Laurence D. Chandler

Larval Susceptibility of an Insecticide-Resistant Western Corn Rootworm (Coleoptera: Chrysomelidae) Population to Soil Insecticides: Laboratory Bioassays, Assays of Detoxification Enzymes, and Field Performance

Abstract Soil insecticides were evaluated in laboratory and field studies against larvae of an insecticide resistant population (Phelps County, NE) of western corn rootworm,Diabrotica virgifera virgiferaLeConte. Insecticide toxicity was evaluated by topical application of technical insecticides to 3rd instars from Saunders County, NE (susceptible) and Phelps County populations. Resistance ratios (LD50Phelps County/LD50Saunders County) for the insecticides methyl parathion, tefluthrin, carbofuran, terbufos, and chlorpyrifos were 28.0, 9.3, 8.7, 2.6 and 1.3, respectively. Biochemical investigation of suspected enzymatic resistance mechanisms in 3rd instars identified significant elevation of esterase activity (alpha and beta naphthyl acetate hydrolysis [3.8- and 3.9-fold]). Examination of 3rd instar esterases by native PAGE identified increased intensity of several isoenzymes in the resistant population. Assays of cytochrome P450 activity (4-CNMA demethylation and aldrin epoxidation) did not identify elevated activity in resistant 3rd instars. Granular soil insecticides were applied at planting to corn, Zea mays L., in replicated field trials in 1997 and 1998 at the same Phelps County site as the source of resistant rootworms for the laboratory studies. In 1997, planting time applications of Counter 20CR, Counter 15 G (terbufos), and Lorsban 15 G (chlorpyrifos) resulted in the lowest root injury ratings (1–6 Iowa scale); 2.50, 2.55, 2.65, respectively (untreated check root rating of 4.55). In 1998, all insecticides performed similarly against a lower rootworm density (untreated check root rating of 3.72). These studies suggest that resistance previously documented in adults also is present in 3rd instars, esterases are possibly involved as resistance mechanisms, and resistance to methyl parathion in adults is also evident in larvae, but does not confer cross-resistance in larvae to all organophosphate insecticides.

Expression and induction of three family 4 cytochrome P450 (CYP4)* genes identified from insecticide-resistant and susceptible western corn rootworms, Diabrotica virgifera virgifera

We have previously determined that cytochrome P450‐based oxidation is involved in resistance to the insecticides methyl parathion and carbaryl in geographically distinct Nebraska western corn rootworm populations. Three new family 4 cytochrome P450 (CYP4) gene fragments (CYP4AJ1, CYP4G18 and CYP4AK1) were cloned and sequenced from insecticide‐resistant and ‐susceptible western corn rootworms. Insecticide bioassays indicated the resistant population employed in this study was significantly resistant to the insecticides methyl parathion and carbaryl. CYP4AJ1 and CYP4G18 were cloned from both genomic PCR and RT‐PCR products, although only CYP4AJ1 contains an intronic region. Alignments of inferred amino acid sequences with other homologous insect CYP4 genes indicates a high degree of similarity. Northern analysis concurrently employing mixed probes representing each of the three rootworm CYP4 fragments identified increased mRNA transcript signals (i) in resistant rootworms and (ii) following induction by the P450 inducer pentamethyl benzene. These results support our previous documentation of P450‐based insecticide resistance and suggest increased CYP4 transcript abundance can serve as a molecular resistance‐associated marker.

Fipronil metabolism, oxidative sulfone formation and toxicity among organophosphate- and carbamate-resistant and susceptible western corn rootworm populations

Fipronil toxicity and metabolism were studied in two insecticide-resistant, and one susceptible western corn rootworm (Diabrotica virgifera virgifera, LeConte) populations. Toxicity was evaluated by exposure to surface residues and by topical application. Surface residue bioassays indicated no differences in fipronil susceptibility among the three populations. Topical bioassays were used to study the relative toxicity of fipronil, fipronila the mono-oxygenase inhibitor piperonyl butoxide, and fipronils oxidative sulfone metabolite in two populations (one resistant with elevated mono-oxygenase activity). Fipronil and fipronil-sulfone exhibited similar toxicity and application of piperonyl butoxide prior to fipronil resulted in marginal effects on toxicity. Metabolism of ( 14 C)fipronil was evaluated in vivo and in vitro in the three rootworm populations. In vivo studies indicated the dominant pathway in all populations to be formation of the oxidative sulfone metabolite. Much lower quantities of polar metabolites were also identified. In vitro studies were performed using sub-cellular protein fractions (microsomal and cytosolic), and glutathione-agarose purified glutathione-S- transferase. Oxidative sulfone formation occurred almost exclusively in in vitro microsomal reactions and was increased in the resistant populations. Highly polar metabolites were formed exclusively in in vitro cytosolic reactions. In vitro reactions performed with purified, cytosolic glutathione-S- transferase (MW = 27kDa) did not result in sulfone formation, although three additional polar metabolites not initially detectable in crude cytosolic reactions were detected. Metabolism results indicate both cytochromes P450 and glutathione-S-transferases are important to fipronil metabolism in the western corn rootworm and that toxic sulfone formation by P450 does not affect net toxicity. # 2000 Society of Chemical Industry

Field-Cage Studies of Beauveria bassiana (Hyphomycetes: Moniliaceae) for the Suppression of Adult Western Corn Rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

The efficacy of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin, was tested as a control agent for adult western corn rootworm, Diabrotica virgifera virgifera LeConte, in walk-in field cages. Suspensions of B. bassiana conidia were applied to corn plants in cages into which laboratory-reared beetles had been released. Beetles were collected at 3 and 5 days post-application and evaluated in the laboratory for mortality. Mortality was 10, 29 and 50%, at rate equivalents of 7 X 10 12 , 2 X 10 13 (two applications), and 5 X 10 13 conidia/ ha, respectively. There was no significant difference in mortality of beetles collected at 3 days compared with 5 days post-application. Mortality due to B. bassiana was 24% when beetles were released into field cages 24 h post-application (5 X 10 13 conidia/ha) compared with 50% when beetles were present during the application. Beetle mortality declined significantly with increasing time from application in feeding assays carried out with leaf samples removed from plants at 0, 12, 24 and 72 h post-application. Mortality of beetles collected from treated plants within cages and maintained in the laboratory was found to overestimate the population decline by 10% when compared with beetle estimates from treated plants within field cages.

Diagnostic Assays Based on Esterase-Mediated Resistance Mechanisms in Western Corn Rootworms (Coleoptera: Chrysomelidae)

Abstract Resistance to methyl-parathion among Nebraska western corn rootworm, Diabrotica virgifera virgifera LeConte, populations is associated with increased hydrolytic metabolism of an organophosphate insecticide substrate. An electrophoretic method to identify resistant individuals based on the staining intensity of esterase isozymes on nondenaturing polyacrylamide gels was developed. Three groups of esterases (I, II, and III) were visible on the gels, but only group II esterase isozymes were intensified in resistant populations. A total of 26 and 31 field populations of western corn rootworms from Nebraska (in 1998 and 1999, respectively) were assessed with nondenaturing polyacrylamide gel electrophoresis (PAGE) assays and diagnostic concentration bioassays. Significant correlations were observed between the two diagnostic assays. Group II esterase isozymes provide a reliable biochemical marker for detection of methyl-parathion resistance in individual western corn rootworms and a tool for monitoring the frequency of resistant individuals in field populations.

Ground Beetle (Coleoptera: Carabidae) Assemblages in a Transgenic Corn-Soybean Cropping System

Abstract Ground beetles often prey on crop pests, and their relative abundance and assemblages vary among cropping systems and pest management practices. We used pitfall traps arranged in transects to study ground beetle assemblages in a large field-scale Bt corn–soybean cropping system for 3 yr. The transgenic corn expressed the Cry1Ab protein targeting lepidopteran pests. Three of the 57 ground beetle species collected accounted for 81% of all individuals captured. Six other species accounted for an additional 14% of all beetles captured. Ground beetles were captured equally in cornfields and soybean fields. They also were captured most frequently at field edges, but many were captured within field centers. Canonical correspondence analysis was used to arrange ground beetles along environmental gradients. Years 2001 and 2002 were the primary variables separating assemblages of ground beetles along the first canonical axis. The second canonical axis further separated the 2000 assemblage of ground beetles. With the effects of year and field removed, ground beetles were classified with respect to crop association and distance into the fields along axes 1 and 2 of a partial canonical correspondence analysis. Based on this analysis, ground beetles occupying the Bt cornfields were separated from those occupying soybean fields along the first canonical axis. The second canonical axis separated beetles occupying the field borders from field interiors. Ground beetles ordinating near the center of the axes may represent habitat generalists, and because of their high relative abundances, continuous seasonal activity, predatory nature, and ability to occupy field centers, they could assist in the biological control of agricultural pests.

Characterization of western corn rootworm (Coleoptera: Chrysomelidae) population dynamics in relation to landscape attributes

Abstract  1 The western corn rootworm (WCR), Diabrotica virgifera virgifera Leconte (Coleoptera: Chrysomelidae), creates economic and environmental concerns in the Corn Belt region of the U.S.A. To supplement the population control tactics of the Areawide Pest Management Program in Brookings, South Dakota, Geographical Information Systems (GIS) were used to examine the spatial relationships from 1997 to 2001 between WCR population dynamics, habitat structure, soil texture and elevation.

Using GIS in Areawide Pest Management: A Case Study in South Dakota

Infestations of corn rootworms (Coleoptera: Chrysomelidae) create economic and environmental concerns in the Corn Belt region of the United States. To supplement the population control tactics of areawide pest management programs, we believe that a better understanding of the spatial relationships between biotic and abiotic or physical factors at the landscape scale is needed. Our research used several geographical information systems (GIS) and spatial analytical techniques to examine relationships between corn rootworm metapopulation dynamics, soil texture, and elevation. Within GIS, several spatially explicit procedures were used that include an interpolation technique, spatial autocorrelation analysis, and contingency analysis. Corn rootworm metapopulation distributions were found to be aggregated and related to soil texture and elevation. We review techniques and discuss our preferences for using particular spatially explicit procedures. The information derived from the spatial analyses demonstrates how GIS can be used in areawide pest management to provide inputs for spatially explicit models to predict future pest populations and formulate more well-informed pest management decisions. The techniques described in this paper could easily be extended to study the spatial dynamics between other pest populations in agricultural landscapes.

Toxicity of insecticide-bait mixtures to insecticide resistant and susceptible western corn rootworms (Coleoptera:: Chrysomelidae)

Organophosphate resistant and susceptible populations of the western corn rootworm, Diabrotica virgifera virgifera LeConte. were subjected to adult feeding bioassays with different combinations of insecticide and a cucurbitacin bait. Five technical grade insecticides (methyl-parathion, carbaryl, fipronil, bifenthrin and indoxacarb) were used in combination with Invite ECt as the feeding stimulant. Differences in susceptibility to the insecticide/bait combinations were observed among the resistant and susceptible populations for methyl-parathion and carbaryl. Susceptibility to fipronil, bifenthrin and indoxacarb was similar among the resistant and susceptible populations. Assays in which response to the insecticide/bait combination was compared with the bait alone indicated that methyl-parathion and bifenthrin were deterrent when compared to other treatments. These results suggest that the efficacy of Invite as a feeding stimulant in combination with certain insecticides may be compromised by previously identified resistance and by insecticides that antagonize the feeding stimulation of the cucurbitacin bait. r 2003 Elsevier Science Ltd. All rights reserved.

Monitoring Western Corn Rootworm (Coleoptera: Chrysomelidae) Susceptibility to Carbaryl and Cucurbitacin Baits in the Areawide Management Pilot Program

Abstract Areawide pest management involves the uniform application of a pest control strategy over wide geographic areas. Therefore, these programs are likely to impose intense selective pressures, and the risk for resistance development among pest species for which areawide management programs are implemented is likely to be high. Pilot studies for areawide management of western corn rootworm, Diabrotica virgifera virgifera LeConte, were conducted from 1996 to 2002 at four different sites across the Corn Belt. This program used cucurbitacin baits to deliver high doses of a traditional neurotoxic insecticide (carbaryl) to individual insects while reducing the overall rate of insecticide use. Because of the concern and potential for resistance evolution, annual assessments of susceptibility to the active ingredient carbaryl were conducted both within the managed area as well as from untreated control areas. Significantly reduced susceptibility to carbaryl based on survival at a diagnostic concentration was detected in three of the four management sites (Kansas, Iowa, and Illinois/Indiana), whereas susceptibility of beetles collected outside the managed areas remained unchanged. Additionally, significantly reduced responsiveness to cucurbitacin baits was observed in beetles collected from the managed area relative to the control area at the same three sites. These results suggest strongly that areawide management has the potential to select for resistance and that a strategy for managing resistance and reducing selective pressure should be proactively implemented.