Repellent Effects of Methyl Anthranilate on Western Corn Rootworm Larvae (Coleoptera: Chrysomelidae) in Soil Bioassays.

Methyl anthranilate (MA), a compound in maize roots that is repellent to western corn rootworm larvae (Diabrotica virgifera virgifera LeConte) was tested in behavioral bioassays in a soil environment. MA prevented larvae from locating roots of a maize seedling, and the repellency strengthened with increasing rates of MA. In a simple push-pull strategy between an MA-treated seedling and an untreated seedling, granules containing 0.1 mg/g MA pushed larvae to the untreated seedling. This push effect increased with dose, with 90% repellency observed for the highest dose tested (100 mg/g). Chemical analysis showed that MA concentrations remained high for 4 wk in dry, sterilized or unsterilized soil, but declined rapidly in moist soil. After 7 d, 50% less MA was recovered in moist, sterilized soil than in dry soil, and only a trace of MA remained in unsterilized moist soil, suggesting that both moisture and microbial activity contributed to the loss of MA. Various (MA) carrier granules were tested in bioassays after aging in moist soil. After 1 d, all of the MA granules were repellent at the 10 mg/g rate and clay granules were also effective at 1 mg/g. After 1 wk, only molecular sieve granules elicited repellency, but that activity disappeared after 2 wk. These results demonstrate that MA is repellent to western corn rootworm larvae in the soil environment and may have potential as a rootworm treatment if formulations can be developed that protect the material from decomposition in the soil.

Methyl Anthranilate as a Repellent for Western Corn Rootworm Larvae (Coleoptera: Chrysomelidae).

Methyl anthranilate was identified as the active compound in extracts of maize (Zea mays L.) roots that were shown to be repellent to neonate western corn rootworm (Diabrotica virgifera virgifera LeConte) larvae. A bioassay-driven approach was used to isolate the active material from diethyl ether extracts of roots from germinating maize seeds. Separation of the extract on a Florisil column yielded an active fraction of 90:10 hexane:diethyl ether. Analysis with gas chromatography-mass spectrometry identified two compounds in the active fraction: indole (2,3-benzopyrrole) and methyl anthranilate (methyl 2-aminobenzoate). When tested in behavioral bioassays, methyl anthranilate elicited a significant (P < 0.05) repellent response at doses of 1, 10, and 100 µg. In subsequent single-choice bioassays, 1, 10, and 100 µg of methyl anthranilate prevented larvae from approaching 10 mmol/mol concentrations of carbon dioxide, which is normally highly attractive to the larvae. Indole, the other compound identified from the active fraction, did not elicit a behavioral response by the larvae. Methyl anthranilate has potential for development as a management tool for western corn rootworm larvae and may be best suited for use in a push-pull control strategy.

Monogalactosyldiacylglycerols as Host Recognition Cues for Western Corn Rootworm Larvae (Coleoptera: Chrysomelidae).

Monogalactosyldiacylglycerol (MGDG) was identified as a host recognition cue for larvae of the western corn rootworm Diabrotica virgifera virgifera LeConte. An active glycolipid fraction obtained from an extract of germinating maize roots was isolated with thin layer chromatography using a bioassay-driven approach. When analyzed with LC-MS (positive ion scanning), the assay-active spot was found to contain four different MGDG species: 18:3-18:3 (1,2-dilinolenoyl), 18:2-18:3 (1-linoleoyl, 2-linolenoyl), 18:2-18:2 (1,2-dilinoleoyl), and 18:2-16:0 (1-linoleoyl, 2-palmitoyl). A polar fraction was also needed for activity. When combined with a polar fraction containing a blend of sugars (glucose:fructose:sucrose:myoinositol), the isolated MGDG elicited a unique tight-turning behavior by neonate western corn rootworm larvae that is indicative of host recognition. In behavioral bioassays where disks treated with the active blend were exposed to successive sets of rootworm larvae, the activity of MGDG increased over four exposures, suggesting that larvae may be responding to compounds produced after enzymatic breakdown of MGDG. In subsequent tests with synthetic blends composed of theoretical MGDG-breakdown products, larval responses to four synthetic blends were not significantly different (P<0.5) than the response to isolated MGDG. GC-MS analysis showed modest increases in the amounts of the 16:0, 18:0, and 18:3 free fatty acids released from MGDG after a 30-min exposure to rootworm larvae, which is consistent with the enzymatic breakdown hypothesis.

Isolation and characterization of host recognition cues in corn roots for larvae of the western corn rootworm (Coleoptera: Chrysomelidae).

Behavioral bioassays were used to isolate compounds from germinating corn roots that elicit a host recognition response (tight-turning behavior) by neonate larvae of the western corn rootworm Diabrotica virgifera virgifera LeConte. When a behaviorally active extract of germinating corn roots was separated into an aqueous partition and a hexane partition, significantly more larvae (P < 0.05) responded to the recombined partitions than to either partition alone, demonstrating that the active material is a blend comprising both polar and nonpolar compounds. When the aqueous partition was separated with reverse-phase solid phase extraction, most of the behavioral activity was retained in the 100% water fraction (F-1). Gas chromatography-mass spectrometry analysis determined that F-1 contained a blend of small sugars, diacids, amino acids, and inorganic compounds. The nonpolar partition was separated on a silica column, and the resulting fractions were tested in combination with F-1 from the aqueous separation. More than 70% of larvae responded to the 100% acetone fraction (fraction B) in combination with F-1, and the response to this treatment was significantly higher than responses to the other nonpolar fractions or to F-1 alone. Methyl esterification of fraction B, followed by gas chromatographic fatty acid methyl ester analysis, confirmed that fraction B primarily consisted of lipids containing fatty acyl groups.

Antixenosis in maize reduces feeding by western corn rootworm larvae (Coleoptera: Chrysomelidae).

SUM2162 is the first known example of a naturally occurring maize, Zea mays L., genotype with antixenosis (nonpreference) resistance to western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), larval feeding. Behavioral responses of neonate western corn rootworm larvae were evaluated in laboratory bioassays with seven maize genotypes selected for native resistance to rootworm feeding damage. Two susceptible maize genotypes and one transgenic (Bacillus thuringiensis) maize genotype were included as controls. In soil bioassays with cut roots, no larvae entered the roots of the resistant variety SUM2162, but at least 75% of the larvae entered the roots of every other maize type. Larvae made significantly fewer feeding holes in the roots of SUM2162 than in all the other maize genotypes, except the isoline control. In feeding bioassays, larval feeding varied significantly among maize genotypes, but there was no significant difference between the resistant varieties and the susceptible controls. There were no significant differences among any of the genotypes in host recognition (search) behavior of larvae after exposure to the roots. Little variation in feeding stimulant blends was observed among maize genotypes, indicating minimal contribution to the observed antixenosis.

Localized search cues in corn roots for western corn rootworm (Coleoptera: Chrysomelidae) larvae.

Cues that elicit a characteristic localized search behavior by neonate larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), were extracted from living corn, Zea mays L., roots with acetone. Larvae were exposed to corn roots or to an acetone extract of corn roots and then transferred into a bioassay arena where their movements were tracked and recorded. After a 5-min exposure to live corn roots, larvae produced highly convoluted tracks that were indicative of a localized search behavior, and these distinctive tracks were also produced by larvae exposed to an acetone extract of corn roots. Larvae exposed to a filter paper control moved in relatively straight paths that were indicative of ranging behavior. Larval tracks were recorded by means of a videocamera and tracking software, and four parameters of movement were used to quantify the tracks: mean turn angle, mean meander, total distance, and maximum distance from origin. For every parameter measured, tracks resulting from exposure to the control were significantly different from tracks resulting from exposure to live corn roots and to all doses of the corn root extract. In a separate experiment, larvae exposed to corn root pieces or corn root juice exhibited the localized search behavior, but larvae exposed to oat root pieces and oat root juice (nonhost) exhibited ranging behavior.

Identification of feeding stimulants in corn roots for western corn rootworm (Coleoptera: Chrysomelidae) larvae.

Using a bioassay-driven approach, we have isolated and identified a blend of compounds from the roots of germinating corn, Zea mays L., that serve as feeding stimulants for neonate western corn rootworm larvae, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). The active blend is a combination of simple sugars (30:4:4 mg/ml glucose:fructose:sucrose in the corn root) plus at least one of the free fatty acids in germinating corn roots (2:5 mg/ml oleic acid:linoleic acid in the corn root). When an extract of germinating corn was partitioned into an ethyl acetate fraction and an aqueous fraction, full feeding occurred only when the two fractions were recombined, indicating that the phagostimulant was comprised of both polar and nonpolar components. Gas chromatography-mass spectrometry analysis of root extracts from germinating corn seedlings revealed a blend of 20 compounds from a variety of chemical classes, including small sugars, diacids, amino acids, inorganic compounds, and free fatty acids. When the major components were tested in feeding bioassays, the sugars and lipids were shown to be essential for feeding by larvae, but the two classes of compounds were only effective when combined. The sugars alone elicited feeding by only 40% of larvae, but the percentage of larvae feeding was increased significantly with the addition of linoleic acid (91.7% larvae feeding) or oleic acid (85.8% larvae feeding). The amino acids alone were not essential elements for feeding by western corn rootworm larvae.

Insecticide enhancement with feeding stimulants in corn for western corn rootworm larvae (Coleoptera: Chrysomelidae).

Amounts of the insecticide thiamethoxam required for 50% mortality of western corn rootworm larvae, Diabrotica virgifera virgifera LeConte, were reduced 100-fold when extracts of germinating corn, Zea mays L., were used to entice neonate larvae to feed on it. In behavioral bioassays, neonate rootworm larvae fed vigorously on filter paper disks treated with liquid pressed from corn roots. Moreover, disks treated with an acetone extract of corn (dried and rewetted with water) also elicited strong feeding from larvae. Larvae wandered away from filter paper disks treated with distilled water without feeding. Dilutions of thiamethoxam were tested in the bioassay alone or with corn extract and the efficacy of this insecticide was improved by the addition of the corn extract. For solutions containing 10 ppm thiamethoxam, 95% larval mortality occurred after 30 min of exposure when corn extract was present, but only 38% mortality occurred when the same concentration of insecticide alone (no feeding stimulants) was tested. Larval mortality after 24 h was significantly higher for corn extract-treated disks with 0.01, 0.1, 1, or 10 ppm insecticide than for the same concentrations without corn extract. Thiamethoxam did not deter larval feeding on corn extract, even at the highest concentration of thiamethoxam tested.

Disruption of host location of western corn rootworm larvae (Coleoptera: Chrysomelidae) with carbon dioxide.

Elevated concentrations of carbon dioxide (CO2) prevented neonate larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, from locating the roots of growing corn in behavioral bioassays conducted in soil tubs. When CO2 was pumped into one end of a soil tub, significantly more larvae were recovered from soil at the treated end than from soil around a growing corn plant at the opposite end of the tub. In controls with ambient air pumped into one end of a soil tub, significantly more larvae were recovered from the soil around the corn plant than from soil on the treated side. Larvae were unable to locate the roots of corn seedlings when CO2-generating materials were mixed into the soil. CO2-concentrations in soil were measured by mass spectrometry with selected ion monitoring at m/z 44. Granules composed of baker's yeast, yeast nutrients, and an organic substrate were prepared as a CO2 source and were tested in larger soil tub bioassays. Significantly fewer larvae were recovered from corn roots in the soil tubs with yeast granules than from corn roots in control soil tubs. The CO2-generating granules produced soil CO2 concentrations between 15.8 and 18.5 mmol/mol (compared with 1.7-2.6 mmol/mol in control tubs), and this was sufficient to prevent larvae from locating corn roots. In field trials, organic and inorganic CO2- generating treatments resulted in root ratings that were significantly lower than for the control plants.

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.

Sex pheromone of caddisflyHesperophylax occidentalis (Banks) (Trichoptera: Limnephilidae).

The main component of the sex pheromone of the caddisflyHesperophylax occidentalis (Banks) (Trichoptera: Limnephilidae) was identified as 6-methylnonan-3-one (enantiomeric composition has not yet been determined). Extracts of adult females elicited strong electroantennogram (EAG) responses from adult male antennae, but elicited significantly smaller EAG responses from adult female antennae. Extracts of adult males did not elicit appreciable EAG responses from antennae of either sex. Racemic 6-methylnonan-3-one was synthesized and elicited EAG responses from male antennae as strong as those obtained with female extracts. In field tests with baited sticky traps near lakes and streams, traps baited with synthetic racemic 6-methylnonan-3-one caught significantly more males than control traps. Female adults contained approximately 1 µg of 6-methylnonan-3-one per individual. Related ketones and alcohols of other chain lengths were also tentatively identified, being present in tiny amounts in female extracts. Extraction of different body parts showed that 6-methylnonan-3-one occurs only in a region near the intersegmental membrane between the fourth and fifth abdominal sternites of the female (no discrete glands were observed). Extracts of males did not contain 6-methylnonan-3-one, nor did pupae of either sex.

Hesperophylax occidentalis (Trichoptera: Limnephilidae): Electroantennogram structure-activity study of sex pheromone component 6-methylnonan-3-one.

Electroantennogram (EAG) analyses ofHesperophylax occidentalis male and female antennae were used to generate dose-response curves for synthetic, racemic 6-methylnonan-3-one and to demonstrate the chemical specificity of male antennal olfactory cells. Male antennae responded to 6-methylnonan-3-one, the main female pheromone component. Females also demonstrated a response to 6-methylnonan-3-one, but a smaller one than males. The chemical specificity of male antennal receptors was determined by comparing EAG activity of synthetic, racemic 6-methylnonan-3-one and a series of structural analogs. The three structural parameters considered, including keto position, chain length, and methyl-group position, were demonstrated to contribute significantly to the reception of synthetic, racemic 6-methylnonan-3-one by male antennae. For straight-chain nonanones with keto groups at different positions, nonan-3-one elicited the greatest EAG responses from male antennae. For straight-chain alkan-3-ones with different chain lengths, the EAG responses of male antennae to nonan-3-one and decan-3-one were the same, but greater than those to other chain lengths. For methyl-branched nonan-3-ones, 6-methylnonan-3-one elicited greater EAG responses from antennae than analogs with the methyl group at another position.

Interfamily variation in comb wax hydrocarbons produced by honey bees.

The hydrocarbons of honeybee comb wax vary significantly between colonies. This variation is explained in part by genetic (familial) differences among colonies. Even though significant differences in wax hydrocarbons exist among families, there is a high level of consistency within and among families in a correlation analysis, indicating structural constancy in comb wax. The significance of these results in interpreting the potential role of comb wax in the nestmate recognition system of the honeybee is discussed.

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.

6-Methoxy-2-benzoxazolinone: A semiochemical for host location by western corn rootworm larvae.

A bioassay-driven sequential fractionation scheme was used to isolate all portions of a crude dichloromethane corn seedling extract behaviorally active to larvae of the western corn rootworm,Diabrotica virgifera virgifera LeConte. 6-Methoxy-2-benzoxazolinone (MBOA) was identified as one of the most important components of an attractive crude corn extract. MBOA was found on or in the intact root tissues by injecting an extract of undamaged roots onto an HPLC immediately after extraction. MBOA was demonstrated to be volatile and functions as a semiochemical in conjunction with carbon dioxide in host location by western corn rootworm larvae, which are oligophagous on the roots of maize and several other species of grasses. Because MBOA occurs almost exclusively in maize and other grasses, it offers a simple way for the larvae to distinguish possible hosts from non-hosts. MBOA has previously been reported as a chemical defense against other insect species. This is the first report in grasses of a secondary compound that is toxic or a deterrent to nonadapted insect herbivores but that is used as a semiochemical in host location by a specialist insect species.

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.

Phytohormone ecology : Herbivory byThrips tabaci induces greater ethylene production in intact onions than mechanical damage alone.

Herbivory byThrips tabaci affected production of the phytohormone ethylene from living onion foliage. Ethylene analysis was performed by gas chromatography on intact onion tissue. Thrips feeding damage and a crushed thrips extract stimulated significantly greater production of eihylene than could be explained by either one-time or semicontinuous mechanical damage alone, suggesting that ethylene-inducing cues may be transferred to the plant during feeding. This is the first demonstration of increased ethylene production from insect-infested intact plants. This study suggests that herbivores affect both the phytohormone physiology and secondary chemistry of living plants because ethylene has been shown to enhance production of defensive phytochemicals.

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.

Quantitative and qualitative variation in male pheromones ofPhragmatobia fuliginosa andPyrrharctia isabella (Lepidoptera: Arctiidae).

The dihydropyrrolizine pheromones, hydroxydanaidal and danaidal, were identified from the scent organs of malePhragmatobia fuliginosa (L.) andPyrrharctia isabella (J.E. Smith). Qualitative and quantitative GLC analyses were conducted on ca. 80 field-collected males of each species. The total pheromone titer was distributed bimodally in each species with most males having either a small amount (< 10 ng) of pheromone or a large amount (1-10 µg inPyrrharctia and 0.3-3 µg inPhragmatobia).Pyrrharctia males in the 1- to 10-µg range had a predominance of hydroxydanaidal, with little if any danaidal. MostPhragmatobia males in the 0.3- to 3-µg range had danaidal with little if any hydroxydanaidal. These compounds elicited a courtship response in sexually receptive females of both species. A bioassay based on this response was used to measure the thresholds of female response to these compounds.Pyrrharctia females were more sensitive to (R)-(-)-hydroxydanaidal than to danaidal.Phragmatobia females were more sensitive to danaidal then to (R)-(-)-hydroxydanaidal.

Sex pheromone biosynthesis in the red-banded leafroller moth, studied by mass-labeling with stable isotopes and analysis with mass spectrometry.

A technique for mass-labeling was developed to study sex pheromone biosynthesis in the red-banded leafroller moth,Argyrotaenia velutinana. With this technique, the pheromone components and all fatty acyl groups in the pheromone gland were analyzed for incorporation of label in the same analytic ran with gas chromatography-mass spectrometry, using chemical ionization and selected ion monitoring (GC-SIM-CI-MS). Sex pheromone glands were incubated with fatty acids or triacylglycerols labeled with at least three deuterium atoms or carbon-13 atoms. The results of these incubations support an interpretation in which hexadecanoate is chain shortened to tetradecanoate, which is desaturated to produce (E)- and (Z)-11-tetradecenoate precursors for the sex pheromone components (E)- and (Z)-11-tetradecen-1-yl acetate. Labeled (E)- and (Z)-11-tetradecenoyl groups in synthetic triacylglycerols were not incorporated into the sex pheromone components, perhaps indicating that this lipid class is not a donor of the immediate fatty acyl precursors in sex pheromone biosynthesis.