Identification of a female-produced sex pheromone of the western corn rootworm

A sex pheromone has been isolated and identified from virgin females of the western corn rootworm (WCR),Diabrotica virgifera virgifera LeConte. The synthesized compound, racemic 8-methyl-2-decanol propanoate, was equal in attraction to the natural pheromone when tested in the field as a trap bait against three taxa ofDiabrotica known to respond to pheromone extracts from female WCR. Five taxa (D. virgifera virgifera; D. virgifera zeae Krysan and Smith, Mexican corn rootworm;D. longicornis barberi Smith and Lawrence, northern corn rootworm;D. longicornis longicornis (Say); andD. porracea Harold) were attracted to traps baited with 8-methyl-2-decanol propanoate. The response of male northern corn rootworms (NCR) in the field peaked at a relatively low concentration of 8-methyl-2-decanol propanoate and then was severely reduced at the higher concentrations tested. Conversely, the response of male WCR in the field continued to increase up to the highest dose tested.     

Semiochemical attractants ofDiabrotica undecimpunctata howardi barber,southern corn rootworm,andDiabrotica virgifera virgifera leconte,the western corn rootworm (Coleoptera: Chrysomelidae)

During the summers of 1984 and 1985, a variety of structurally related benzenoid compounds was evaluated in sweet corn plots as attractants for adult southern corn rootworms (SCR), western com rootworms (WCR), and northern corn rootworms (NCR). Field response to the volatiles was measured by beetle counts on baited cylindrical sticky traps placed inside the corn plots at a height of l m above ground level. SCR adults were attracted late in the season (last week of August through September, 1984 and 1985) to numerous aromatic compounds, including phenylacetaldehyde, benzyl acetone, phenethyl alcohol, phenyl acetate, indole, veratrole, methyl eugenol, methyl isoeugenol, eugenol, and isoeugenol. Although many compounds attracted SCR adults late in the season, only veratrole, phenylacetaldehyde, and chavicol were significantly active in early and middle August 1985. WCR adults were attracted to a different group of compounds, namely estragole,trans-anethole, and indole. Estragole (4-methoxy-1-allylbenzene) was an effective WCR attractant from corn tasseling in early August 1985, until the end of the trapping period in late September and early October 1985. Indole andtrans-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. Traps baited with 100 mg of estragole caught an average of 20 times more WCR adults than unbaited control traps, and the females outnumbered the males in the baited traps. Estragole dosage tests were conducted in three sweet corn plots on different dates in 1985 and the minimum effective dose ranged between 5 and 30 mg/trap. Field tests with structural analogs revealed the importance of the site of unsaturation in the allylic side chain of estragole and the effect of different ring substituents on WCR response. The phenylpropanoids, eugenol and isoeugenol, significantly attracted NCR adults, even though these beetles were in low abundance in the test corn plots. Field tests indicate there is no cross-species response by WCR and NCR adults to their related phenylpropanoid attractants. However, in late August, SCR adults do respond to some WCR and NCR attractants (indole and several eugenol analogs). Electroantennographic analysis of SCR males revealed they can perceive peripherally a wide range of benzenoid compounds.     

Corn volatiles as attractants for northern and western corn rootworm beetles (Coleoptera: Chrysomelidae:Diabrotica spp.)

Synthetic corn volatiles and selected analogs were tested in commercial corn fields for attractiveness to feral northern (NCR,Diabrotica barberi) and western corn rootworm beetles (WCR,D. virgifera virgifera). Two new attractants, geranylacetone and -terpineol, were identified among corn terpenes and compared at four stages in crop development with the phenylpropanoid standards cinnamyl alcohol and 4-methoxy-cinnamaldehyde, with each component at 30 mg/trap. Dose-response relationships (0.1–100 mg/trap) and efficacy of two-component blends (30 mg/component) were also examined. More beetles were captured on traps baited with (+)- than (–)--terpineol, but the difference was statistically significant only for WCR. Captures with geranylacetone or (+)--terpineol were directly proportional to the logarithm of the attractant dose. WCR females were attracted to as little as 0.1 mg of either compound. WCR males required 1.0 mg of (+)--terpineol and were not attracted to geranylacetone at any dose. NCR required 0.3 mg of either attractant and showed less marked response differences between the sexes than did WCR. Geranylacetone and cinnamyl alcohol were equally effective attractants, whereas (+)--terpineol was significantly less attractive to WCR but more attractive to NCR than was 4-methoxycinnamaldehyde. Corn terpenes and phenylpropanoid standards produced similar seasonal response patterns in that captures tended to rise in each case as the season progressed, except during silking when no compound was attractive. Mixing corn terpenes or phenylpropanoid standards synergized responses of WCR females, but (+)--terpineol suppressed attraction of NCR females to geranylacetone.Mention of a proprietary product or company name does not imply endorsement by the U.S. Department of Agriculture.     

Single and blended maize volatiles as attractants for diabroticite corn rootworm beetles

Synthetic maize volatiles and analogs dispensed singly and blended were tested for attractiveness to western (WCR, Diabrotica virgifera virgifera) and northern corn rootworm beetles (NCR, D. barberi) in maize fields. Newly identified attractants included syn-benzaldoxime, especially for NCR, and -caryophyllene for WCR females. (±)-Linalool was more effective than was (–)-linalool. Myrcene, (+)--pinene, and (–)--pinene were unattractive. Adding methyl salicylate to (±)-linalool, (+)--terpineol, or -ionone appeared to synergistically increase capture of WCR females, but dispensing the terpenes in binary blends did not. Dose–response data for methyl salicylate, (±)-linalool, and a blend of both compounds confirmed the synergy. -Caryophyllene, but not (–)--pinene, added to the latter blend produced a further synergistic increase in WCR female capture that did not vary with sesquiterpene dose from 1.0 to 100 mg. Indole addition to the same blend caused an increase in WCR female captures indicative of synergy, assuming that each did not individually lure different segments of the WCR female population. The green leaf volatiles (Z)-3-hexenyl acetate and (Z)-3-hexen-1-ol were unattractive alone and had no influence on efficacy of traps baited with 3.3 mg each of (±)-linalool, methyl salicylate, and -caryophyllene. The latter mixture captured about half as many WCR females as did 10 mg of 4-methoxycinnamaldehyde, a potent WCR attractant standard. Substituting -ionone for (±)-linalool yielded a ternary blend that captured more beetles than did the aldehyde and was unaffected by aldehyde addition. Olive oil, which has been used to sustain attractant volatilization, did not affect captures. The results show that the blending of maize volatiles has the potential to greatly improve efficacy of lures having promising applications in corn rootworm population management.     

Response of northern corn rootworm,Diabrotica barberi Smith and Lawrence,to stereoisomers of 8-methyl-2-decyl propanoate

The four stereoisomers of 8-methyl-2-decyl propanoate were tested in South Dakota for attractiveness to the northern corn rootworm,Diabrotica barberi Smith and Lawrence (NCR). Only the 2R,8R configuration was attractive to the NCR. Inhibition of the NCR response to 2R,8R occurred when either the 2S,8R or 2S,8S isomers were components of the pheromone source. The 2R,8S configuration elicited no behavioral activity in the NCR.Coleoptera: ChrysomelidaeMention of a commercial or proprietary product does not constitute an endorsement by the USDA.Deceased.     

Dichloromethane attracts diabroticite larvae in a laboratory behavioral bioassay

A two-choice laboratory behavioral bioassay was used to demonstrate that dichloromethane elicits the dose-dependent attraction of secondinstar western and southern corn rootworms. Preliminary data suggest that second-instar banded cucumber beetles are also attracted to dichloromethane. An eluotropic series of 10 materials, including distilled water, ethanol, methanol, acetone, ethyl dichloroacetate, dichloromethane, diethyl ether, benzene, hexadecane, and hexane, was tested for attraction of western corn rootworm larvae. Dichloromethane was the only one attractive at all doses tested, and orthogonal comparisons revealed a quadratic trend (convex) for responses of larvae to increasing dose. Benzene and hexadecane also attracted larvae, but significantly fewer than dichloromethane, and only at three doses and one dose, respectively. Orthogonal comparisons revealed no linear or quadratic trend for responses of larvae to increasing doses of either compound. Dichloromethane is the first organic compound demonstrated to attract western corn rootworm larvae in the absence of carbon dioxide. Carbon dioxide has previously been reported to attract western corn rootworm larvae either independently or when combined with other organic compounds, and the sensitivity of our bioassay was tested by demonstrating the dose-dependent attraction of western corn rootworm larvae to carbonated water as a carbon dioxide source. We have also demonstrated the attraction of southern corn rootworm larvae to carbon dioxide and propose that carbon dioxide and dichloromethane behave analogously when they interact with chemoreceptor sites on larvae.     

Electrophysiologically and Behaviorally Active Volatiles of Buffalo Gourd Root Powder for Corn Rootworm Beetles

The dried, powdered roots of buffalo gourd, Cucurbita foetidissima, were tested in a cornfield and shown to attract adult northern and southern corn rootworm beetles. Coupled gas chromatography–electroantennography (GC-EAG) analyses of headspace samples of the root powder showed several GC-EAG-active compounds on the antennae of female northern, southern, and western corn rootworms. Among other techniques, solid-phase microextraction and GC-mass spectrometry identified the following GC-EAG-active compounds: hexanol, nonanal, 1-octen-3-ol, benzaldehyde, benzyl alcohol, (E)-3-octen-2-one, (E,E)-3,5-octadien-2-one, and (E,Z)-3,5-octadien-2-one. EAG dose–response studies of several of the identified root powder volatiles also were performed and compared with results from known attractants. Field tests of synthetic root powder volatiles in commercial cornfields showed that northern corn rootworm adults were attracted to (E,E)-3,5-octadien-2-one. The antennae of the Diabrotica species and the field tests showed specificity for different geometrical isomers of 3,5-octadien-2-one, with a behavioral preference for (E,E)-3,5-octadien-2-one. In addition, we have shown that the efficacy of buffalo gourd root powder as a feeding stimulant and arrestant can be enhanced for northern and western corn rootworm adults by augmenting buffalo gourd root powder with additional (E,E)-3,5-octadien-2-one.     

Behavioral responses of western corn rootworm larvae to volatile semiochemicals from corn seedlings

Corn seedling volatiles collected cryogenically are highly attractive to western corn rootworm larvae,Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), in a laboratory bioassay. Carbon dioxide is known as an attractant for western corn rootworm larvae, and the amount of carbon dioxide in the cryogenic collections was measured with an infrared gas analyzer. In a choice test between a source containing carbon dioxide alone and a source containing corn seedling volatiles with an equal amount of carbon dioxide (verified by infrared gas analysis), western corn rootworm larvae chose the corn volatile source significantly more often than the side with carbon dioxide alone. This indicates that carbon dioxide is only one of the volatiles from corn seedlings that is behaviorally important and that other compounds of behavioral importance are present as well.     

Isolation of corn semiochemicals attractive and repellent to western corn rootworm larvae

Dichloromethane extracts of germinating corn are significantly attractive to western corn rootworm larvae in choice tests with equal levels of carbon dioxide present on both sides of the choice. Two fractions that are significantly attractive and two fractions that are significantly repellent to larvae were isolated from these extracts of germinating corn by gas chromatography and silica gel chromatography. In a separate set of experiments, Porapak N was used to collect headspace volatiles from germinating corn; significantly more larvae were attracted to aliquots of these extracts in singlechoice tests without added carbon dioxide present than to solvent controls.Coleoptera: Chrysomelidae.     

Field Capture of Northern and Western Corn Rootworm Beetles Relative to Attractant Structure and Volatility

We used field assays to study attraction of feral northern and western corn rootworm beetles (Diabrotica barberi and D. virgifera virgifera) to a series of mostly nitrogenous and benzenoid synthetic compounds allied with host plant and floral aromas. Vaporization rates were obtained for most field-tested compounds and selected additional lures under both ideal and field-representative, but constant, conditions. Although many test compounds showed at least trace activity for one or both species, methyl benzoate and some of its derivatives, notably methyl anthranilate and methyl 4-methoxybenzoate, merited emphasis as effective new lures for females. Structural alteration of methyl benzoate had consistently negative effects on northern corn rootworm captures despite variable effects on release rate, whereas western corn rootworm was more strongly attracted to methyl anthranilate and methyl 4-methoxybenzoate than to the considerably more volatile parent compound. Phenylacetaldoxime was attractive to females of both species, but no more so than syn-benzaldoxime, included as reference. Release rate was disproportionately low for benzaldoxime, as well as other nitrogenous lures, under field compared with ideal conditions. The attractiveness of salicylaldoxime to northern corn rootworm, despite its low field release rate, and the unattractiveness of methyl salicylate, having a methyl ester in place of the oxime group, similarly highlighted importance of the oxime moiety for reactivity of this species.     

Long-chain free fatty acids: Semiochemicals for host location by western corn rootworm larvae

A bioassay-driven sequential fractionation scheme was used to isolate fractions of a crude dichloromethane maize seedling extract behaviorally active to larvae of the western corn rootworm,Diabrotica virgifera virgifera LeConte. (Z,Z)-9,12-Octadecadienoic (linoleic) acid, (Z)-9-octadecenoic (oleic) acid, and octadecanoic (stearic) acid were identified from a purified fraction of maize extract that was attractive to western corn rootworm larvae in choice tests with equal levels of carbon dioxide on both sides of the choice. When synthetic linoleic, oleic, and stearic acids were tested together in the amounts and proportions found in the attractive fraction (1000, 800, and 300 ng of linoleic, oleic, and stearic acids, respectively), significantly more western corn rootworm larvae were found on the side with synthetic free fatty acids plus carbon dioxide than on the side with carbon dioxide alone. Results of the choice-test bioassays were not significantly different when the synthetic blend of free fatty acids was substituted for the purified maize fraction. Neither the purified extract nor the synthetic blend was behaviorally active in preliminary single-choice experiments without carbon dioxide. Linoleic, oleic, and stearic acids were also tested individually in the choice test bioassay with carbon dioxide on both sides of the choice to determine a dose-response curve. Linoleic and oleic acid each had one dose that was significantly attractive in conjunction with carbon dioxide on both sides of the choice, but stearic acid was not active in the doses tested.     

Identification of a volatile attractant forDiabrotica andAcalymma spp. from blossoms ofCucurbita maxima duchesne

Fractionation of headspace volatiles fromCucurbita maxima blossoms by high-performance liquid chromatography resulted in the isolation of a single component which was highly active in an electroantennogram bioassay onDiabrotica undecimpunctata howardi antennae. This compound was identified as indole by gas chromatography-mass spectrometry. Field-trapping bioassays were conducted which indicated that indole is a potent attractant of the western corn rootworm,D. virgifera virgifera, and the striped cucumber beetle,Acalymma vittatum. The southern corn rootworm,D. u. howardi, did not respond, despite its strong EAG response. The sex ratio ofD. v. virgifera found in indole-baited traps varied seasonally. Males were trapped in abundance in late July and later September, 1983, while females were more abundant August and early September. The effectiveness of indole as aD. v. virgifera attractant also varied seasonally. A prolonged period of depressed trap catches occurred in early August 1983, during the silking and tasseling period of the corn in the field where trapping was carried out.Coleoptera: Chrysomelidae.     

Antifeedant effects of proteinase inhibitors on feeding behaviors of adult western corn rootworm (Diabrotica virgifera virgifera)

Low-molecular-weight peptidyl proteinase inhibitors (PIs) including leupeptin, calpain inhibitor I, and calpeptin were found to be potent antifeedants for adult western corn rootworm (WCR) against the phagostimulation of cucurbitacin B (Cuc B) or a corn pollen extract (CPE). Leupeptin was the strongest (ED₅₀ = 0.36 and 0.55 nmol/disk for Cuc B and CPE, respectively) among PIs tested with an antifeedant potency much stronger than the steroid progesterone (ED₅₀ = 2.29 and 5.05 nmol/disk for Cuc B and CPE, respectively), but slightly less than the reference alkaloid, strychnine (ED₅₀ = 0.17 and 0.37 nmol/disk for Cuc B and CPE, respectively). All active PIs contain a di- or tripeptidyl aldehyde moiety, indicating that PIs exert their antifeedant effects by covalent interaction with putative sulfhydryl (SH) groups on taste receptors as do these PIs with cysteine proteinases. However, opposite inhibition potency against Cuc B versus CPE by two thiol-group reducing agents, DTT and L-cysteine, and the results with other cysteine-modifying reagents obscure the net functional role of SH groups at WCR taste chemoreceptors. Surprisingly, the model phagostimulant for diabroticites, Cuc B, was more easily counteracted by these feeding deterrents than the stimulants present in CPE. Three-dimensional structure–antifeedant relationships for the PIs suggest that a novel taste chemoreception mechanism exists for these peptidyl aldehydes or that they fit partially into a strychnine binding pocket on protein chemoreceptors. Favorable economic benefit may be achieved if PIs are discovered to be useful in adult WCR control, since both pre- and postingestive sites would be targeted.     

Control of low-density gypsy moth (Lepidoptera: Lymantriidae) populations by mating disruption with pheromone

This four-year study demonstrated that low-density populations of the gypsy moth,Lymantria dispar (L.), were effectively suppressed by annual aerial application of 75 g of racemic disparlure per hectare formulated in plastic laminate flakes. These tests also showed that, when plots were treated with 150 g of pheromone per hectare in 1990 only and left untreated for the following three years, populations continued to be suppressed in 1991–1993 as compared with the controls. Although none of the plots were treated in 1994, population assessment continued and showed that the gypsy moth population density remained low in the plots that had been treated annually for the preceding four years. The laminate flakes released an average of 0.48 g disparlure per day per hectare from each of the two applications in 1990, and 0.72 g per day per hectare from the single application in each of the following three years (1991–1993). Only 27–40% of the applied pheromone dose was emitted during male moth flight.     

Identification of Semiochemicals Associated with Jeffrey Pine Beetle, Dendroctonus jeffreyi

Analysis of frass extracts and aerations from adult beetles confirmed earlier studies identifying 1-heptanol as a principal pheromone constituent produced by female Dendroctonus jeffreyi Hopkins. The aerations also indicated that males produced 33% (+) and 67% (–)-frontalin, previously unreported for this species, and >99% (+)-exo-brevicomin. Both males and females were responsive to these compounds in electrophysiological studies. Laboratory bioassays and field trapping experiments indicated that males and females were attracted to a mixture of 1-heptanol plus the principal host resin component heptane, but females were less attracted than males. Inclusion of racemic exo-brevicomin at 0.1% concentration in the traps increased the response of females. Inclusion of racemic frontalin above a 1% concentration in the normally attractive lures blocked arrival of beetles to the traps. While the female-produced pheromone components and host synergists are very different between the sibling species D. jeffreyi and Dendroctonus ponderosae Hopkins, the principal male-produced compounds are the same, and the response of the female beetles to those compounds appears to be similar.     

Analysis of long-range reproductive behavior of maleDiabrotica virgifera virgifera leconte andD. Barberi Smith and Lawrence to stereoisomers of 8-methyl-2-decyl propanoate under laboratory conditions

Behavioral analysis of male attraction to stereoisomers of 8-methyl-2-decyl propanoate was conducted withDiabrotica virgifera virgifera andD. barberi using a flight tunnel.D. barberi males were attracted to the 2R,8R isomer only, and the response was inhibited by the addition of 2S,8R.D. v. virgifera males were also attracted to the 2R,8R isomer and were neither attracted nor inhibited by any of the other isomers. Males attracted to the lures displayed a series of behaviors characterized by a slow hovering flight upwind towards the odor source. While significant numbers of males of both species landed on the pheromone lure, none attempted copulation, indicating that other cues are necessary to induce this behavior.     

Behavioral responses of western corn rootworm larvae to naturally occurring and synthetic hydroxamic acids

Hydroxamic acids have been shown to be toxic to many pest insects and pathogens. In this study, the behavioral responses of western corn rootworm larvae to naturally occurring and synthetic hydroxamic acids were investigated. In a choice test between corn roots treated with hydroxamic acids and roots treated with distilled water (control), western corn rootworm larvae chose to burrow into the control roots significantly more often than compoundtreated roots. In addition, when corn roots were treated with different hydroxamic acids in a designed searching-behavior test, neonate larvae of western corn rootworm responded by significantly reducing the number of turns, while the area searched and locomotor rate significantly increased. The responses were dependent on the concentrations of the test compounds. These results suggested that hydroxamic acids were acting as behavior-modifying and possibly feeding-deterrent chemicals.     

Pheromone-mediated behavior of the gypsy moth

The pheromone-mediated behavior of gypsy moth males was studied in both natural and simulated populations in central Pennsylvania. Feral males released into 50-m-diam plots, each with 2 feral females around the perimeter, oriented initially to trees and not to females. Neither exposure to virgin females nor exposure to wicks baited with approx 6 mg disparlure affected the subsequent sexual activity of males released into the 0.2-hectare plots. Males released into untreated plots, following 24 hr exposure in an area treated with approx 37 g/hectare of microencapsulated disparlure, located and mated with feral females within 4 min after release. None of the released males was caught in disparlure-baited Delta traps. In the disparlure-treated plot none of the females was mated. Males within this treated plot continued to search actively but did not settle down on the bark surface and initiate short-range (< 15 cm) search behavior. In plots testing the effect of various ratios of baited wicks to virgin females on disruption, there was no evidence of mating disruption due to point-source confusion. There were no significant differences in the responses of feral males to either virgin females or the various portions of Hercon wicks placed out in 0.2-hectare plots. In a series of tests using feral virgin females given various treatments to alter their physical and chemical characteristics (i.e., removed wings, denuded abdomen, washed in xylene, etc.), all females elicited the full range of sexual behavior responses of the male moths in natural populations. Apparently, males stimulated by pheromone are capable of using a number of different additional stimuli to initiate and terminate short-range sexual behavior patterns. Lymantria dispar (L.); Lymantriidae: Lepidoptera.Authorized for publication on April 19, 1976 as paper no. 5066 in the journal series of the Pennsylvania Agricultural Experiment Station, Experiment Station Project No. 2044.     

Gypsy moth mating disruption: Dosage effects

Small (1-hectare) plots in a dense gypsy moth (Lymantria dispar) infestation were treated with 5, 50, or 500 g racemic disparlure, and effects on male trap catch and mating behavior were studied. Capture of males in traps baited with 1, 10, 100, or 1000 g (+)-disparlure declined as disruptant dosages increased. Traps with high levels of attractant caught moths when capture in those with lower baitings was dramatically reduced. While all disruptant dosages reduced trap catch, it was reducd at least 95% at all attractant levels at the 500-g disruptant application rate. Visual estimates indicated that male moth density was similar in treated and control plots; female mating success was reduced 6.5, 34.5, and 84% in plots with 5, 50, and 500 g/ hectare disruptant, respectively. The duration of precopulatory and copulatory periods was similar for all females that were observed mating, regardless of disruptant treatment. It is proposed that reduced trap catch and female mating success are due to effects of atmospheric synthetic disparlure (disruptant) camouflaging natural attractant point sources.Lepidoptera: Lymantriidae.     

(R *,S *)-5-hydroxy-4-methyl-3-heptanone male-produced aggregation pheromone ofSitophilus oryzae (L.) andS. zeamais motsch

A male-produced aggregation pheromone, common to the rice weevil,Sitophilus oryzae, and the maize weevil,S. zeamais, was isolated and identified from hexane extracts of highly absorbent paper disks exposed individually to young virgin male weevils. A combination of preparative column and gas-liquid chromatography of disk extracts yielded purified natural pheromone. When analyzed by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry, the structure of the natural pheromone, sitophilure, proved to be (R ^*,S ^*)-5-hydroxy-4-methyl-3-heptanone, of unknown enantiomeric composition. Synthetic racemic pheromone was highly attractive to males and females of both rice and maize weevils. In addition, both sexes of the granary weevil,S. granarius (L.) were attracted to the racemic preparation.