The Effect of Western Corn Rootworm (Coleoptera: Chrysomelidae) and Water Deficit on Maize Performance Under Controlled Conditions.

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is the most important insect of maize, Zea mays L., but knowledge of its interaction with water deficit on maize production is lacking. A series of greenhouse experiments using three infestation levels of the western corn rootworm, D. virgifera virgifera, under well-watered, moderately dry, and very dry soil moisture levels were conducted to quantify the interaction of western corn rootworm and soil water deficit on B73×Mo17 maize growth and physiology. Three separate experiments were conducted. Soil moisture regimes were initiated 30 d postplanting for experiments using neonate and second-instar larvae and 30 d postinfestation in the experiment using eggs. In the neonate and second-instar experiments, there were no significant differences among western corn rootworm levels in their effects on leaf water potential, shoot dry weight, and root dry weight. The interaction of western corn rootworm and soil moisture significantly impacted the larval recovery in the neonate experiment, but no other significant interactions were documented between soil moisture levels and rootworm infestation levels. Overall, the results indicate that under the conditions of these experiments, the effect of water deficit was much greater on plants than the effect of western corn rootworm and that the interactions between water deficit and western corn rootworm levels minimally affected the measured parameters of plant performance.

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.

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.

Maize phenology affects establishment, damage, and development of the western corn rootworm (Coleoptera: Chrysomelidae).

The effects of maize (Zea mays L.) phenology on establishment and adult emergence of the western corn rootworm (Diabrotica virgifera virgifera LeConte) and plant damage to maize was evaluated in field trials in 2001 and 2002 and in the greenhouse. Although neonate western corn rootworm larvae were able to initially establish on maize roots during anthesis and early reproductive stages, these older roots were apparently unsuitable for complete western corn rootworm larval development. The number of western corn rootworm beetles that emerged from eggs that hatched during anthesis to early reproductive stages was significantly fewer than the number of beetles that eclosed from V4 to V11. Plant damage was also lowest from larvae that eclosed during anthesis to early reproductive stages. Potential causal mechanisms and implications of these data in terms of potential management strategies in the future are discussed.

Response of Maize Hybrids With and Without Rootworm- and Drought-Tolerance to Rootworm Infestation Under Well-Watered and Drought Conditions.

Anecdotal data in the past have suggested that the effect of the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), on maize yield is greater under drought and the effect of drought is greater under rootworm infestations, but no field experiments have controlled both moisture and rootworm levels. Field studies were conducted in 2012, 2013, and 2014 with treatments in a factorial arrangement of western corn rootworm infestation levels, and maize hybrids (with and without tolerance to drought and rootworm feeding). The experiment was repeated under well-watered and drought conditions in adjacent plots. Leaf water potential and stomatal conductance data suggested significant plant stress was achieved in the drought plots toward the end of the season each year and maize hybrids only played a minor role. In particular, in 2012 and 2013 yield was dramatically lower for the drought experiment than for the well-watered experiment. However, the impacts of rootworm infestation level and maize hybrids on water potential, stomatal conductance, and yield were variable across years and between experiments. In fact, the only year that the main effect of rootworm infestation levels significantly impacted yield was in 2014, when an extremely high infestation level was added and this was only for the well-watered portion of the experiment. Overall, rootworm infestation level played a relatively minor role in maize productivity and it did not appear that soil moisture level influenced that to a large degree.

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.

Effect of Cry3Bb1-expressing transgenic corn on plant-to-plant movement by western corn rootworm larvae (Coleoptera: Chrysomelidae).

Dispersal of larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, in specific combinations of transgenic corn expressing the Cry3Bb1 protein and nontransgenic, isoline corn was evaluated in a 2-yr field study. In total, 1,500 viable western corn rootworm eggs were infested in each subplot. Each year, plant damage and larval recovery were evaluated among four pedigree combinations (straight transgenic; straight nontransgenic corn; nontransgenic corn with a transgenic central, infested plant; and transgenic corn with a nontransgenic central, infested plant) on six sample dates between egg hatch and pupation. For each subplot, the infested plant, three successive plants down the row (P1, P2, and P3), the closest plant in the adjacent row of the plot, and a control plant were sampled. The number of western corn rootworm larvae recovered from transgenic rootworm-resistant plants adjacent to infested nontransgenic plants was low and not statistically significant in either 2001 or 2002. In 2001, significantly fewer larvae were recovered from transgenic rootworm-resistant plants than from nontransgenic plants when both were adjacent to infested, nontransgenic plants. In 2002, significantly more neonate western corn rootworm larvae were recovered from nontransgenic plants adjacent to infested, transgenic rootworm-resistant plants than nontransgenic plants adjacent to infested, nontransgenic plants on the second sample date. Together, these data imply that both neonate and later instar western corn rootworm larvae prefer nontransgenic roots to transgenic rootworm-resistant roots when a choice is possible. However, when damage to the infested, nontransgenic plant was high, western corn rootworm larvae apparently moved to neighboring transgenic rootworm-resistant plants and caused statistically significant, although only marginally economic, damage on the last sample date in 2001. Implications of these data toward resistance management plan are discussed.

Role of egg density on establishment and plant-to-plant movement by western corn rootworm larvae (Coleoptera: Chrysomelidae).

The effect of egg density on establishment and dispersal of larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, was evaluated in a 3-yr field study. Implications of these data for resistance management plans for Bt crops are discussed. Viable egg levels of 100, 200, 400, 800, and 1600 eggs per infested plant were evaluated in 2000, 2001, and 2002. A 3200 viable egg level was also tested in 2001 and 2002. All eggs were infested on one plant per subplot in a field that was planted to soybean, Glycine max (L.), in the previous year. For each subplot, the infested plant, three plants down the row, the closest plant in the adjacent row of the plot, and a control plant at least 1.5 m from any infested plant (six plants total) were sampled. In 2000, there were five sample dates between egg hatch and pupation, and in 2001 and 2002, there were six sample dates. On each sample date, four replications of each egg density were sampled for both larval recovery and plant damage. Initial establishment on a corn plant seemed to not be density-dependent because a similar percentage of larvae was recovered from all infestation rates. Plant damage and, secondarily, subsequent postestablishment larval movement were density-dependent. Very little damage and postestablishment movement occurred at lower infestation levels, but significant damage and movement occurred at higher infestation rates. Movement generally occurred at a similar time as significant plant damage and not at initial establishment, so timing of movement seemed to be motivated by available food resources rather than crowding. At the highest infestation level in 2001, significant movement three plants down the row and across the 0.76 m row was detected, perhaps impacting refuge strategies for transgenic corn.

Evaluation of conventional resistance to European corn borer (Lepidoptera: Crambidae) and western corn rootworm (Coleoptera: Chrysomelidae) in experimental maize lines developed from a backcross breeding program.

Plant resistance is a promising control method for the two most damaging insect pests of maize, Zea mays L.: the European corn borer, Ostrinia nubilalis (Hübner), and the western corn rootworm Diabrotica virgifera virgifera LeConte. Fifteen experimental lines of maize, derived from a backcross breeding program designed to introgress resistance to European corn borer from Peruvian maize into two U.S. Corn Belt adapted inbred lines, were evaluated for resistance to European corn borer and western corn rootwonrm. The experimental lines were in the second generation of backcrossing. All experimental lines were resistant to leaf blade feeding by European corn borer. These lines had low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, a chemical commonly associated with leaf blade feeding resistance, indicating that this was not the mechanism of resistance to leaf blade feeding in these lines. Eleven experimental lines were resistant to leaf sheath and collar feeding by European corn borer. Useful sources of European corn borer ovipositional nonpreference and root feeding resistance to western corn rootworm were not identified. Some of the lines evaluated in this study may provide useful sources of resistance to both leaf blade and leaf sheath and collar feeding by European corn borer.

Enantiomeric composition of grandisol and grandisal produced byPissodes strobi andP. nemorensis and their electroantennogram response to pure enantiomers.

Grandisol (cis-2-isopropenyl-1-methylcyclobutaneethanol) and its corresponding aldehyde, grandisal, were previously isolated and identified as aggregation pheromone components forPissodes strobi (Peck) andP. nemorensis Germar, but the enantiomeric ratios produced by these insects were not previously determined. We isolated grandisol and grandisal from males of bothP. strobi andP. nemorensis. The insect-produced grandisol was derivatized with trifluoroacetic anhydride, and the enantiomeric composition was determined by gas chromatography on an optically active cyclodextrin glass capillary column. The insect-produced grandisal was first reduced to grandisol before derivatization.P. nemorensis produced nearly 100% (1R,2S)-grandisol and nearly 100% (1S,2R)-grandisal.P. strobi produced 99% (1R,2S)-grandisol and approximately 60% (lR,2S)-grandisal. In electroantennogram (EAG) studies with liveP. nemorensis andP. strobi, no significant differences were found between the responses of males and females to racemic grandisol, racemic grandisal, or the 1R,2S and 1S,2R enantiomers of grandisol and grandisal, which is consistent with previous assertions that these compounds are aggregation pheromones. Although no studies to date withP. strobi have demonstrated a behavioral response to grandisol and grandisal,P. strobi antennae detected all enantiomers of grandisol and grandisal tested in EAG tests. The antennae of P.nemorensis responded significantly more to (1R,2S) grandisal than to (1S,2R)-grandisal, despite producing only (1S,2R)-grandisal.

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.

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.

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.

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.

Phenotypic versus marker-assisted selection for stalk strength and second-generation European corn borer resistance in maize.

Maize ( Zea mays L.) stalk lodging is breakage of the stalk at or below the ear, which may result in loss of the ear at harvest. Stalk lodging is often intensified by the stalk tunneling action of the second-generation of the European corn borer (2-ECB) [Ostrinia nubilalis (Hübner)]. Rind penetrometer resistance (RPR) has been used to measure stalk strength and improve stalk lodging resistance, and quantitative trait loci (QTL) have been identified for both RPR and 2-ECB damage. Phenotypic recurrent selection (PS) increases the frequency of favorable alleles over cycles of selection. Several studies have indicated that marker-assisted selection (MAS) is also a potentially valuable selection tool. The objective of this study was to compare the efficiency of PS versus MAS for RPR and 2-ECB. Marker-assisted selection for high and low RPR was effective in the three populations studied. Phenotypic selection for both high and low RPR was more effective than MAS in two of the populations. However, in a third population, MAS for high RPR using QTL effects from the same population was more effective than PS, and using QTL effects from a separate population was just as effective as PS. Marker-assisted selection for resistance and susceptibility to 2-ECB using QTL effects from the same population was effective in increasing susceptibility, but not in increasing resistance. Marker-assisted selection using QTL effects from a separate population was effective in both directions of selection. Thus, MAS was effective in selecting for both resistance and susceptibility to 2-ECB. These results demonstrated that MAS can be an effective selection tool for both RPR and 2-ECB resistance. These results also validate the locations and effects of QTL for RPR and 2-ECB resistance identified in earlier studies.

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.

Laboratory and field tests with the synthetic sex pheromone of threeMatsucoccus (1) pine bast scales.

Matsunone, (2E,4E)-4.6,10,12-tetramethyl-2,4-tridecadien-7-one, is the primary sex pheromone ofMatsucoccus resinosae, M. thunbergianae, andM. matsumurae. The synthetic compound was tested in the laboratory and in field tests with all three species, and significantly more males responded to synthetic matsuone than to controls. The attraction of synthetic matsuone to males in field tests was not significantly different from that of virgin females at the same matsuone release rate. An analog of matsuone previously shown to be biologically active was also tested in the laboratory withM. resinosae andM. thunbergianae, and field tested withM. resinosae. The active analog required a dose approximately 100 times greater than matsuone to yield maximum laboratory bioassay response, and in field tests, attraction was not significantly different from controls at doses at which matsuone was significantly attractive.