Management of Insect Pests with Bt Crops in the United States.

Genetically engineered corn and cotton that produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) have been used to manage insect pests in the United States and elsewhere. In some cases, this has led to regional suppression of pest populations and pest eradication within the United States, and these outcomes were associated with reductions in conventional insecticides and increased profits for farmers. In other instances, pests evolved resistance to multiple Bt traits, compromising the capacity of Bt crops to manage pests and leading to increased feeding injury to crops in the field. Several aspects of pest biology and pest-crop interactions were associated with cases where pests remained susceptible versus instances where pests evolved resistance. The viability of future transgenic traits can be improved by learning from these past outcomes. In particular, efforts should be made to delay resistance by increasing the prevalence of refuges and using integrated pest management.

Up-regulation of apoptotic- and cell survival-related gene pathways following exposures of western corn rootworm to B. thuringiensis crystalline pesticidal proteins in transgenic maize roots.

BACKGROUND: Resistance of pest insect species to insecticides, including B. thuringiensis (Bt) pesticidal proteins expressed by transgenic plants, is a threat to global food security. Despite the western corn rootworm, Diabrotica virgifera virgifera, being a major pest of maize and having populations showing increasing levels of resistance to hybrids expressing Bt pesticidal proteins, the cell mechanisms leading to mortality are not fully understood. RESULTS: Twenty unique RNA-seq libraries from the Bt susceptible D. v. virgifera inbred line Ped12, representing all growth stages and a range of different adult and larval exposures, were assembled into a reference transcriptome. Ten-day exposures of Ped12 larvae to transgenic Bt Cry3Bb1 and Gpp34/Tpp35Ab1 maize roots showed significant differential expression of 1055 and 1374 transcripts, respectively, compared to cohorts on non-Bt maize. Among these, 696 were differentially expressed in both Cry3Bb1 and Gpp34/Tpp35Ab1 maize exposures. Differentially-expressed transcripts encoded protein domains putatively involved in detoxification, metabolism, binding, and transport, were, in part, shared among transcripts that changed significantly following exposures to the entomopathogens Heterorhabditis bacteriophora and Metarhizium anisopliae. Differentially expressed transcripts in common between Bt and entomopathogen treatments encode proteins in general stress response pathways, including putative Bt binding receptors from the ATP binding cassette transporter superfamily. Putative caspases, pro- and anti-apoptotic factors, as well as endoplasmic reticulum (ER) stress-response factors were identified among transcripts uniquely up-regulated following exposure to either Bt protein. CONCLUSIONS: Our study suggests that the up-regulation of genes involved in ER stress management and apoptotic progression may be important in determining cell fate following exposure of susceptible D. v. virgifera larvae to Bt maize roots. This study provides novel insights into insect response to Bt intoxication, and a possible framework for future investigations of resistance mechanisms.

Linking land use patterns and pest outbreaks in Bt maize.

Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of maize in the United States and is an invasive pest in Europe. Maize is the only agricultural crop on which western corn rootworm larvae can survive and this insect requires two consecutive years of maize cultivation to complete its life cycle. Transgenic maize producing insecticidal proteins derived from the bacterium Bacillus thuringiensis (Bt) is often used to manage rootworm populations. The first Bt trait, Cry3Bb1, was introduced in 2003, but larval resistance to this toxin appeared in northeastern Iowa in 2009. Rootworm management occurs on a field-by-field basis, but adult rootworm may disperse among fields. It is known that growing consecutive years of Cry3Bb1 maize within a field can lead to resistance, but the relationship of the surrounding landscape to the development of resistance is unknown. Using geospatial tools and publicly available land-use data, we examined circular areas (buffers) surrounding fields that had previously experienced high levels of rootworm injury to Cry3Bb1 maize and rootworm resistance to Cry3Bb1 maize (problem fields). We calculated the proportion of area inside each buffer planted to maize continuously for 1-9 yr, and compared these values to those for randomly selected control points throughout the state. We also calculated the proportion of the state planted to maize for at least three consecutive years for 2003 through 2018, and its relationship with the annual value of maize. We found that areas surrounding problem fields had significantly more continuous maize compared to controls, with the most continuous maize within 1.6 km of problem fields. We also found that the cultivation of continuous maize in Iowa increased significantly between 2003 and 2018, and this was correlated with average annual price of maize. We hypothesize a scenario in which continuous cultivation of Cry3Bb1 maize in local landscapes, driven in part by the increased value of maize, facilitated selection for Cry3Bb1 resistance. These results suggest that land use in areas surrounding problem fields affect the rate of resistance evolution and approaches for resistance management can be enhanced by taking a landscape-level perspective.

Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize.

The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin.

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

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.

Inheritance and Fitness Costs of Resistance to Cry3Bb1 Corn by Western Corn Rootworm (Coleoptera: Chrysomelidae).

Transgenic crops that produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) are widely planted to manage pest insects. One of the primary pests targeted by Bt corn in the United States is western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). Cry3Bb1 corn for management of western corn rootworm was commercialized in 2003, and beginning in 2009, populations of western corn rootworm with field-evolved resistance to Cry3Bb1 corn were found in Iowa. Here we quantify the magnitude, inheritance, and fitness costs of resistance to Cry3Bb1 corn in two strains (Hopkinton and Cresco) derived from field populations that evolved resistance to Cry3Bb1 corn. For Hopkinton, we found evidence for complete resistance to Cry3Bb1 corn and nonrecessive inheritance. Additionally, no fitness costs of Cry3Bb1 resistance were detected for Hopkinton. For Cresco, resistance was incomplete and recessive, and we detected fitness costs affecting developmental rate, survival to adulthood, and fecundity. These results suggest that variation may exist among field populations in both the inheritance and accompanying fitness costs of resistance. To the extent that field populations exhibit nonrecessive inheritance and a lack of fitness cost, this will favor more rapid evolution of resistance than would be expected when resistance is functionally recessive and is accompanied by fitness costs.

Effects of Pyramided Bt Corn and Blended Refuges on Western Corn Rootworm and Northern Corn Rootworm (Coleoptera: Chrysomelidae).

The western corn rootworm, Diabrotica virgifera virgifera LeConte, and the northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), are major pests of corn (Zea mays L). Several transgenic corn events producing insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) kill corn rootworm larvae and reduce injury to corn roots. However, planting of Bt corn imposes selection on rootworm populations to evolve Bt resistance. The refuge strategy and pyramiding of multiple Bt toxins can delay resistance to Bt crops. In this study, we assessed the impact of four treatments--1) non-Bt corn, 2) Cry3Bb1 corn, 3) corn pyramided with Cry3Bb1 and Cry34/35Ab1, and 4) pyramided corn with a blended refuge--on survival, time of adult emergence, and size of western and northern corn rootworm. All treatments with Bt corn led to significant reductions in the number of adults that emerged per plot. However, at one location, we identified Cry3Bb1-resistant western corn rootworm. In some cases Bt treatments reduced size of adults and delayed time of adult emergence, with effects most pronounced for pyramided corn. For both species, the number of adults that emerged from pyramided corn with a blended refuge was significantly lower than expected, based solely on emergence from pure stands of pyramided corn and non-Bt corn. The results of this study indicate that pyramided corn with a blended refuge substantially reduces survival of both western and northern corn rootworm, and as such, should be a useful tool within the context of a broader integrated pest management strategy.

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

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 rootworm-active 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.

Effect of Seed Blends and Soil-Insecticide on Western and Northern Corn Rootworm Emergence from mCry3A+eCry3.1Ab Bt Maize.

Seed blends containing various ratios of transgenic Bt maize (Zea mays L.) expressing the mCry3A+eCry3.1Ab proteins and non-Bt maize (near-isoline maize) were deployed alone and in combination with a soil applied pyrethroid insecticide (Force CS) to evaluate the emergence of the western corn rootworm, Diabrotica virgifera virgifera LeConte, in a total of nine field environments across the Midwestern United States in 2010 and 2011. Northern corn rootworm, Diabrotica barberi Smith & Lawrence emergence was also evaluated in four of these environments. Both western and northern corn rootworm beetle emergence from all Bt treatments was significantly reduced when compared with beetle emergence from near-isoline treatments. Averaged across all environments, western corn rootworm beetle emergence from 95:5, 90:10, and 80:20 seed blend ratios of mCry3A+eCry3.1Ab: near-isoline were 2.6-, 4.2-, and 6.7-fold greater than that from the 100:0 ratio treatment. Northern corn rootworm emergence from the same seed blend treatments resulted in 2.8-, 3.2-, and 4.2-fold more beetles than from the 100:0 treatment. The addition of Force CS (tefluthrin) significantly reduced western corn rootworm beetle emergence for each of the three treatments to which it was applied. Force CS also significantly delayed the number of days to 50% beetle emergence in western corn rootworms. Time to 50% beetle emergence in the 100% mCry3A+eCry3.1Ab treatment with Force CS was delayed 13.7 d when compared with western corn rootworm beetle emergence on near-isoline corn. These data are discussed in terms of rootworm resistance management.

Effects of entomopathogens on mortality of western corn rootworm (Coleoptera: Chrysomelidae) and fitness costs of resistance to Cry3Bb1 maize.

Fitness costs can delay pest resistance to crops that produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt), and past research has found that entomopathogens impose fitness costs of Bt resistance. In addition, entomopathogens can be used for integrated pest management by providing biological control of pests. The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of maize and is currently managed by planting of Bt maize. We tested whether entomopathogenic nematodes and fungi increased mortality of western corn rootworm and whether these entomopathogens increased fitness costs of resistance to Cry3Bb1 maize. We exposed western corn rootworm larvae to two species of nematodes, Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and Steinernemafeltiae Filipjev (Rhabditida: Steinernematidae), and to two species of fungi, Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) (strain GHA) and Metarhizium brunneum (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) (strain F52) in two assay types, namely, seedling mat and small cup. Larval mortality increased with the concentration of H. bacteriophora and S. feltiae in the small cup assay, and with the exception of S. feltiae and B. bassiana in the seedling mat assay, mortality from entomopathogens was significantly greater than zero for the remaining entomopathogens in both assays. However, no fitness costs were observed in either assay type for any entomopathogen. Increased mortality of western corn rootworm larvae caused by these entomopathogens supports their potential use in biological control; however, the lack of fitness costs suggests that entomopathogens will not delay the evolution of Bt resistance in western corn rootworm.

Effect of maize lines on larval fitness costs of Cry1F resistance in the European corn borer (Lepidoptera: Crambidae).

Crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) are widely planted and enable management of key insect pests while reducing the use of conventional insecticides. However, the evolution of Bt resistance could diminish these benefits. Fitness costs of Bt resistance occur in the absence of Bt toxin when individuals with resistance alleles show a reduction in fitness relative to susceptible individuals, and they can delay the evolution of resistance. Ecological factors including host-plant variety can affect the magnitude of fitness costs, and consequently, the degree to which fitness costs delay resistance. In this study, we measured fitness costs of resistance to Bt toxin Cry1F in the European corn borer Ostrinia nubilalis Hübner (Lepidoptera: Crambidae) using Cry1F-resistant and Cry1F-susceptible strains sharing a similar genetic background. Fitness costs were tested on three lines of maize, Zea mays L., by measuring larval survival and development in two greenhouse experiments with plants in either the vegetative or reproductive stage. Both experiments showed that maize line significantly affected larval survival and developmental rate. However, larval survival, mass, and developmental rate did not differ between the Cry1F-resistant and susceptible strains, indicating a lack of fitness costs of resistance to Cry1F for the larval fitness components measured in this experiment. Future experiments should test for fitness costs of Cry1F resistance affecting survival to adulthood and adult life-history parameters.

Larval density-dependent mortality of western corn rootworm (Coleoptera: Chrysomelidae) in Bt and non-Bt maize and implications on dose calculations†.

BACKGROUND: Transgenic crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) have been used to manage insect pests for nearly 30 years. Dose of a Bt crop is key to assessing the risk of resistance evolution because it affects the heritability of resistance traits. Western corn rootworm (Diabrotica virgifera virgifera, LeConte), a major pest of maize, has evolved resistance to all commercially available Bt traits targeting it, and threatens resistance to future transgenic traits. Past research shows the dose of Bt maize targeting western corn rootworm can be confounded by larval density-dependent mortality. We conducted a 2-year field study at two locations to quantify larval density-dependent mortality in Bt and non-Bt maize. We used these results to calculate dose for our method and compared it to three previously published methods. Additionally, adult emergence and root injury were analyzed for predicting initial egg density. RESULTS: Increased pest density caused greater proportions of larvae to die in Bt maize than in non-Bt maize. All methods for calculating dose produced values less than high-dose, and stochastic variation had the greatest impact on dose at high and low pest densities. Our method for calculating dose did not produce values positively correlated with pest density while the three other methods did. CONCLUSION: To achieve the most accurate calculation of dose for transgenic maize targeting western corn rootworm, density-dependent mortality should be taken into account for both transgenic and non-transgenic maize and assessed at moderate pest densities. © 2024 Society of Chemical Industry.

Effects of combining soil-applied insecticide and Bt corn for integrated pest management and resistance management of western corn rootworm (Coleoptera: Chrysomelidae).

The western corn rootworm, (Diabrotica virgifera virgifera LeConte, Coleoptera: Chrysomelidae), is a serious pest of corn (Zea mays Linnaeus, Cyperales: Poaceae) in the midwestern United States. Management practices for corn rootworm larvae include crop rotation, transgenic corn producing insecticidal toxins from the bacterium Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt), and soil-applied insecticides. The extent to which combining soil-applied insecticide with Bt corn would be beneficial from the perspective of insect resistance management (IRM) or integrated pest management (IPM) remains uncertain. We conducted a 3-yr field study to characterize the implications of combining a soil-applied insecticide and Bt corn for IRM and IPM of western corn rootworm. Experimental treatments were Bt corn, a soil-applied insecticide, the combination of these factors, and an experimental control in which both factors were absent. Data were collected on root injury to corn by rootworm, survival to adulthood, adult size, and emergence time for western corn rootworm. We found that mortality caused by the soil-applied insecticide was insufficient to delay resistance to Bt corn. While combining Bt corn and a soil-applied insecticide may provide a short-term economic benefit, additional research is needed to determine appropriate economic thresholds for combining these tactics. Additionally, combining a soil-applied insecticide and Bt corn would not be sustainable over multiple growing seasons because of its potential to rapidly select for Bt resistance. In general, a more sustainable IRM strategy for rootworm management would include using crop rotation and alternating between non-Bt corn with soil-applied insecticide and Bt corn without soil-applied insecticide.

Entomopathogenic fungi in cornfields and their potential to manage larval western corn rootworm Diabrotica virgifera virgifera.

Entomopathogenic ascomycete fungi are ubiquitous in soil and on phylloplanes, and are important natural enemies of many soil-borne arthropods including larval western corn rootworm, Diabrotica virgifera virgifera, which is a major pest of corn. We measured the prevalence of Beauveria bassiana and Metarhizium anisopliae sensu lato in ten cornfields in Iowa, USA by baiting with larval insects. B. bassiana and M. anisopliae s.l. were present in 60% ± 6.3% and 55% ± 6.4% of soil samples, respectively. Subsequent laboratory bioassays found that some M. anisopliae s.l. strains collected from cornfields killed a greater proportion of D.v. virgifera larvae than a standard commercial strain.

Abundance and distribution of western and northern corn rootworm (Diabrotica spp.) and prevalence of rotation resistance in eastern Iowa.

The western corn rootworm Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) and the northern corn rootworm Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae) are major pests of corn (Zea mays L.). Historically, crop rotation has been an effective management strategy, but both species have adapted to crop rotation in the Midwest. For both species in eastern Iowa, we measured abundance and prevalence of rotation resistance using sticky traps and emergence cages in fields of corn and soybean (Glycine max L.). Based on currently available data, we calculated the economic thresholds for these pests at two Diabrotica spp. per trap per day in cornfields and 1.5 D. v. virgifera per trap per day in soybean fields. The economic injury level of rotation-resistant D. barberi was determined to be 3.5 adult insects per emergence cage per year. Peak abundance of rootworm adults in cornfields was below economic thresholds in the majority of fields sampled, suggesting that management of rootworm larvae in continuous cornfields may not always be necessary. Rotation-resistant D. barberi was found throughout eastern Iowa using emergence cages in first-year cornfields, however, the abundance was below levels expected to impose economic injury in 14 of 17 fields evaluated. The presence of rotation-resistant D. v. virgifera, as measured by the occurrence of this insect in soybean fields, occurred only in northeastern Iowa and was also below the economic threshold. These data suggests that crop rotation remains a viable pest management strategy in eastern Iowa.

Effects of Rag1 on the preference and performance of soybean defoliators.

The Rag1 gene confers antibiotic resistance to soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), and in 2010, varieties expressing Rag1 were released for commercial use in the United States. We do not know how Rag1 varieties will influence the broader community of defoliating insects that inhabit soybean fields. In 2010 and 2011, the preference and performance of pest insects that defoliate soybeans [Glycines max (L.) Merr] were tested using Rag1 and aphid-susceptible varieties. Three coleopterans and four lepidopterans were used: northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae); southern corn rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae); bean leaf beetle, Ceratoma trsifurcata Förster (Coleoptera: Chrysomelidae); fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae); corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae); soybean looper, Chrysodeix includens (Walker) (Lepidoptera: Noctuidae); and velvet-bean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae). The preference of insects was evaluated in choice and no-choice tests using Rag1 and susceptible soybeans. Lepidopterans also were evaluated on Rag1 leaves using four nutritional indices: relative growth rate, approximate digestibility, and efficiency of conversion of ingested material. In the majority of preference tests, no effect of Rag1 was detected, and in cases where preferences were found, there was no consistent pattern of preference for Rag1 vs. susceptible leaf tissue. Helicoverpa zea demonstrated a preference for resistant leaf tissue, but this was dependent on the genetic background of the variety. Evaluations of nutritional indices indicated that three species of Lepidoptera, S. frugiperda, H. zea, and A. gemmatalis, displayed reduced conversion efficiency for Rag1 soybeans, suggesting effects of antibiosis.

Effect of Bt maize and soil insecticides on yield, injury, and rootworm survival: implications for resistance management.

A 2-yr field experiment was conducted to determine the effects on Diabrotica spp. (Coleoptera: Chrysomelidae) of an insecticidal seed treatment (Poncho 1250, (AI)/clothianidin) and a granular insecticide (Aztec 2.1G, (AI)/tebupirimphos and cyfluthrin) alone and in combination with maize producing the insectidical toxin Cry3Bb1 derived from the bacterium Bacillus thuringiensis (Bt). Yields for Bt maize plots were significantly greater than for non-Bt maize; however, insecticides did not significantly affect yield. Insecticides significantly decreased root injury in non-Bt maize plots, but there were no significant differences in root injury between Bt maize with or without either insecticide. Maize producing the Bt toxin Cry3Bb1 and the soil-applied insecticide Aztec significantly decreased survival of western corn rootworm (Diabrotica virgifera virgifera LeConte), while only Bt maize significantly decreased survival of the northern corn rootworm (Diabrotica barberi Smith & Lawrence). For both species, Bt maize and each of the insecticides delayed emergence. In the absence of density-dependent mortality, Bt maize imposed 71 and 80% reduction in survival on the western corn rootworm and the northern corn rootworm, respectively. The data from this study do not support combining insecticide with Bt maize because the addition of insecticide did not increase yield or reduce root injury for Bt maize, and the level of rootworm mortality achieved with conventional insecticide was likely too low to delay the evolution of Bt resistance. In addition, delays in emergence from Bt maize combined with insecticides could promote assortative mating among Bt-selected individuals, which may hasten resistance evolution.

Interactions among Bt maize, entomopathogens, and rootworm species (Coleoptera: Chrysomelidae) in the field: effects on survival, yield, and root injury.

A 2 yr field study was conducted to determine how a blend of entomopathogens interacted with Bt maize to affect mortality of Diabrotica spp. (Coleoptera: Chrysomelidae), root injury to maize (Zea maize L.) and yield. The blend of entomopathogens included two entomopathogenic nematodes, Steinernema carpocapsae Weiser and Heterorhabditis bacteriophora Poinar, and one entomopathogenic fungus, Metarhizium brunneum (Metschnikoff) Sorokin. Bt maize (event DAS59122-7, which produces Bt toxin Cry34/35Ab1) decreased root injury and survival of western corn rootworm (Diabrotica virgifera virgifera LeConte) and northern corn rootworm (Diabrotica barberi Smith & Lawrence) but did not affect yield. During year 1 of the study, when rootworm abundance was high, entomopathogens in combination with Bt maize led to a significant reduction in root injury. In year 2 of the study, when rootworm abundance was lower, entomopathogens significantly decreased injury to non-Bt maize roots, but had no effect on Bt maize roots. Yield was significantly increased by the addition of entomopathogens to the soil. Entomopathogens did not decrease survival of corn rootworm species. The results suggest that soil-borne entomopathogens can complement Bt maize by protecting roots from feeding injury from corn rootworm when pest abundance is high, and can decrease root injury to non-Bt maize when rootworm abundance is low. In addition, this study also showed that the addition of entomopathogens to soil contributed to an overall increase in yield.

Applying an integrated refuge to manage western corn rootworm (Coleoptera: Chrysomelidae): effects on survival, fitness, and selection pressure.

The refuge strategy can delay resistance of insect pests to transgenic maize producing toxins from Bacillus thuringiensis (Bt). This is important for the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), because of its history of adaptation to several management practices. A 2-yr study across four locations was conducted to measure the effects of integrated refuge (i.e., blended refuge) on western corn rootworm survival to adulthood, fitness characteristics, and susceptibility to Bt maize in the subsequent generation. The treatments tested in this study were as follows: a pure stand of Bt maize (event DAS-59122-7, which produces Bt toxins Cry34Ab1/Cry35Ab1), a pure stand of refuge (non-Bt maize), and two variations on an integrated refuge consisting of 94.4% Bt maize and 5.6% non-Bt maize. Within the two integrated refuge treatments, refuge seeds received a neonicotinoid insecticidal seed treatment of either 1.25 mg clothianidin per kernel or 0.25 mg thiamethoxam per kernel. Insects in the pure stand refuge treatment had greater survival to adulthood and earlier emergence than in all other treatments. Although fecundity, longevity, and head capsule width were reduced in treatments containing Bt maize for some site by year combinations, Bt maize did not have a significant effect on these factors when testing data across all sites and years. We found no differences in susceptibility of larval progeny to Bt maize in bioassays using progeny of adults collected from the four treatments.

Inheritance and Fitness Costs of Laboratory-Selected Resistance to Gpp34/Tpp35Ab1 Corn in Western Corn Rootworm (Coleoptera: Chrysomelidae).

Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn and is currently managed with corn hybrids that produce insecticidal proteins derived from the bacterium Bacillus thuringiensis (Bt). Bt corn kills rootworm larvae and reduces larval feeding injury to corn roots. The Bt protein Gpp34/Tpp35Ab1, previously named Cry34/35Ab1, has been widely used in transgenic Bt corn for management of western corn rootworm, and field-evolved resistance has been found in some populations. In the United States, the refuge strategy is used to manage Bt resistance, with refuges of non-Bt host plants serving as a source of Bt-susceptible individuals, which in turn reduce the frequency of homozygous resistant individuals within a population. As such, the dominance of resistance strongly influences resistance evolution, with faster evolution of resistance when resistance is not recessive. Additionally, selection for resistance by a Bt crop leads to the accumulation of resistance alleles within refuge populations, thereby reducing the capacity of refuges to delay resistance. However, fitness costs can remove resistance alleles from refuge populations and preserve the dynamic of refuges producing Bt-susceptible genotypes. Bt-susceptible and Gpp34/Tpp35Ab1-resistant western corn rootworm were used to quantify the inheritance and fitness costs of resistance. We found that Gpp34/Tpp35Ab1 resistance was not recessive and had the accompanying fitness costs of slower developmental rate to adulthood and lower egg viability. This research will help improve insect resistance management by providing a better understanding of the risk of western corn rootworm evolving resistance to transgenic corn that produces Gpp34/Tpp35Ab1.