Lance J. Meinke

Western corn rootworm (Diabrotica virgifera virgifera LeConte) population dynamics

1 The western corn rootworm Diabrotica virgifera virgifera LeConte is a major insect pest of field maize, Zea mays L. Larvae can cause substantial injury by feeding on maize roots. Larval feeding may destroy individual roots or root nodes, and reduce plant growth, stability, and yield. Costs associated with managing corn rootworms in continuous maize are annually one of the largest expenditures for insect management in the United States Corn Belt.

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.

Modeling Evolution of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) to Transgenic Corn With Two Insecticidal Traits

ABSTRACT A simulation model of the population dynamics and genetics of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), was created to evaluate the use of refuges in the management of resistance to transgenic insecticidal corn, Zea mays L., expressing one or two toxin traits. Hypothetical scenarios and a case study of a corn hybrid pyramided with existing toxins are simulated. In the hypothetical situations, results demonstrated that evolution is generally delayed by pyramids compared with deployment of a single-toxin corn hybrid. However, soil insecticide use in the refuge reduced this delay and quickened the evolution of resistance. Results were sensitive to the degree of male beetle dispersal before mating and to the effectiveness of both toxins in the pyramid. Resistance evolved faster as fecundity increased for survivors of insecticidal corn. Thus, effects on fecundity must be measured to predict which resistance management plans will work well. Evolution of resistance also occurred faster if the survival rate due to exposure to the two toxins was not calculated by multiplication of two independent survival rates (one for each insect gene) but was equivalent to the minimum of the two. Furthermore, when single-trait and pyramided corn hybrids were planted within rootworm-dispersal distance of each other, the toxin traits lost efficacy more quickly than they did in scenarios without single-trait corn. For the case study involving transgenic corn expressing Cry34/35Ab1 and Cry3Bb1, the pyramid delayed evolution longer than a single trait corn hybrid and longer than a sequence of toxins based on at least one resistance-allele frequency remaining below 50%. Results are discussed within the context of a changing transgenic corn marketplace and the landscape dynamics of resistance management.

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.

Molecular phylogeny of Diabrotica beetles (Coleoptera: Chrysomelidae) inferred from analysis of combined mitochondrial and nuclear DNA sequences

The phylogenetic relationships of thirteen Diabrotica (representing virgifera and fucata species groups) and two outgroup Acalymma beetle species (Coleoptera: Chrysomelidae) were inferred from the phylogenetic analysis of a combined data set of 1323 bp of mitochondrial DNA (mtDNA) cytochrome oxidase subunit 1 (COI) and the entire second internal transcribed spacer region (ITS‐2) of nuclear ribosomal DNA of 362 characters. Species investigated were D. adelpha, D. balteata, D. barberi, D. cristata, D. lemniscata, D. longicornis, D. porracea, D. speciosa, D. undecimpunctata howardi, D. u. undecimpunctata, D. virgifera virgifera, D. v. zeae, D. viridula, and outgroup A. blandulum and A. vittatum. Maximum parsimony (MP), minimum evolution (ME), and maximum likelihood (ML) analyses of combined COI and ITS‐2 sequences clearly place species into their traditional morphological species groups with MP and ME analyses resulting in identical topologies. Results generally confer with a prior work based on allozyme data, but within the virgifera species group, D. barberi and D. longicornis strongly resolve as sister taxa as well as monophyletic with the neotropical species, D. viridula, D. cristata and D. lemniscata also resolve as sister taxa. Both relationships are not in congruence with the prior allozyme‐based hypothesis. Within the fucata species group, D. speciosa and D. balteata resolve as sister taxa. Results also strongly supported the D. virgifera and D. undecimpunctata subspecies complexes. Our proposed phylogeny provides some insight into current hypotheses regarding distribution status and evolution of various life history traits for Diabrotica.

Susceptibility of Nebraska Western Corn Rootworm (Coleoptera: Chrysomelidae) Populations to Bt Corn Events

ABSTRACT Transgenic plants have been widely adopted by growers to manage the western corn rootworm, Diabrotica virgifera virgifera LeConte, in field corn. Because of reduced efficacy in some Nebraska fields after repeated use of Cry3Bb1-expressing hybrids, single plant bioassays were conducted in 2012 and 2013 to characterize the susceptibility of western corn rootworm populations to the rootwormactive proteins Cry3Bb1, mCry3A, and Cry34/35Ab1. Results demonstrate that there are heritable differences in susceptibility of Nebraska western corn rootworm populations to rootworm-active Bt traits. Proportional survival and corrected survival data coupled with field histories collectively support the conclusion that a level of field resistance to Cry3Bb1 has evolved in some Nebraska populations in response to selection pressure and that cross-resistance exists between Cry3Bb1 and mCry3A. There was no apparent cross-resistance between Cry34/35Ab1 and either Cry3Bb1 or mCry3A. The potential implications of these results on current and future corn rootworm management strategies are discussed.

Early Detection and Mitigation of Resistance to Bt Maize by Western Corn Rootworm (Coleoptera: Chrysomelidae)

Abstract Transgenic Bt maize that produces less than a high-dose has been widely adopted and presents considerable insect resistance management (IRM) challenges. Western corn rootworm, Diabrotica virgifera virgifera LeConte, has rapidly evolved resistance to Bt maize in the field, leading to local loss of efficacy for some corn rootworm Bt maize events. Documenting and responding to this resistance has been complicated by a lack of rapid diagnostic bioassays and by regulatory triggers that hinder timely and effective management responses. These failures are of great concern to the scientific and agricultural community. Specific challenges posed by western corn rootworm resistance to Bt maize, and more general concerns around Bt crops that produce less than a high-dose of Bt toxin, have caused uncertainty around current IRM protocols. More than 15 years of experience with IRM has shown that high-dose and refuge-based IRM is not applicable to Bt crops that produce less than a high-dose. Adaptive IRM approaches and pro-active, integrated IRM-pest management strategies are needed and should be in place before release of new technologies that produce less than a high-dose. We suggest changes in IRM strategies to preserve the utility of corn rootworm Bt maize by 1) targeting local resistance management earlier in the sequence of responses to resistance and 2) developing area-wide criteria to address widespread economic losses. We also favor consideration of policies and programs to counteract economic forces that are contributing to rapid resistance evolution.

Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) Larval Feeding Behavior on Transgenic Maize (MON 863) and Its Isoline

Diabrotica species (Coleoptera: Chrysomelidae) larval behavior studies have posed a challenge to researchers because of the subterranean life cycle of this pest. To fully understand how the western corn rootworm, Diabrotica virgifera virgifera LeConte, injures the maize, Zea mays L., root system, its behavior must be studied. For example, larvae that can detect an area of the root that has a lower amount of toxin, whether from an insecticide or a transgenic maize plant, have an increased chance of survival. This study assessed D. v. virgifera larval feeding behavior on rootworm-susceptible maize and maize containing a biotechnology-derived trait (MON 863) with resistance to D. v. virgifera first instar feeding. Maize plants were grown in a medium that allowed for direct observation and measurements during feeding of larval stadia. Neonates were placed on maize seedlings, and data were taken at 3, 6, 9, and 12 d postinfestation on resistant and susceptible maize. On rootworm-susceptible maize, neonate larvae aggregated at the root tips and began actively feeding, and then they moved to older root tissue. Conversely, some larvae that ingested Cry 3Bb1 from the resistant maize exhibited no movement. Other larvae on the resistant maize moved continuously, sampling root hairs or root tissue but not actively feeding. The continuously moving larvae had visibly empty guts, suggesting possible nonpreference for the resistant root. This study contributes to our understanding of D. v. virgifera larval behavior and provides insight into questions surrounding the potential evolution of behavioral and biochemical resistance to Cry3Bb1.

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

Western Corn Rootworm (Coleoptera: Chrysomelidae) Dispersal and Adaptation to Single-Toxin Transgenic Corn Deployed With Block or Blended Refuge

ABSTRACT A simulation model of the temporal and spatial dynamics and population genetics of western corn rootworm, Diabrotica virgifera virgifera LeConte, was created to evaluate the use of block refuges and seed blends in the management of resistance to transgenic insecticidal corn (Zea mays L.). This Bt corn expresses one transgenic corn event, DAS-59122-7, that produces a binary insecticidal protein toxin (Cry34Ab1/Cry35Ab1) and provides host-plant resistance. The model incorporates the latest information about larval and adult behavior. Results of this modeling effort indicate that the seed-blend scenarios in many cases produced equal or greater durability than block refuges that were relocated each year. Resistance evolved in the most likely scenarios in 10–16 yr. Our standard analysis presumed complete adoption of 59122 corn by all farmers in our hypothetical region, no crop rotation, and 100% compliance with Insect Resistant Management (IRM) regulations. As compliance levels declined, resistance evolved faster when block refuges were deployed. Seed treatments that killed the pest when applied to all seeds in a seed blend or just to seeds in Bt corn blocks delayed evolution of resistance. Greater control of the pest population by the seed treatment facilitated longer durability of the transgenic trait. Therefore, data support the concept that pyramiding a transgenic insecticidal trait with a highly efficacious insecticidal seed treatment can delay evolution of resistance.