-341 (1984), Lampman, et al., J. Econ. Entomol., 80:1137-1142 (1987), Lampman, et al., J. Chem. Ecol., 13:959-975 (1987).
Geographical variation in the response of D.
barberi and D. cristata to eugenol is apparently minimal based on the similar attraction to eugenol analogs recorded in central Illinois, Ohio, (Ladd, J. Econ.
Entomol., 77:339-341 (1984) and in South Dakota (Yaro, et al., Environ. Entomol., 16:126-128 (1987). Although the two specie~ responded identically to eugenol and two chemical analogs in a previous test (Yaro, et al., supra), the data presented here demonstrates that D.
cristata also shares some chemosensory adaptations with D. v. virgifera. In the prairie tests, D. cristata was moderately attracted to ~-ionone and 4-methoxycinnamaldehyde, whereas D. barberi adults did not respond to these compounds. These same compounds were potent attractants of D. v. virgifera when evaluated in corn field tests. Female D. barberi also appear more responsive to eugenol-type lures than female D. cristata (Yaro, et al., supra). Even though D. cristata responds to attractants for D. v. virgifera, the highest trap catches in this study were with attractants for D.
baberi (i.e., eugenol and cinnamyl alcohol). The a~

attraction of D. barberi and D. cristata to cinnamyl alcohol in prairie and corn habitats was unexpected, as previous tests had shown attractants for D. barberi require a methoxy and hydroxy group on a phenyl ring with a three-carbon side chain (Ladd, J. Econ. Entomol., 77:339-341 (1984), Lampman, et al., J. Chem. Ecol., 13:959-975 (1987)). Both Diabrotica species responded to compounds that lacked these structural moieties (i.e., cinnamyl alcohol, E~-methoxycinnamaldehyde, and ~-ionone).
All of the Diabrotica species examined to date are attracted to chemically related cyclic compounds, however, each species displays a distinctive pattern of response when exposed to a broad spectrum of candidate lures. The adaptation of D. cristata and D. barberi to eugenol-type analogs may have arisen in a Nearctic ancestor as conjectured by Yaro, et al., supra; however, the attraction of Diabrotica species from two distinct taxonomic groups (fucata and virgifera (Wilcox, Coleopterorum Catalogus Supplementa (Chrysomelidae:
Galerucinae, Luperini), Pars 78, Fasc. 3. 2nd ed. Dr. W.
Junk, s'Gravenhagen, Netherlands, 1972) to a variety of structurally related compounds suggest the chemosensory response to phenylpropanoid and cyclic semiochemicals is - of older evolutionary origin, perhaps parallel to the generalized response of this group to cucurbitacins (Metcalf, J. Chem. Ecol., 12:1109-1124 (1986)). In this regard, it is interesting that attractants for D.
barberi, D. cristata, D. v. virgifera, and D. u. howardi (such as cinnamyl alcohol, ~-ionone, and the components -of the TIC mixture) have been isolated from the floral volatiles of Cucurbita maxima cultivars (Andersen, J.
Chem. Ecol., 12:687-699 (1986), Metcalf & Lampman unpublished data). Furthermore, D. barberi, D.
virgifera, and Acalymma vittata, the striped cucumber beetle, are attracted to sticky traps baited with 21~24~g5 flowers from "Blue Hubbard", a cultivar of C. maxima (McAuslane, _ al., Proc. Entomol. Soc. Ont., 117:49-57 (1986), Metcalf & Lampman unpublished data). An evaluation of the olfactory responses of additional Diabrotica species, particularly those with different host plant affinities and geographical distributions, to a variety of semiochemicals and volatile constituents of the host plants of adults, may further elucidate the adaptive significance of the chemosensory response to these natural products and the evolution of chemoreception in this group.

Evaluation of Twenty-four Compounds Including Estragole and Estragole Analogues as Attractants:
Six of the twenty-four chemical compounds evaluated as attractants were prepared as described. 4-methoxy-l-propylbenzene (IV, Table 1) was prepared from 4-propylphenol and dimethylsulfate, b.p. 215-216C, lit.
210-214C (Klages, Ber. Deut. Chem. Gesell., 32:1437-1441 (1899)). 4-methoxybenzyl methyl ether (V) was prepared from 4-methoxybenzyl alcohol and dimethyl sulfate, b.p. 128-130/30 mm, lit. 107-108/15 mm (Anglande, Compt. Rend. Acad. Sci., 210:52-54 (1940)).
4-methoxypheny} ethyl ether ~VI) was prepared from 4-methoxyphenol and diethyl sulfate, m.p. 34-5, lit. 36-7 (Robinson, et al., J. Chem. Soc., 1926:392-401 (1926)). 4-methoxycinnamaldehyde (XI) was prepared from anisaldehyde and acetaldehyde, m.p. 58 (Vorlander, et al., J. Prakt. Chem., 229:237-247 (1929)). 4-methoxycinnamic acid methyl ester (XIV) was prepared from the acid and anhydrous methanol, m.p. 87-89, lit.
89 (Perkin, J. Chem. Soc., 34:409-452 (1881)). 4-(4'-methoxyphenyl)-3-butene-2-one (XVI) was obtained from 4-methoxybenzaldehyde and acetone, m.p. 72-3, lit. 72-3 2~4~8~

(Einhorn, et al., Justus Liebig's Ann. Chem., 243:362-378 (1888)). These compounds were all ~98% pure as determined by gas chromatography-mass spectrometry. The remaining 18 chemicals described in the tables were purchased from Aldrich Chemical Co., Milwaukee, Wisc.
and were 97-99% pure.
Test chemicals were applied by capillary micropipettes to cotton dental wicks that were attached to tops of 1.0 liter cylindrical paper cartons covered with TangleTrap~, Grand Rapids, Mich. The trapping techniques used were as previously described in Example 1. Cinnamyl alcohol, 4-methoxycinnamaldehyde, cinnamyl acetate, and 4-(4'methoxyphenyl)-3-butene-2-one which are solids at ambient temperature were prepared as standard w/v solutions in acetone prior to dosing the cotton wicks.- All treatments were made at a dosage of 100 mg or 100 ~L of attractant per trap. Control traps had untreated wicks. Four replicates of treated and untreated traps were positioned randomly 10 meters apart on top of 1 m high posts along the edges of 25-50 ha.
fields of hybrid corn (Zea m~y~), infested with adult NCR, SCR, and WCR beetles. Several experiments detailed in the Tables were made in a 0.4 ha plot of sweet corn (Illini X-tra Sweet).
Attraction of Diabrotica species adults of the sticky traps was measured by the mean number of beetles caught after 1 day (ca. 24 hours) exposure. The significance of the treatments was determined by analysis of variance and the individual means were separated by Duncan's multiple range test (Statistical Package for the Social Sciences, Nie, et al., 2nd. ed.
McGraw-Hill, New York (1975). The data was logarithmically transformed for statistical analysis.
Means and standard deviations are presented for the untransformed data. Significance levels were set at P=0.05 for all statistical analyses.

20241~5 The response of WCR adults in corn to sticky traps baited with estragole or 4-methoxy-1-allylbenzene (I) and 7 analogues combining ~ara-methoxyphenyl moieties with 2 and 3 atom side chains is shown in Table 7.

2û2~08S

_ Ll , In v a~
d _ _ _ o O U~ ~ ~ ~ -c ~_I _I ~ a~ t~ ~1 1 o V---- _ _ _ _ V ~1 .r1 O~
V :~ ~ ~ U U ~ ~C V
C

~ O
+~ ~ z ~ E~ ~ ~ ~ ~ Z U
- ~ ~ U
C ~ ~ u~ I~ O U~
~ V
V ~D t~ ~1 ~D ~ ~r ~ C
~ ~ C
s ~ V
Ll C
V ~O ~
C) 11 ~ oq :~, ~ -- -- C' -_ ~
Y ~ l ~ s U ~ w o ~P ~ ~ d~ clP d ` V _I C
,, V ~ O a~ o v " -- _1 ~ ~ ~ ~ ~ ~ ro~ E S
~o~ ~ _ _ _ _ _ _ _ ,.,,~
o ~ e~ V

3 C~ ~ +1 +I tl +1 -H +1 +1 +1 ~ S n 3 ~: o o C
bq C~ _I ~a u F~ 3 t` --~ ~ u~ t` o ~n c _~ ~ 0 ~ o r~ Cl E
a~ ~ ~ ~ ~ ~ ~ 3 0 C ` U
o ~ ~,, _ V _ 0 0 1., _ _ Q~ ^ ~ "O
0 ~ S ~ _I W~ O
C-~l C _ _ V 11~ -1 v11 :~
~ -- 8~ ~ ~ ~ S ~ C P~ V
O C ~L~ N C C V V 0E-- ~1 ~
u-~l c c ~ ~ ~ V
_1 N ~ ~) 11~ ~ N ¢ N ~ C 3 _I V ~
C ~ ~ N C 1~ ¢ C C;~ C S ~1 0 0 C V
L~ ~ U cU ~U _I U ~ ¢ ~ z v ~u ~ u au ~ m _l :, o ~ ~ s x ~ u s u au ~ v v ~ a u o c ~u _I u E N~ ~U
0 0 ~ U _l11 S ~ P~ ~ O ~ IE 'I v S
O E ~ ~ ~ ~ U ~ ~ ~ U - U ~ ~4 C v 3 -1 V5 ~--~ U O) O ~C U O :C ~1O--~ ~ ~ 0 U _ _~ ~C ~ U N ~, ~ V ~
~C~: C .OC ~ 4 0 ~ ~ 0 1 1 U I ~ 0~ U ~ ~ S
S V ~ 1 U 0 ~ O _1U--~ ~Q U S O S ~1V ~
) v C U ~ O C I ~1 0 UO Q. ~0~ ~ V
O V ~ ~ 0 ~ ~ ~ C C C a~ - v O V -- ~0 C ~ XU ~ ~X :~ X U X
E U~ C :q ~ O U v OI O U O:~: O C~ O I O U U ~
~ _ ~ _I U--~ I s ~: I _u s I s~r s ~ v _l e E o I _~r v _ ~ J I V ~r V V S e ~
c ~ o~ v E E E e E E E ~ ~ e ~ o C I I H I I I I H I E~ ~ ~ ~ Z
~- O ~ H ~ H ~ ~ ~ ~t H ~ H ~ ~1 ~ U H --H -- H --H --~ --~ -- ~ ~ 0 2az40~

Estragole is an effeetive kairomonal attractant for WCR
adults in eorn. Lampman, et al., J. Chem. ECQ1 ., 13:959-975 (1987). Attraetion of WCR adults decrease markedly with a shift in the position of the C=C bond from l-propenyl to 2-propenyl as in trans-anethole (II), and is lost through saturation of the double bond to 4-methoxy-l-propylbenzene (IV) (Table 7). Substitution of an oxygen atom for earbon in the side ehain, as in 4-methoxyphenyl ethyl ether (VI), resulted in loss of attraetion and aetivity was only marginally retained in 4-methoxybenzyl methyl ether (V) (Table 7). These ~ ehanges suggest that the interaetion of estragole with - the WCR antennal reeeptors is unusually specific, as attraetant studies with methyl eugenol and the oriental fruit fly have shown that highly attraetive bioisosterie moleeules ean be formed by saturation of the C=C bond in the naturally oeeurring kairomone or by substitution of oxygen atoms (Mitchell, et al., Environ. Entomol., 14:176-181 (1985). The intermediate attractivity of 4-methoxyphenylaeetonitrile (VII) (Table 1) and the slight attraetivity of 4-methoxy-1-vinylbenzene (III) supports the necessity of a 3 atom unsaturated side ehain.
Role of methoxy group. The data in Table 8 demonstrate that a ~ara-methoxy phenyl group is important for the speeific attraetion of WCR adults.

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.. Il e ~ . U c 51 ~ c--D . -- Q 51 r~ 51 ~ t-- u~C U
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a~ . Ll I I ~ ~OU ~r U 1~ V
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11~ S C H H H ~ C Z
~) O ~ O ~ HX ~ H ~ H I--I ~ ~
4 U H -- :~ H-- X X ~ X X ~ 1~1 ra a u 2~24~

The unsubstituted eompounds, allylbenzene (VIII), cinnamonitrile (X) and cinnamaldehyde (XII), were unattraetive or only weakly attraetive to WCR
adults. However, cinnamonitrile (X) and cinnamaldehyde (XII) were highly attractive to SCR adults, the latter being the most effeetive attraetant yet found for this species. Conver~ely, estragole or 4-methoxy-1-allylbenzene (I), 4-methoxycinnamonitrile (IX), and 4-methoxycinnamaldehyde (XI) were highly attractive to WCR
(Table 8). Experiments with pairs of eandidate - attractants with and without para-methoxy groups were repeated on several oeeasions with similar results (Table 9).

8` 5:

~ ~ V
U ~ ~ U ~ ~ ~ ~ ~ v V . . . . ~, o ~ o , ~ ~

In ~ ~ v V ~ -' O?
, ~ t` U o ,` ~ o o ~ ~
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v c ~ c on ~ ~ ~ o t~ o E s ~
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V S C
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v O -- _~ _ o ~ Dq O
--~ m -- ~ c ~ ~0 ~: U ~ ~ I o ~ 11 "-- o ~ U ~ V C
3c .m-- -- -v ---c ~ mE----o --~ O ~ O ~ Y ~
,1 o~ --~ ~ --v ~ 3_~v C J --~ U~ ~ ~ O C
S ~ um~ c ~u ~ U ~ C~ I~
v ~ _~ ~-- 11o c 11 ~o I
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~r~1 ~ ~ ~-- C.):r: C~ C v .OC L C ~~0~ 1l 0 ~a ~
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E UJ ~ c C~ O ~o~I O _~ U-~ I O ~ ~) I U U r`
I s~ E ~ S ~ ~E~r S
c--O E O~ v ~ v S U ~ v o:~S C ~ _~
C ~ ~ C ~ V C ~ V Ct~ ~--1--C Q.-~ ~ v E c E ~ C E ~ ~ ~ ~ ~ C
1.~ C ~ ~ I E ~H I Y H S W
~1 `~ OH ~r1--~ U H
V H '--~ UX -- X --X -- X --X --2~-4~1~5 Shifting the position of the methoxy group to 2-methoxycinnamaldehyde (XIII) ~Table 8) resulted in a substantial decrease in attraction for WCR. This information suggests that WCR adults are evolutionarily attuned for maximal response to phenylpropanoid kairomones with para-methoxy groups, e.g., estragole, in contrast to SCR adults that respond to unsubstituted phenylpropanoid moieties, e.g., cinnamaldehyde.
10Role of C=C unsaturation. Most of the kairomonal attractants for WCR, SCR, and NCR have side chains incorporating a C=C double bond, e.g., estragole and anethole for WCR (Lampman, et al., J. Chem. Ecol., 13:959-975 (1987)), eugenol and isoeugenol for NCR
15(Ladd, J. Econ. Entomol., 77:339-341 (1984)) and cinnamaldehyde for SCR (Tables 7, 9, 10). Saturation of the C=C bond of estragole, as in 4-methoxy-1-propylbenzene (IV), eliminated attraction for WCR (Table 7) and saturation of cinnamaldehyde as in phenylpropanaldehyde (XXII) substantially decreased attraction to SCR and WCR (Table 10).

nJ ~ ta U D t~ ~1 D; ~ --I
~ O _l O ~ ~ _l O _~ O
v g +l ~t l +l ~ ~ +l , C
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c _ C Z
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z c ~ u rq cJ o ~ ~ ~ o O ~ ~ ~ ~ U ~ :¢ U ~ ~ ~ ~ s 11 ~ V 11 ~ C 11 6 U ~ 11 E~ ~ ~ U C .,i V
C~ U ~ ~U~ 6 mU~C~ U ~C m ~ U C >~ U~ aS' ~C C~
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~ ~1 ~ _I ~D~ u~C ~OU~ DU ~ U--C 6 0 ~ U ~DU ~ U-- U-- U--6 SU C
H U -- ~ H X X H ~1 V 4 U X X ~ X X X X

2~4~8~

This requirement for an unsaturated side ehain does not appear to be clear eut for NCR, as (Ladd, J.
Econ. Entomol., 77:339-341 (1984)) found that the saturated analogue of eugenol, 2-methoxy-4-propylphenol, was approximately as attractive as eugenol or isoeugenol for this species.
Importance of carbonyl groups. The data of Table 3 shows the influence of three types of earbonyl (C=O) eontaining groups on the attraetion of WCR and SCR. Only the aldehydes, C(O)H (XI and XII) were attraetive to either WCR or SCR respeetively, and the esters C(O)OCH3 (XIV and XV) and the ketones C(O)CH3 (XVI and XVII) were unattractive to either species. The nitriles (IX and X) provide interesting examples of bioisosterism with aldehydes (XI and XII) (Table 2), as 4-methoxycinnamonitrile (IX) was almost as attraetive as 4-methoxycinnamaldehyde (XI) to WCR, and cinnamonitrile (X) approached cinnamaldehyde (XII) in attractivity to SCR. Saturation of the C=C bond of cinnamonitrile, as in phenylpropionitrile (XXIII), decreased attractivity to both WCR and SCR (Table 10).
Cinnamyl alcohol as an NCR attractant. The speeies specifie attraction of Diabrotica species to phenylpropanoids is apparently assoeiated with the nature of the phenyl substitution, e.g., para-methoxy for WCR, unsubstituted for SCR, and 3-methoxy-4-hydroxy for NCR (Ladd, et al., J. Econ. Entomol., 76:1049-1051 (1983), Ladd, J. Econ. Entomol., 77:339-341 (1984), Lampman, et al., J. Chem. Ecol., 13:959-975 (1987), Lampman, _ al., Environ. Entomol., in press (1988)).
Neither cinnamaldehyde (XII) nor cinnamonitrile (X) were signifi~antly attractive to NCR adults (Table 10).
Therefore, the marked attractivity of einnamyl aleohol (XVIII) to this species (Tables 10, 11) was unexpeeted and demonstrates another facet of singularities of phenylpropanoid attraction to the three rootworm 2~4~5 speeies. Cinnamyl aeetate (XIX) was also moderately attraetive to NCR although it was not attractive to SCR
or WCR (Table 10). SCR adults were weakly attraeted to einnamyl aleohol in one test (Table 11). Both cinnamic aeid (XX) and its methyl ester (XXI) were also unattraetive to all three speeies of rootworms (Table - 10). Further evaluation (Table 11) showed that cinnamyl aleohol (XVIII) was as attractive to NCR as was eugenol and this result was eorroborated several times during August and September (Lampman, et al., Environ.
Entomol., in press (1988)). 4-methoxyeinnamaldehyde (XI) proved to be unattraetive to both NCR and SCR
(Table 11).

2~240~5 - -u 8 o ~
. . .
U _ o UUl Z Z
~111 0 N
y O ~ O O
U N_I
Il~ 8 O4 g O _~
O Z Z

; O _~

~ --~N
_ O Cl~ ~) N .U
O
8,~ ~ ~ C
_~ N ~
~.0N N O ~1 gZ ~ +l ~ H
C O
L~ ~ N t~
U ~U-l N O S
8 ~ ~ U C
N N (~
N C ' U ~ ~ ~ ~ . C
N U In N O U'l N
C 8 N ~ 8 8 ~
~rl O O N U~ U7 t~ I

UU, 3 H ~ C t~
O :~Z
O ~ O O 1~_1 8 ~
O u- ~ O q~
0 N r _ S ~
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5! 1~ D
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0~ :Lo~~ ~oU ~1 _~
U ~ _ I ~ ~ U U-- ,C C
_ . ,., g " ~ 3 20240~5 4-methoxycinnamaldehyde as a WCR attractant.
This compound (XI, Table 8) is a very effective kairomonal attractant for WCR adults, but is essentially unattractive to NCR and SCR adults (Tables 8, 9, 11).
It is however, attractive to the closely related Diabrotica cristata (Harris) (Lampman, et al., Environ.
Entomol., in press (1988)). Cylindrical sticky traps baited with logarithmically decreasing amounts of 4-methoxycinnamaldehyde were used to determine the limitof response (LR) for attraction of WCR adults in corn.
Significant attraction was obtained with 4-methoxycinnamaldehyde present on cotton wicks at 0.03 mg, the approximate LR value. Similar experiments with other WCR kairomonal attractants determined LR values for indole of 1 mg (Andersen, et al., J. Chem. Ecol., 12:687-699 (1986)) and estragole of about 3 mg (Lampman, et al., J. Chem. Ecol., 13:959-975 (1987)). Thus 4-methoxycinnamaldehyde is about 100-fold more effective than these previously described WCR attractants, and its activity approaches that of the WCR female sex pheromone 8-methyl-2-decylpropanoate which attracted male WCR at 5 ug per sticky trap (Andersen, et al., J. Chem. Ecol., 12:687-699 (1986)). Unlike the sex pheromone, 4-methoxycinnamaldehyde is effective in attracting bothmale and female WCR and the combined male/female sex ratio of beetles trapped during August and September of the second year varied from 0.2 to 1.3. Thus this kairomone can be useful for attracting WCR to poison baits for control just prior to oviposition. 4-methoxycinnamaldehyde was used routinely to monitor WCR
populations during the summer of the second year and consistently attracted several times as many WCR adults as did estragole at equivalent dosages (see Table 8).
In experiments to determine the longevity of attraction, wicks treated with 100 mg of 4-methoxycinnamaldehyde were transferred to fresh sticky 2~240~ --traps daily and the 24 hour trap catch determined. The original treated wicks remained highly attractive over a 16 day period of exposure to August sunlight and through two heavy rainstorms.
4-methoxycinnamaldehyde has been identified as a constituent of the essential oils of a variety-of plants, e.g., Agastache rugosa (Fujita, et al., Nippon Kagaku Zasshi, 78:1541-1542 (1957); chem. Absts., 53:22754 (1959)), Orthodon methylchavicoliferum (ueda~
et al., Nippon Kagaku Zasshi, 77:1308-1310 (1956); Chem.
- Absts., 53:22754g (1959)), and Ocimum basilicum (Pogany, et al., Perfume Essential Oil Rev., 59:558-865.t (1968)) all of the family Lamiaceae; Acorus gramineus (Araceae, Fujita, et al., Yakugaku Zasshi, 91:132-133 (1971);
Chem. Absts., 74:130278k (1971)), Artemesia dracunculus (Carduaceae, Thieme, et al., Pharmazie, 27:255-265 (1972)), Limnophila rugosa (Scropulariaceae, Argwal, _ al., Ind. J. Pharmacol., 37:99-100 (1975)) and Sphaeranthus indicus (Compositae, Baslac, Perfumery Essential Oil Record, 50:765-768 (1959)). The presence of 4-methoxycinnamaldehyde in these essential oils was consistently associated with the presence of much larger quantities of estragole.
Species-specific attraction to phenylpropanoids. All of the Diabrotica species studied are attracted to structurally related phenylpropanoids and several synthetic derivatives. Within the spectrum of response to these compounds there are both specific responses to individual compounds and significant overlaps in the patterns of response among the several species. Species specific attraction is associated with two major alterations in phenylpropanoid structures:
first, the nature of the substituents on the phenyl ring 35~ where 4-methoxy provides maximal attraction to WCR, as in estragole (Lampman, et al., J. Chem. Ecol., 13:959-975 (1987)), and 3-methoxy-4-hydroxy provides maximaI

2~24~85 attraction to NCR, as in eugenol (Ladd, et al., J. Econ.
Entomol., 76:1049-1051 (1983); Ladd, J. Econ. Entomol., 77:339-341 (1984)). The feral D. cristata appears to represent an intermediate type of response in preferring eugenol but also in responding to several WCR
attractants (Lampman, et al., Environ. Entomol., in press (1988)). The second important structural modification is the nature of the unsaturated side chain where both WCR and SCR respond most strongly to be unsaturated aldehyde and NCR to the unsaturated alcohol. Here again, D. cristata is strongly attracted to cinnamyl alcohol but also displays a significant response to the WCR attractant, 4-methoxycinnamaldehyde (Lampman, et al., Environ. Entomol., in press (1988)).
It is interesting that all four species of Diabrotica show significant attractant response to the mixture of squash blossom volatiles, 1,2,4-trimethoxybenzene, indole, and cinnamaldehyde (TIC) (Lampman, et al., J.
Econ. Entomol., 80:1137-1142 (1987); Lampman, et al., Environ. Entomol., in press (1988)).

.

Preparation and Use of Poison Baits Containing Cucurbitacins, Volatile Attractants and Insecticides:
This example relates to the use of cucurbitacins, powerful feeding stimulants which are not volatile and therefore ineffective as long-range - attractants, in combination with volatile attractants, such as eugenol, indole and veratrole in poison baits which greatly enhance the distance over which the baits act. A determination of optimal bait formulation, choice of insecticide, rate of application, and mode of application for efficient use to control adult corn rootworms was made.

2~2~!~8~

The oxygenated tetracyclic triterpenoid cucurbitacins, bitter and toxic substances characteristic of the cucurbitaceae, mediate a classic example of co-evolutionary interaction between plants and insects. Cucurbitacins (cucs), allomones that originally served to protect the plants from attack by herbivores, have become kairomones for a large group of diabroticite beetles (Chambliss, et al., Science, 154:1392-1393 (1966), Sharma, et al, Environ. Entomol., 2:154-156 (1973), Howe, _ al., Environ. Entomol., 5:1043-1048 (1976), Metcalf, et al., Proc. Natl. Acad.
Sci. USA, 77:3769-3772 (1980), Metcalf, et al., Environ.
Entomol., 11:921-927 (1982), Ferguson, et al., J. Econ.
Entomol., 76:47-51 (1983), Metcalf, Nat. Hist. Surv., 33:175-198 (1985), Metcalf, J. Chem. Ecol., 12:1109-1124 (1986). The adul~ beetles detect cucurbitacins in nanogram quantities by specific sensory receptors on the maxillary palpi and respond by arrest and compulsive feeding. These kairomones influence the behavior of a number of important crop pests including the banded cucumber beetle (BCB), Diabrotica balteata LeConte; the northern corn rootworm (NCR), Diabrotica barberi Smith &
Lawrence; the southern corn rootworm (SCR), Diabrotica undecimpunctata howardi Barber, and its western relative, D. u. undecimpunctata Mannerheim; the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte; the striped cucumber beetle (SCR), Acalymma vittatum, (F.) r and its western relative, Acalymma trivittatum (Mannerheim). This specific response by diabroticite beetles to the cucurbitacin kairomones has considerable potential for the integrated pest management of cucumber beetles and corn rootworms - (Rhodes, et al., J. Am. Soc. Hortic. Sci., 105:838-842 (1980); Metcalf, et al., Cucurbit Genet. Coop. Rep., 4:37-38 (1981); Metcalf, et al., Cucurbit Genet. Coop.
Rep., 6:79-81 (1983); Metcalf, J. Chem. Ecol., 12:1109-202~08~
.

1124 (1985); and Metcalf, et al., Canadian Patent No.
1,195,922 (1985)). The latter reference disclosed the addition of dried bitter Cucurbitale fruit (as opposed to the blossoms and/or blossom volatiles) as a good source of cucurbitacins. Cucurbitacins are not volatile. Because they are not volatile, they are ineffective as long-range attractants of insects.
Cucurbitacins are arrestants that act as kairomones to arrest diabroticites. "Arrested" insects initiate compulsive feeding responses. That is, they stop dead in their tracks and compulsively feed on the fruits.
The laboratory experiments described below were conducted with a colony of SCR adults and with WCR
adults from a nondiapausing race obtained from the USDA, Northern Grain Insects Research Laboratory, Brookings, South Dakota. The field experiments were conducted with normal Illinois populations of SCR, NCR, WCR, and SCB
found in cultivated cucurbits (Cucurbita species) and corn (Zea mays) grown on the University Vegetable Crops Farm, Urbana, Illinois). Cucurbita fruit for bait production was grown on the University of Illinois Vegetable Crops Farm. C. foetidissima roots were provided by W.P. Bemis, University of Arizona.
Dried Cucurbita Fl baits were produced from the fruits of the hybrids AND x MAX and TEX x PEP
(Rhodes, et al., J. Am. Soc. Hortic. Sci., 105:838-842 (1980)) and from the roots of C. foetidissima. The fruits were split, partially air-dried, then thoroughly dried in a forced-air oven at 70C, and ground in a Wiley mill. The completely dry fruits of AND x MAX
contained 8.9% solids and those of TEX x PEP contained 9.1% slids. C. foetidissima roots averaged 22.7% solids (Berry, et al., J. Agric. Food Chem., 26:345-346 (1978)). The proportions of the ground baits passing through a screen (2 mm) were as follows: AND X MAX, 34%; TEX x PEP, 70%; and C. foetidissima, 98%. ~aits 202~08!~

formulated from TEX x PEP F2 fruits grown in Ari20na were supplied by American Cyanamid; 44% passed through a 2-mm screen.
A noncucurbit carrier, 10-mesh corn grits (71%
of which passed through a 2-mm screen), was obtained from Illinois Cereal Mills~ Paris, Illinois (designated as pesticide carrier grits, 980). This carrier was impregnated with different levels of cucurbitacins by addition of varying amounts of a chloroform extract of AND x MAX fruit containing 0.12~ cucurbitacins B-and D
(Metcalf, et al., Proc. Natl. Acad. Sci. USA, 77:3769-3772 (1980), Metcalf, et al., Environ. Entomol., 11:921-927 (1982)). The cucurbitacin-containing areas were scraped from plates, extracted in anhydrous methanol, centrifuged, and the W absorption at 210 nm determined by W spectrophotometry. Content of cucurbitacins was quantified from standard curves of pure cucurbitacins B, D, E, and I, and E-glycoside (Metcalf, et al., Proc.
Natl. Acad. Sci. USA, 77:3769-3772 (1980), Metcalf, et al., Environ. Entomol., 11:921-927 (1982)).
To determine which cucurbitacins were active as kairomones to diabroticite beetles, thin-layer plates of silica gel with fluorescent indicator on polyethylene terephthalate (Eastman Chromogram, Eastman Kodak, Rochester, New York) were developed from the chloroform extracts of dried cucurbit baits and exposed to the feeding of 100 SCR or WCR beetles for 2-4 d (Metcalf, et al., Proc. Natl. Acad. Sci. USA, 77:3769-3772 (1980), Metcalf, et al., Environ. Entomol., 11:921-927 (1982)). The beetles fed avidly on areas with active cucurbitacins with a sensitivity of detection of ca.
0.01 llg.
The various dried, ground, and insecticide-treated baits were given preliminary field evaluation by placing uniform amounts in petri-dish halves (150-mm diameter) that were placed at random on the ground in 2Q2~085 cucurbit and corn rows heavily infested ith SCR, WCR, and SCB adults. Ten dishes were used for each treatment; the maximum dosage of dried baits used was 300 mg per dish, equivalent to 17 kg/ha. Mortality counts of adult beetles in the dishes were made after 20-40 h.
~ Evaluations of the various bait formulations were also made by broadcast applications to 0.04-ha plots of sweet corn. Weighed samples of the baits were broadcast over the tops of the corn plants so that a portion of the bait was retained on leaves and silks.
Before treatment, the total number of rootworm beetles on the corn plants in each of the plots was estimated by counting the number on each of 50 plants and extrapolating the count to the entire plot (precount).
Twenty-four hours after treatment, dead rootworm beetles on the ground were counted in each plot and expressed as a ratio of the precount (percentage killed). Three-day post-treatment counts on the corn were also determined (total number of beetles per plot) and were expressed as a ratio of the precount (percentage of reduction).
Different rates of application of methomyl-impregnated bait were further evaluated in 0.02-ha sweet-corn plots in an analogous manner with 24-h posttreatment counts of dead beetles on the ground. Statistical evaluations were made with Duncan, Biometrics, 11:1-42 (1955) multiple range test (P=0.05).
The role of cucurbitacin kairomones as arrestants and feeding stimulants for SCR beetles was unequivocally demonstrated by the petri-dish tests summarized in Table 12.

~4085 Effect of cucurbitacins (cucs), indole, and eugenol additives to corn-grit baits with 0.1% methomyl in petri-dish tests against D. u. howardi adults in cucurbit plots.
No. dead and Test Bait additive(s)moribund beetles (xiSD) 1 0 Cucs - A None Oa 0.0036% eucs 7.9 i ll.la 0.012% cues 25.9 i 18.5ab 0.036% cucs 37.9 i 23.3bc 0.120% cucs 65.9 i 28.4c Cucs, indole, or eugenol B None 1.2 i 2.Sa 0.1% eugenol Oa 0.120% cucs 22.~ i 18.3b 0.120% eues + 1% eugenol86.0 i 50.3e 0.120% eues + 1% indole9.S i 4.lb Means followed by the same letters are not signifieantly different (P>0.05; Dunean's (l9SS) multiple range test). Test A, 3 Oetober; test B, 24 August.

- 2024~

Corn-grits bait with 0.1% methomyl was ineffective, but the addition of 0.0036-0.120% cucurbitacins increased the kill proportionately. The effective dose range (Table 12) is equivalent to 67-202 g/ha of cucur-bitacins. This range corresponds to previously reported field experiments in which AND x MAX and TEX x PEP baits with 0.1% methomyl were broadcast at 11-33 kg/ha or 60-240 of cucurbitacins for effective control of diabroticites (Metcalf, _ al., Cucurbit Genet. Coop.
Rep., 4:37-38 (1981),-Metcalf, et al., Cucurbit Genet.
Coop. Rep., 6:79-81 (1983)).
While the volatile additives eugenol and indole, are attractive to diabroticite beetles on cylindrical sticky traps (Ladd, et al., J. Econ.
Entomol., 76:1049-1051 (1983), Andersen, et al., J.
Chem. Ecol., 12:687-699 (1986), Lampman, et al., J.
Chem. Ecol., 13:959-975 (1987)), the combination of attractants with corn-grits bait containing 0.1%
methomyl are ineffective (Table 12). However, in the presence of cucurbitacin arrestants, the incorporation of 1% eugenol improved the efficiency to kill of SCR
beetles from 3- to 4-fold with the corn-grits bait (Table 12). Corresponding data from petri-dish tests with TEX x PEP bait showed that the incorporation of 1%
eugenol into this bait produced a similar 4-fold increase in the kill of SCR beetles, but did not improve the efficiency of kill for WCR and SCB beetles. Indole was ineffective in improving bait performance, but the addition of 1% veratrole to the TEX x PEP bait increased the kill of SCR beetles in petri-dish tests from a mean (+SD) of 27.9+53 dead beetles for 10 dishes with 0.1%
methomyl alone to 63.1+39.9 dead beetles with the bait containing veratrole.
Bait applications to evaluate various insecticides. The relative effectiveness of a variety of insecticides incorporated at 0.01-0.1% (wt/wt) in TEX

x PEP dry ground bait was compared. The following insecticides were tested: bendiocarb, carbaryl, carbouran, methomyl, oxamyl; dimethoate, malathion, isofenphos, phospholan, and mephospholan; and permethrin, cypermethrin, fenvalerate, and flucythrinate. In l-to 3.3-m2 plots of corn and cucurbits, the carbamates methomyl, carbofuran, carbaryl, and bendiocarb at 0.1~ were more effective than the pyrethroids permethrin, cypermethrin, fenvalerate, and flucythrinate at 0.01%. Isofenphos was the most effective of the organophosphorus insecticides evaluated in both corn and cucurbits. The overall greater effectiveness of the carbamates in these small-plot tests is attributed to their rapid kill and lack ofrepellent effect. The pyrethroids were somewhat repellent to the rootworm beetles.
In larger scale field evaluations in sweet corn, broadcast applications of TEX x PEP (F2) bait, with both the 0.1% methomyl and 0.1% isofenphos baits produced comparable results. Apparently, both carbamates and organophosphorus insecticides are effective toxicants in cucurbitacin baits.
- OptLmum insecticide concentration, efficiency of application, and duration of effectiveness of - baits. Although adult corn rootworm beetles frequent corn leaf sheaths, silks, and tassels during the daytime and are seldom seen on the ground, the cucurbitacin baits applied directly to the ground were surprisingly effective. Baits broadcast over the tops of corn plants were also effective at comparable rates. Both modes of bait application were highly selective. No dead predators were found after ground treatment, and dead coccinellids were observed only occasionally after broadcase treatment.
Evaluation of TEX x PEP F2 bait broadcast over several corn plots at five different combinations of ` 20240~

methomyl concentration and rates of application showed that 0.1~ methomyl bait applied at 33 kg/ha was optimally effective. The application rate for methomyl alone was 33.6 g/ha, and this rate of application is much more efficient than the conventional dosage of 1,121 g/ha of carbaryl for aircraft sprays to control adult corn rootworm beetles. An equal amount of methomyl (33.6 g/ha) in bait applied at 3.3 kg/ha was less effective because of inefficient distribution of bait particles over the treated area. Reduction of the total amount of methomyl in the bait from 112 g/ha to llg/ha while the amount of bait was kept constant at 11 kg/ha decreased the kill of rootworm beetles, although large numbers of dead beetles were found on the ground in the treated plot.
Application of AND x MAX bait containing 0.1%
methomyl at 33 kg/ha to sweet-corn plots heavily infested with corn rootworm adults resulted in substantial reduction of adult beetles after 1 d (WCR, 98%; SCR, 56%; NCR, 100%). The lower apparent reduction of SCR beetles in this test compared with the previous was probably due to continued invasion of the small plots by SCR beetles. A reduction in the numbers of beetles per plot was also observed after 2 d, and incremental numbers of dead and moribund beetles were found on the ground after 3, 7,-and 11 d. This indicates that the poison baits may have potential for killing diabroticites for substantial periods of time after a single application.
The foregoing results demonstrate some of the possibilities for regulating insect behavior by incorpo~ation of plant kairomones into toxic baits.
Cucurbitacins from dried bitter squash fruits or roots were very effective in arresting the several species of rootworm beetles and in causing them to feed on baits poisoned with a variety of insecticides. With rapidly 202~ 8~

acting insecticides such as methomyl, there was little or no survival from exposure to the baits.
The diabroticites are susceptible to a wide variety of organophosphorus, carbamate, and pyrethroid insecticides that could be incorporated into bitter cucurbita baits (Chio, et al., J. Econ. Entomol., 71:289-393 (1978)). Factors influencing the choice of insecticide include intrinsic beetle susceptibility, repellence of beetle feeding, solubility in water, and photostability as it affects persistence of the toxic bait under the influence of sunlight and moisture.
Although carbamate insecticides consistently had the lowest LD50 values for the important pest species of rootworms (Chio, et al., J. Econ. Entomol., 71:289-393 (1978)), carbamate resistance is evident especially with SCR (Chio, et al., J. Econ. Entomol., 71:289-393 (1978)). Because of the very small amount of insecticide required for effective baits (Metcalf, Nat.
Hist. Surv., 33:175-198 (1985)), considerations of human and environmental toxicity and of the cost of insecticide are less critical than for conventional spray treatments.
The cucurbitacins were effective arrestants and feeding stimulants in such poisoned baits when broadcast at rates of 40-240 g/ha. The cucurbitacins seemed about equally effective as kairomones whether present as naturally occurring substances in dried cucurbita fruits or when extracted and impregnated in pelleted corn grits. Volatile attractants such as eugenol or veratrole improved the effectiveness of the cucurbitacin-containing baits for SCR by attracting the beetles from a distance. Such artificial kairomone baits seem to have potential for practical control of adult diabroticite beetles.

202~û85 _ 59 Cucurbita Blossom Aroma and Diabrotica Rootworm Beetle Attraction:
As described inter alia, recent research has shown that many diabroticite species found on cucurbits including the striped cucumber beetle (SCB) Acalymma vittata, the spotted cucumber beetle (SCR) D.
undecimpunctata howardi, the western corn rootworm (WCR) D. virgifera virgifera, and the northern corn rootworm (NCR) D. barberi, are attracted to olfactory cucs. The preponderance of previously described attractants are phenylpropanoids or closely related compounds e.g., eugenol, estragole, and 4-methoxycinnamaldehyde (Lampman, R.L., and Metcalf, R.L., J. Econ. Entomol., 80:1137-1142 (19873; t~mp~n, R.L., Metcalf, R.L., and Andersen, J.F., J. Chem. Ecol., 13:959-975 (1987)).
The attraction of Diabrotica beetles to the odors of Cucurbita blossoms was examined independent of visual and contact cucs, such as color, size, shape, and cucurbitacin content. Thirty grams of blossoms from cv.
Dickinson Field (C. moschata) and cv. Blue Hubbard (C.
maxima) were placed inside paper cartons, the top covered with cheesecloth (preventing contact with the blossoms), and the outside of the trap coated with sticky material. The traps were placed in a field of Blue Hubbard squash for a period of sixty minutes after which they were collected and the total number of-WCR, SCR and SCB trapped were counted.
As shown in Table 13 below, the attraction of WCR and SCB beetles to the isolated Blue Hubbard blossoms demonstrates that blossom odor alone plays an important role in the distribution of these rootworm beetles. The apparent lack of response from SCR adults is probably due to the extremely low number of beetles present in the field.

- 202~8~

Mean number of rootworm beetles in sticky traps baited with Cucurbita blossoms WCR SCR SCB
control 60 min. 6.8 + 7.5 0.5+ 0.6 1.8 _ 0.5 10 C. maxima 60 min. 86.0+ 30.6 3.0_ 1.4 11.8 + 4.5 C. moschata 60 min. 11.3~ 6.2 0.5+ 1.5 2.8 + 1.5 Over 40 individual volatile chemicals have been isolated from Cucurbita maxima blossoms and circa 25 of these have been unequivocally identified (Andersen, J.F., J. Agric. Food Chem., 35:60-62 (1987);
Andersen, J.F. and Metcalf, R.L., J. Chem. Ecol., 12:687-699 (1986)). The major odor components were evaluated as potential attractants for adult Diabrotica species using cylindrical sticky traps baited with dental cotton wicks (Metcalf, R.L., and Lampman, R.L., Paper submitted to J. Econ. Entomol., (1988)) containing from 0.01 mg to 200 mg of volatile compound.
The effective attractants show a linear log-dosage response and the attractive compounds have been rated according to their limit of response (LR), i.e.
the least amount of compound producing significant attraction over a 24 hour period vis-a-vis unbaited control traps as follows:

Amount [in mgs] Rating 30-100 +1 10-30 +2 3-10 ~3 1-3 +4 2024~
-The results are based on the means of four replicate traps and were significantly different from the control traps at P=0.01 by Duncan's multiple range test (Nie, et al., supra). A summary of the results obtained are shown in Table 14.

Summary of attraction of rootworm beetles to sticky traps baited with individual components of squash blossom volatiles Attractant Rating Volatile: NCR SCR WCR
green volatiles:
l-hexanol 0 0 0 l-hexanal 0 trans-2-hexenol 0 +1 +1 cis-3-hexenol 0 0 +1 trans-2-hexenal 0 0 0 aromatics:
1,4-dimethoxybenzene 0 0 0 1,2,4-trimethoxybenzene 0 +1 +1 benzyl alcohol 0 0 0 benzaldehyde 0 0 phenylethanol 0 +1 0 phenylacetaldehyde 0 +2 0 -~-methoxybenzyl alcohol 0 0 0 ~-methoxybenzaldehyde 0 0 0 phenyl propanoids:
indole +4 cinnamyl alcohol +3 +2 +1 trans-cinnamaldehyde 0 +4 +3 terpenoids:
a-ionone ~-ionone +4 nerolidol 0 0 0 202408~

As is clear from the results shown in Table 14, the majority of the C. maxima blossom volatiles are unnattractive when tested singularly. The green volatiles, especially trans-2-hexenol and cis-3-hexenol, are marginally attractive, as are the aromatic compounds, 1,2,4-trimethoxybenzene, phenylethanol, and phenylacetaldehyde for SCR. Trans-cinnamaldehyde is highly attractive to SCR and moderately attractive to WCR, but not appreciably attractive to NCR. Trans-cinnamyl alcohol is highly attractive to NCR, although only slightly attractive to SCR and WCR. Indole is highly attractive to WCR (LR 1 mg), but not appreciably attractive to NCR and SCR. The terpenoid, B-ionone~ is highly attractive to WCR (LR 3 mg), but unattractive to NCR and SCR. Its isomer ~-ionone is completely unattractive to all three Diabrotica species. In contrast, the most effective volatile attractants yet identified for the respective species and their LR
values are eugenol for NCR (LR 10 mg), estragole for WCR
(LR 3 mg), and 4-methoxycinnamaldehyde for WCR (LR 0.03 mg) Metcalf, R.L., and Lampman, R.L., Paper submitted to J. Econ. Entomol., (1988).

Evaluation of Seven Compounds -Including Phenylethanol Analogues as Attractants:
Test chemicals (purchased from Aldrich 3 Chemical Co.; Milwaukee, WI) were applied by capillary micropipettes to cotton dental wicks that were attached to tops of 1.0 liter cylindrical paper cartons covered with TangleTrap~, Grand Rapids, Mich. The trapping techniques used were as previously described in Example 1. Unless otherwise indicated, all treatments were made at a dosage of 100 mg or 100 ~L of attractant per trap. Control traps had untreated wicks. Four 202~0~5 -replicates of treated and untreated traps were positioned randomly 10 meters apart on top of 1 m high posts along the edges of 25-50 ha. fields of hybrid corn (Zea maYs), infested with adult NCR, SCR, and WCR
beetles. Several experiments detailed in the Tables were made in a 0.4 ha plot of sweet corn (Illini X-tra Sweet).
Attraction of Diabrotica species adults of the sticky traps was measured by the mean number of beetles caught after 1 day (ca. 24 hours) exposure. The significance of the treatments was determined by analysis of variance and the individual means were separated by Duncan's multiple range test (Statistical Package for the Social Sciences, Nie, et al., 2nd. ed.
McGraw-Hill, New York (1975). The data was logarithmically transformed for statistical analysis.
Means and standard deviations are presented for the untransformed data. Significance levels were set at P=0.05 for all statistical analyses.
The response of NCR and WCR adults in corn to sticky traps baited with 4-methoxyphenylethanol, 4-methylphenylethanol, 4-chlorophenylethanol, 4-fluorophenylethanol, 4-nitrophenylethanol, p-aminophenylethanol, control (untreated), phenyl ethanol,and 4-methoxycinnamaldehyde is shown in Table 15, below.
This experiment demonstrates that 4-methoxyphenylethanol is much more attractive to NCR than phenylethanol and that attraction is by far the greatest with para-substitution with a methoxy group. Despite the presence in this field of large numbers of Western corn rootworm adults, as evidenced by the high catch obtained using 4-methoxycinnamaldehyde (a WCR
attractant)~ WCR were not attracted by any of the phenylethanols.

202~a85 Mean number of northern corn rootworm (NCR) and western corn rootworm (WCR) per trap (+ standard deviation);
four replicates; 24 hour evaluation.

Treatment NCR WCR

4-methoxyphenylethanol187.7 i62.5d* 5.7 i 2.8a 4-methylphenylethanol 89.2 + 7.8c 12.2 i 5.6a 4-chlorophenylethanol 30.0 + 7.4b 13.7 i 3.9a 4-fluorophenylethanol 38.0 + 6.2b 14.0 i 12.3a 4-nitrophenylethanol 7.7 t 2.7a 7.9 i 5.la 4-aminophenylethanol 10.5 + 2.la 8.5 + 3.4a control (untreated) 7.5 + 3.3a 7.2 + 1.5a phenyl ethanol 50.0 +20.9b 18.7 ~ 10.2a 4-methoxycinnamaldehyde6.4 + 4.9a 567.5 +111.6b * means in the same column followed by the same letter are not significantly different (p=0.05) by Duncan's multiple range test.

202i~85 The results shown in Table 16, below, demonstrate that the para-isomer of methoxyphenylethanol is mueh more attraetive to NCR than the ortho and meta-isomers, and that para-methoxyphenylethanol is a more effective attractant than einnamyl aleohol.

Mean number of Northern eorn rootworm (NCR) and Western eorn rootworm (WCR) per trap (+ standard deviation);
four replieates, 24 hour evaluation.
Treatment NCR WCR

4-methoxyphenylethanol60.3 ~ 26.8b* 3.5 + 2.la 1 3-methoxyphenylethanol7.2 + 3.4a 3.7 + 2.la 2-methylphenylethanol13.5 + 9.3a 6.0 + S.la einnamyl aleohol 50.7 + 50.9b 4.0 ~ 1.6a eontrol (untreated)4.7 + l.9a 2.2 + 1.9 * means in the same column followed by the same letter are not signifieantly different (p=0.05) by Dunean's multiple range test.

2G2~Q8~

The results shown in Table 17 demonstrate that 4-methoxyphenyl ethanol is the most effective and specific NCR attractant evaluated. Increase in side-chain length and removal of 4-methoxy group decreased activity.

Mean number of Northern corn rootworm (NCR) and Western corn rootworm (WCR) per trap (i standard deviation);
four replicates, 24 hour evaluation.
Treatment NCR WCR

phenylethanol 12.2 + 5.Sb*9.2 + 9.2a phenylpropanol 36.2 +12.2c 4.0 + 2.9a 4-methoxyphenylethanol56.7 + 7.5c 6.0 + 4.2a 4-methoxyphenylpropanollS.S + 6.2b 3.2 + 2.2a cinnamaldehyde lS.S + 3.8b 56.0 + 34.6b 4-methoxycinnamaldehyde2.5 + l.9a 360.2 + 181.3c control (untreated) 3.5 + l.Oa 4.0 + 1.6a * means in the same column followed by the same letter are not significantly different (p=O.OS) by Duncan's multiple range test.

20~0~5 The results shown in Table 18, below demonstrate that 4-methoxyphenylethanol is much more attractive to NCR than cinnamyl alcohol and further demonstrate that levels as low as 0.1 mg of 4-methoxyphenylethanol are attractive to NCR.

Mean number of Northern corn rootworm (NCR) per trap (+ standard deviation); four replicates, 24 hour evaluation.
Treatment NCR

cinnamyl alcohol lS 30 mg 15.2 + 4.2c*
10 mg 8.5 + 2.1bc 3 mg 4.0 + 0.8ab 1 mg 3.5 + 2.4a control (untreated) 2.2 + 1.2a 4-methoxyphenyl ethanol 3 mg 72.0 ~ 10.8d 1 mg 41.0 + 23.4c 0.3 mg 32.7 + 9.4c 0.1 mg 16.2 + 1.2b control (untreated) 4.5 + 0.6a *-means in the same column followed by the same letter are not significantly different (p=0.05) by Duncan's multiple range test.

20~4~:g~

A comparative experiment was undertaken to compare the effective time duration of attraction for a single 100 mg dosage of attractant. The attractants selected were eugenol, cinnamyl alcohol, phenyl propanol, phenylethyl amine, and 4-methoxyphenylethanol. Eugenol and cinnamyl alcohol were chosen for this comparison study because these two compounds have been shown to be effective attractants for NCR. The results are shown in Table 19, below.

Effective time duration of attraction from single 100 mg dosage of attractant on cotton wicks on cylindrical sticky traps; treated cotton wicks are transferred to a new cylindrical sticky trap at the indicated interval after original treatment. Mean number (control subtracted) of Northern corn rootworm (NCR) per trap;
four replicates.
Days Treatment phenyl eugenol cinnamyl phenyl ethyl 4-methoxy alcohol propanol amine phenylethanol 1 88.2 52.0 76.5 85.9 134.5 2- 67.3 65.3 115.3125.3 231.8 314.3 17.3 38.8 39.4 93.1 418.0 19.5 25.0 27.5 51.5 520.7 17.7 41.8 155.4 128.9 629.9 7.7 10.5 44.2 49.2 730.2 10.4 10.7 38.4 76.7 835.5 11.0 29.5 17.5 99.0 1110.0 9.8 4.5 3.8 64.8 The results of this comparative study demonstrate that all compounds tested are effective attractants for NCR. ~owever, 4-methoxyphenylethanol remains a highly effective attractant for the greatest length of time.
Another comparative study was undertaken to compare the effectiveness of various amine derivatives including: benzylamine, phenylethylamine, ~-20~4085 , .

phenylpropylamine, phenylbutylamine, 4-methoxyphenyl-ethylamine and cinnamyl alcohol. These compounds were chosen because they are structural analogues of the phenyl alkanols incorporating the nitrogen atom with an unshared pair of electrons, analogous to those in the phenyl alkanols.

Mean number of Northern corn rootworm (NCR) per trap (+
standard deviation); four replicates; 24 hour evaluation.
Treatment NCR

benzylamine 57.5 + 29.6b*
phenylethylamine 206.7 + 32.3d phenylpropylamine 131.5 + 16.5c phenylbutylamine 12.2 + 6.la 4-methoxyphenylethylamine24.2 + 15.4a cinnamyl alcohol 127.0 ~ 24.2b control (untreated) 10.5 + 7.9a * means in the ~ame column followed by the same letter are not significantly different (p=0.05) by Duncan's multiple range test.

This experiment demonstrates that phenylethylamine and phenylpropylamine are most effective as attractants for NCR.

2G2408~

The foregoing illustrative examples relate to lures for attracting the adult form of Diabrotica species and to the use of these lures individually or in mixtures, or in combination with insecticides and/or compulsive feeding stimulants. While the present invention has been described in terms of specific methods and compositions, it is understood that variations and modifications will occur to those skilled in the art upon consideration of the present invention.
Accordingly it is intended in the appended claims to cover all such equivalent variations which come within the scope of the invention as claimed.

Claims (6)

1. An attractant for northern corn rootworm comprising a compound selected from the group consisting of: 4-methylphenylethanol, 4-chlorophenylethanol, 4-fluorophenylethanol, phenylethanol, phenylpropanol, 4-methoxyphenylethanol, 3-methoxyphenylethanol, 2-methoxyphenylethanol, 4-methoxyphenylpropanol, phenylethylamine, and phenylpropylamine.

2. In a method for attracting northern corn rootworm, the improvement comprising the step of employing an effective amount of a compound selected from the group consisting: 4-methylphenylethanol, 4-chlorophenylethanol, 4-fluorophenylethanol, phenylethanol, phenylpropanol, 4-methoxyphenylethanol,

3-methoxy phenylethanol, 2-methoxyphenylethanol, 4-methoxyphenylpropanol, phenylethylamine, and phenylpropylamine.

3. A toxic bait for northern corn rootworm comprising effective amounts of each of:
a northern corn rootworm volatile attractant comprising an attractant selected from the group consisting of: 4-methylphenylethanol, 4-chlorophenylethanol, 4-fluorophenylethanol, phenylethanol, phenylpropanol, 4-methoxyphenylethanol, 3-methoxyphenylethanol, 2-methoxyphenylethanol, 4-methoxyphenylpropanol, phenylethylamine, and phenylpropylamine;
a diabroticide; and a Diabrotica species non-volatile compulsive feeding stimulant.

4. The toxic bait of claim 3, wherein said diabroticide is an insecticide selected from the group consisting of: organophosphates, carbamates, and pyrethroids.

5. The toxic bait of claim 3, wherein said compulsive feeding stimulant comprises a cucurbitacin.

6. The toxic bait of claim 3, wherein said compulsive feeding stimulant is selected from the group consisting of: AND x MAX and TEX x PEP hybrid Cucurbita fruits, and Cucurbita foetidissima roots.