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 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.

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.

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.

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.

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.

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.

Adaptation by western corn rootworm (Coleoptera: Chrysomelidae) to Bt maize: inheritance, fitness costs, and feeding preference.

We examined inheritance of resistance, feeding behavior, and fitness costs for a laboratory-selected strain of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), with resistance to maize (Zea maize L.) producing the Bacillus thuringiensis Berliner (Bt) toxin Cry3Bb1. The resistant strain developed faster and had increased survival on Bt maize relative to a susceptible strain. Results from reciprocal crosses of the resistant and susceptible strains indicated that inheritance of resistance was nonrecessive. No fitness costs were associated with resistance alleles in the presence of two entomopathogenic nematode species, Steinernema carpocapsae Weiser and Heterorhabditis bacteriophora Poinar. Larval feeding studies indicated that the susceptible and resistant strains did not differ in preference for Bt and non-Bt root tissue in choice assays.

Western corn rootworm and Bt maize: challenges of pest resistance in the field.

Crops genetically engineered to produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) manage many key insect pests while reducing the use of conventional insecticides. One of the primary pests targeted by Bt maize in the United States is the western corn rootworm, Diabrotica virgifera virgifera LeConte. Beginning in 2009, populations of western corn rootworm were identified in Iowa, USA that imposed severe root injury to Cry3Bb1 maize. Subsequent laboratory bioassays revealed that these populations were resistant to Cry3Bb1 maize, with survival on Cry3Bb1 maize that was three times higher than populations not associated with such injury. Here we report the results of research that began in 2010 when western corn rootworm were sampled from 14 fields in Iowa, half of which had root injury to Cry3Bb1 maize of greater than 1 node. Of these samples, sufficient eggs were collected to conduct bioassays on seven populations. Laboratory bioassays revealed that these 2010 populations had survival on Cry3Bb1 maize that was 11 times higher and significantly greater than that of control populations, which were brought into the laboratory prior to the commercialization of Bt maize for control of corn rootworm. Additionally, the developmental delays observed for control populations on Cry3Bb1 maize were greatly diminished for 2010 populations. All 2010 populations evaluated in bioassays came from fields with a history of continuous maize production and between 3 and 7 y of Cry3Bb1 maize cultivation. Resistance to Cry34/35Ab1 maize was not detected and there was no correlation between survival on Cry3Bb1 maize and Cry34/35Ab1 maize, suggesting a lack of cross resistance between these Bt toxins. Effectively dealing with the challenge of field-evolved resistance to Bt maize by western corn rootworm will require better adherence to the principles of integrated pest management.

Field-evolved resistance to Bt maize by western corn rootworm.

BACKGROUND: Crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) are planted on millions of hectares annually, reducing the use of conventional insecticides and suppressing pests. However, the evolution of resistance could cut short these benefits. A primary pest targeted by Bt maize in the United States is the western corn rootworm Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). METHODOLOGY/PRINCIPAL FINDINGS: We report that fields identified by farmers as having severe rootworm feeding injury to Bt maize contained populations of western corn rootworm that displayed significantly higher survival on Cry3Bb1 maize in laboratory bioassays than did western corn rootworm from fields not associated with such feeding injury. In all cases, fields experiencing severe rootworm feeding contained Cry3Bb1 maize. Interviews with farmers indicated that Cry3Bb1 maize had been grown in those fields for at least three consecutive years. There was a significant positive correlation between the number of years Cry3Bb1 maize had been grown in a field and the survival of rootworm populations on Cry3Bb1 maize in bioassays. However, there was no significant correlation among populations for survival on Cry34/35Ab1 maize and Cry3Bb1 maize, suggesting a lack of cross resistance between these Bt toxins. CONCLUSIONS/SIGNIFICANCE: This is the first report of field-evolved resistance to a Bt toxin by the western corn rootworm and by any species of Coleoptera. Insufficient planting of refuges and non-recessive inheritance of resistance may have contributed to resistance. These results suggest that improvements in resistance management and a more integrated approach to the use of Bt crops may be necessary.