Gerald E. Wilde

Effect of Bt Corn for Corn Rootworm Control on Nontarget Soil Microarthropods and Nematodes

Abstract The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of corn in Kansas. Planting Bt corn hybrids resistant to this pest is being tested as a method to control the larval stage of corn rootworms. These hybrids express Cry3Bb1 toxin and are expected to only directly impact chrysomelids and possibly related taxa. Soil samples were examined to evaluate the effect of Bt corn for corn rootworm control on soil microarthropods and nematodes in Kansas in 2000 and 2001. Soil samples from soil close to Bt corn and to its isoline were taken on three occasions (early, mid, and late season) from eight locations in 2000 and three locations in 2001. Soil mites and Collembola were extracted using a modified Tullgren high-gradient extractor. Nematodes were extracted using a centrifugal-flotation procedure. In general, numbers of soil mites (Prostigmata, Mesostigmata, and Oribatei), Collembola, and nematodes were similar in soil planted with Bt corn and soil planted with its isoline.

Detectability of Coleopteran-specific Cry3Bb1 Protein in Soil and Its Effect on Nontarget Surface and Below-Ground Arthropods

Abstract Corn engineered to produce the Cry3Bb1 protein from Bacillus thuringiensis (Bt) kumamotoensis has provided unprecedented control for corn rootworm (Diabrotica spp.). However, the Bt protein may be released in soil by root exudates or decaying plant residues that may affect soil organisms. Field studies were conducted to determine the abundance of surface and below-ground nontarget arthropods in fields planted with Bt or non-Bt corn for the first year or planted over 3 consecutive yr. Results of these studies showed that there were no significant differences in numbers of surface and below-ground arthropods in soil planted with Bt and non-Bt corn at any of the studied locations. Enzyme-linked immunosorbent assay (ELISA) showed no detectable Cry3Bb1 protein in any of the soil samples collected in a field planted with a Bt corn hybrid and its non-Bt isogenic hybrid for the first year or planted over 3 consecutive yr near Manhattan, KS. However, a small amount of Cry3Bb1 protein (3.38–6.89 ng/g dry soil) was detected in the soil samples collected from an area near plants in a Bt corn field that was planted for the first year near Scandia, KS. These findings indicate that the Cry3Bb1 protein released from root exudates or decaying plant residues does not persist and is rapidly broken down in the soil. The rapid degradation of Cry3Bb1 in soil results in none or trace amounts of protein being detected by ELISA.

No Effect of Bacillus thuringiensis Corn and Bacillus thuringiensis on the Predator Orius insidiosus (Hemiptera: Anthocoridae)

Abstract Laboratory feeding studies were conducted to determine the effects on Orius insidiosus nymphs of feeding on 1-d-old European corn borer, Ostrina nubilalis (Hübner), larvae that had ingested a diet containing Bt toxins. A commercial formulation of Bacillus thuringiensis subsp kurstaki (Dipel ES) was incorporated into a meridic diet used to feed European corn borer larvae; they then were offered as food to O. insidiosus nymphs. Immediately after adult eclosion, O. insidiosus sex was determined, body weight and length were measured, and developmental time was calculated. Another feeding study was conducted to determine the effect of Bt corn silk on mortality of immature O. insidiosus. Fresh silks of Bt and non-Bt corn plants were offered to O. insidiosus nymphs until they reached adulthood. Mortality counts were made daily. Finally, visual counts of O. insidiosus were made on Bt and non-Bt corn in fields at three locations in Kansas. The numbers of O. insidiosus nymphs and adults were recorded on 40 plants per location on two sampling dates. No significant differences occurred in developmental time, body weight, or body length of mature O. insidiosus or mortality of immature O. insidiosus when reared on European corn borer larvae that had fed on a diet containing Dipel ES. The nymphs feeding only on Bt or non-Bt corn silk suffered 100% mortality. No significant difference occurred in mortality of immature O. insidiosus when they were fed on Bt or non-Bt silk one day and on corn earworm eggs the next day. Numbers of O. insidiosus adults and nymphs in fields of Bt corn and non-Bt corn did not differ significantly in most cases. Our results suggest that Bt corn does not have a significant effect on the predator O. insidiosus.

Effect of Bt Corn Expressing the Cry3Bb1 Toxin for Corn Rootworm Control on Aboveground Nontarget Arthropods

Abstract The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of corn in Kansas. Planting Bt corn hybrids resistant to this pest is being tested for control of the larval stage of corn rootworms. These hybrids express the Cry3Bb1 toxin and are expected to only directly impact chrysomelids and possibly related taxa. A 2-yr study was conducted to evaluate the effect of Bt corn expressing the Cry3Bb1 toxin on foliar and ground-dwelling nontarget arthropods in Kansas. Visual inspections of adult and immature Coleomegilla maculata (DeGeer), Orius insidiosus (Say), Hippodamia convergens Gurin-Meneville, and Scymnus spp. occurring in Bt corn and its isoline were made at eight locations in Kansas in 2000 and three locations in 2001. Pitfall traps were installed at three locations in 2000 and two locations in 2001 to collect ground-dwelling arthropods in Bt corn and its isoline. In general, no significant differences in numbers of C. maculata, O. insidiosus, H. convergens, and Scymnus spp. were detected between Bt corn and its non-Bt isoline. There were also no significant differences in number of insects collected in pitfall traps installed in plots of Bt corn and its isoline. Bt corn for corn rootworm control had no deleterious effects on beneficial and other nontarget arthropods sampled in this study.

Evidence of Evolving Carbaryl Resistance in Western Corn Rootworm (Coleoptera: Chrysomelidae) in Areawide-Managed Cornfields in North Central Kansas

Abstract Susceptibility of adult populations of the western corn rootworm, Diabrotica virgifera virgifera LeConte, to carbaryl was determined by a survey in 1996 before the implementation of an areawide management program near Scandia in north central Kansas. Subsequently, the susceptibility of western corn rootworm adults to carbaryl has been monitored throughout the program from 1997 to 2000 in both control and managed areas. In 1996, adults were highly susceptible to carbaryl with a mean LC50 value of 0.64 μg/vial. This value was comparable to those for adults collected from other regions within Kansas. However, adult susceptibility to carbaryl decreased rapidly within the managed area, where the cucurbitacin-carbaryl-based bait SLAM has been used as the primary tool to control adults in this project since 1997. In 1999, adults collected from the managed area were 9- and 20-fold less susceptible to carbaryl at the LC50 and LC90 levels, respectively, than those evaluated in 1996. In contrast, adults collected from the control area were only 2- and 3-fold less susceptible to carbaryl at the LC50 and LC90 levels, respectively, than adults evaluated in 1996. Although field adult populations of western corn rootworm were relatively low in 2000, evaluations showed trends similar to those in 1999 regarding their carbaryl susceptibility in the managed and control areas. These results provide evidence that western corn rootworm has been evolving carbaryl resistance rapidly in response to the use of SLAM in areawide-managed cornfields near Scandia.

Effect of Insecticides Used in Corn, Sorghum, and Alfalfa on the Predator Orius insidiosus (Hemiptera: Anthocoridae)

Abstract Orius insidoisus (Say) is an important predator in corn, sorghum, and alfalfa. Foliar insecticides commonly used on corn (permethrin, bifenthrin, and fipronil); sorghum (chlorpyrifos, carbofuran, dimethoate, and cyfluthrin); and both crops (λ-cyhalothrin and ethyl parathion) were evaluated in 1998 and 1999 for their residual effects on O. insidiosus by caging adults on treated plants at several time intervals: at application (day 0) and 2, 3, and 6 d after application. In addition, imidacloprid, fipronil, and thiamethoxam used as seed treatments on corn and sorghum were tested for their effects on O. insidiosus by caging adults on plants in the presence and absence of greenbugs, Schizaphis graminum (Rondani). Finally, six of the same insecticides that also are used on alfalfa were evaluated in the field for their effects on O. insidiosus and other insects. On day 0, ethyl parathion, bifenthrin, and ]lambda]-cyhalothrin on corn caused significantly higher mortality to O. insidiosus than permethrin and fipronil. Ethyl parathion and carbofuran on sorghum caused significantly higher mortality than chlorpyrifos, dimethoate, and λ-cyhalothrin, which differed significantly from the control. Mortality with cyfluthrin did not differ significantly from that in the control. Insecticides had no significant effects on O. insidiosus 3 and 6 d after application in 1998 with the exception of permethrin on day 3. Similar patterns of mortality were observed in 1999 experiments. No significant differences in mortality of adults occurred with fipronil and thiamethoxam in the presence and absence of greenbugs. Imidachloprid caused significantly higher mortality to O. insidiosus adults than thiamethoxam or fipronil in some instances when greenbugs were not supplied as food. In alfalfa, the insecticides caused significant mortality to most of the insects evaluated. Ethyl parathion, permethrin, chlorpyrifos, and cyfluthrin caused significantly higher mortality to O. insidiosus than carbofuran and λ-cyhalothrin, which differed significantly from the control in 1998. In 1999, all treatments significantly reduced O. insidiosus numbers and did not differ significantly from each other.

Effect of Corn Hybrids Expressing the Coleopteran-Specific Cry3Bb1 Protein for Corn Rootworm Control on Aboveground Insect Predators

Field and laboratory studies were conducted to determine the effect of transgenic Bacillus thuringiensis (Bt) corn, Zea mays L. (YieldGard Rootworm), expressing the Cry3Bb1 protein on aboveground nontarget insect predators (minute pirate bug, ladybird beetles, and carabids). Visual counts of adult and immature Orius insidiosus (Say), Coleomegilla maculata (DeGeer), Hippodamia convergens Gurin-Meneville, and Scymnus spp. occurring in Bt corn and its non-Bt isoline were made at Manhattan, KS, in 2002 and at Manhattan and Scandia, KS, in 2003. No significant differences were found between the Bt corn and non-Bt isoline plots in the abundance (number per plant) of O. insidiosus, C. maculata, H. convergens, and Scymnus spp. Field predation on Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) egg masses was also observed during the silking stage of corn at Manhattan and Scandia in 2003. No significant differences were observed among treatments in predation rate for predators with chewing versus sucking mouthparts. Two laboratory studies determined the effect of Cry3Bb1 protein expressed in Bt corn pollen on C. maculata and carabids. The larvae of C. maculata were reared on Bt pollen, non-Bt pollen, or greenbugs, Schizaphis graminum (Rondani). The duration of larval and pupal stages, developmental time from egg hatch to adult emergence, percentage of survival, and elytra length were compared among treatments. There were no significant differences in developmental time of larvae fed pollen or greenbugs during their first two instars. However, significantly prolonged development of the third (1 d) and fourth instars (2 d) was observed for larvae fed greenbugs only. Total time for larval development was significantly longer for larvae that fed on greenbugs versus larvae fed on pollen. No significant differences were observed among treatments in the percentage of larvae that pupated or pupal stage duration. Larvae that fed on greenbugs had higher pupal and adult weights compared with pollen-fed larvae. However, pupal and adult weights did not vary between the Bt and non-Bt pollen treatments. No significant differences occurred in longevity and elytra length of beetles among all treatments. Two carabid species, Harpalus caliginosus F. and Harpalus pensylvanicus DeGeer, were reared on moistened dog food sprinkled with Bt or non-Bt corn pollen. No significant differences in mortality of H. caliginosus and H. pensylvanicus were detected among any of the treatments. There was no significant effect of Bt pollen on fecundity and egg viability of H. caliginosus. Our studies showed that YieldGard Rootworm had no effect on the selected coleopteran predators; therefore, this Bt corn hybrid could be used in an integrated pest management system.

Effect of Bt Corn Expressing the Cry3Bb1 Toxin on Western Corn Rootworm (Coleoptera: Chrysomelidae) Biology

Abstract In Kansas, the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is one of the most economically important pests of corn. Crop losses and control costs attributed to Diabrotica spp. reach

Purification and Kinetic Analysis of Acetylcholinesterase From Western Corn Rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

1 billion annually in the U.S. Recently, Bt corn hybrids resistant to western corn rootworm larval damage have been field-tested for rootworm control. This study evaluated the effect of a specific Bt corn (YieldGard Rootworm®) for corn rootworm control on larval survival. Elytra length, fecundity, percent egg hatch, and longevity of adult D. v. virgifera that survived exposure to Bt corn in the larval stage were also evaluated. Two groups of D. v. virgifera adults were assessed. One group had emerged from Bt and non-Bt corn roots; the other group was obtained from a colony with no larval exposure to Bt corn and fed Bt or non-Bt corn silk and ears. Bt corn significantly reduced the number of D. v. virgifera beetles emerging from corn roots. In general, Bt corn had no significant effect on longevity, elytra length, and female fecundity of D. v. virgifera beetles emerging from Bt corn plants. Bt corn had no significant effect on longevity and female fecundity of D. v. virgifera beetles feeding only as adults on Bt corn silk and ears. In general, Bt corn had no effect on number of D. v. virgifera beetles feeding in the field on corn silk and on silk damage caused by adults. Generally, the results of this study demonstrated that Bt corn (MON 863) expressing the Cry3Bb1 toxin was extremely effective in reducing damage caused by D. v. virgifera larvae and had no significant effects on the longevity of beetles emerging from YieldGard Rootworm® corn.

Genotypic interaction between resistance genes in wheat and virulence genes in the Hessian fly Mayetiola destructor (Diptera: Cecidomyiidae)

Acetylcholinesterase (AChE, EC 3.1.1.7) was purified from western corn rootworm (WCR, Diabrotica virgifera virgifera) beetles by affinity chromatography. The purification factor reached over 20,000-fold with a specific activity of 169.5 mumol/min/mg and a yield of 23%. The Vmax values for hydrolyzing acetylthiocholine (ATC), acetyl-(beta-methyl) thiocholine (A beta MTC), propionylthiocholine (PTC), and S-butyrylthiocholine (BTC) were 184.8, 140.5, 150.2, and 18.8 mumol/min/mg, respectively, and K(m) values were 19.7, 18.5, 14.1, and 11.0 microM, respectively. The first three substrates showed significant inhibition to the AChE at higher concentrations, whereas BTC showed inhibition at the concentrations of 0.25-2 nM but activation at > 4 mM. AChE activity was almost completely inhibited by 1 microM eserine and BW284C15, respectively, but only 12% of AChE activity were inhibited by ethopropazine at the same concentration. These results suggested that the purified AChE from WCR was a typical insect AChE. Insecticides or their oxidative metabolites, chlorpyrifos-methyl oxon, carbofuran, carbaryl, malaoxon, and paraoxon, used in in vitro kinetic study exhibited high inhibition to AChE purified from WCR. However, chlorpyrifos-methyl oxon and carbofuran showed at least 36- and 4-fold, respectively, higher inhibitory potency than the remaining insecticides examined. Results from our in vitro inhibition of AChE agreed quite well with the previously published in vivo bioassay data.