Field-evolved resistance to Bt maize by western corn rootworm: predictions from the laboratory and effects in the field

Crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) provide an effective management tool for many key insect pests. However, pest species have repeatedly demonstrated their ability to adapt to management practices. Results from laboratory selection experiments illustrate the capacity of pest species to evolve Bt resistance. Furthermore, resistance has been documented to Bt sprays in the field and greenhouse, and more recently, by some pests to Bt crops in the field. In 2009, fields were discovered in Iowa (USA) with populations of western corn rootworm, Diabrotica virgifera virgifera LeConte, that had evolved resistance to maize that produces the Bt toxin Cry3Bb1. Fields with resistant insects in 2009 had been planted to Cry3Bb1 maize for at least three consecutive years and as many as 6years. Computer simulation models predicted that the western corn rootworm might evolve resistance to Bt maize in as few as 3years. Laboratory and field data for interactions between western corn rootworm and Bt maize indicate that currently commercialized products are not high-dose events, which increases the risk of resistance evolution because non-recessive resistance traits may enhance survival on Bt maize. Furthermore, genetic analysis of laboratory strains of western corn rootworm has found non-recessive inheritance of resistance. Field studies conducted in two fields identified as harboring Cry3Bb1-resistant western corn rootworm found that survival of western corn rootworm did not differ between Cry3Bb1 maize and non-Bt maize and that root injury to Cry3Bb1 maize was higher than injury to other types of Bt maize or to maize roots protected with a soil insecticide. These first cases of field-evolved resistance to Bt maize by western corn rootworm provide an early warning and point to the need to apply better integrated pest management practices when using Bt maize to manage western corn rootworm.     

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.     

Assessment of fitness costs in Cry3Bb1‐resistant and susceptible western corn rootworm (Coleoptera: Chrysomelidae) laboratory colonies

Abstract Maize production in the United States is dominated by plants genetically modified with transgenes from Bacillus thuringiensis (Bt). Cry3Bb delta endotoxins expressed by Bt maize specifically target corn rootworms (genus Diabrotica) and have proven highly efficacious. However, development of resistance to Bt maize, especially among western corn rootworm (Diabrotica virgifera virgifera) populations, poses a significant threat to the future viability of this pest control biotechnology. The structured refuge insect resistance management (IRM) strategy implemented in the United States for Bt maize adopts a conservative approach to managing resistance by assuming no fitness costs of Bt resistance, even though these trade‐offs strongly influence the dynamics of Bt resistance within numerous agricultural pest species. To investigate the effects of Bt resistance on fitness components of western corn rootworm, we compared survivorship, fecundity and viability of five Bt‐resistant laboratory lines reared on MON863 (YieldGard Rootworm), a Bt maize product that expresses Cry3Bb1 delta endotoxin, and on its non‐transgenic isoline. Analysis of performance on the isoline maize demonstrated no fitness costs associated with Bt resistance. In fact, resistant lines emerged approximately 2–3 days earlier than control lines when reared on both MON863 and the isoline, indicating that selection for Bt resistance resulted in a general increase in the rate of larval development. In addition, resistant lines reared on Bt maize displayed higher fecundity than those reared on the isoline, which may have significant management implications. These data will be valuable for formulating improved IRM strategies for a principal agricultural pest of maize.     

Delaying corn rootworm resistance to Bt corn

Transgenic crops producing Bacillus thuringiensis (Bt) toxins for insect control have been successful, but their efficacy is reduced when pests evolve resistance. To delay pest resistance to Bt crops, the U.S. Environmental Protection Agency (EPA) has required refuges of host plants that do not produce Bt toxins to promote survival of susceptible pests. Such refuges are expected to be most effective if the Bt plants deliver a dose of toxin high enough to kill nearly all hybrid progeny produced by matings between resistant and susceptible pests. In 2003, the EPA first registered corn, Zea mays L., producing a Bt toxin (Cry3Bb1) that kills western corn rootworm, Diabrotica virgifera virgifera LeConte, one of the most economically important crop pests in the United States. The EPA requires minimum refuges of 20% for Cry3Bb1 corn and 5% for corn producing two Bt toxins active against corn rootworms. We conclude that the current refuge requirements are not adequate, because Bt corn hybrids active against corn rootworms do not meet the high-dose standard, and western corn rootworm has rapidly evolved resistance to Cry3Bb1 corn in the laboratory, greenhouse, and field. Accordingly, we recommend increasing the minimum refuge for Bt corn targeting corn rootworms to 50% for plants producing one toxin active against these pests and to 20% for plants producing two toxins active against these pests. Increasing the minimum refuge percentage can help to delay pest resistance, encourage integrated pest management, and promote more sustainable crop protection.     

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.     

Larval mortality and development for rotation‐resistant and rotation‐susceptible populations of western corn rootworm on Bt corn

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is one of the most economically important insect pests threatening the production of corn, Zea mays (L.), in the United States. Throughout its history, this insect has displayed considerable adaptability by overcoming a variety of pest management tactics, including the cultural practice of annual crop rotation. Since first reported in Illinois in the late 1980s, populations of the rotation‐resistant western corn rootworm have spread over a wide area of the eastern Corn Belt. Currently, little information is available concerning the interaction of rotation resistance with the use of genetically modified corn expressing insecticidal toxins from Bacillus thuringiensis Berliner (Bt), a popular tactic for preventing larval injury and its associated yield loss. The goal of this greenhouse experiment was to determine whether rotation‐resistant and rotation‐susceptible western corn rootworm larvae differ with respect to survival or development when exposed to single‐ or dual‐toxin (pyramided) Bt corn. Individual corn plants were infested with 225 near‐hatch eggs at the V5 (five leaf collar) growth stage. Larvae developed undisturbed on the root systems for 17 days, after which they were recovered using Berlese–Tullgren funnels. Surviving larvae were counted to estimate mortality, and head capsule widths were measured to assess development. Rotation‐resistant and rotation‐susceptible larvae had statistically similar mean levels of mortality and head capsule widths when exposed to both single‐toxin (Cry3Bb1 or Cry34/35Ab1) and pyramided (Cry3Bb1+ Cry34/35Ab1) Bt corn, suggesting that these two populations do not differ with respect to survival or development when exposed to Bt corn. Additionally, the statistically similar mean levels of mortality for larvae exposed to single‐toxin and pyramided Bt corn suggest that pyramided Bt hybrids containing the Cry3Bb1 and Cry34/35Ab1 toxins do not result in additive mortality for western corn rootworm larvae. Implications for management of this economically important pest are discussed.     

Minnesota field population of western corn rootworm (Coleoptera: Chrysomelidae) shows incomplete resistance to Cry34Ab1/Cry35Ab1 and Cry3Bb1

In the United States of America, the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is commonly managed with transgenic corn (Zea mays L.) expressing insecticidal proteins from the bacteria Bacillus thuringiensis Berliner (Bt). Colonies of this pest have been selected in the laboratory on each commercially available transformation event and several resistant field populations have also been identified; some field populations are also resistant. In this study, progeny of a western corn rootworm population collected from a Minnesota corn field planted to SmartStax^® corn were evaluated for resistance to corn hybrids expressing Cry3Bb1 (event MON88017) or Cry34/35Ab1 (event DAS‐59122‐7) and to the individual constituent proteins in diet‐overlay bioassays. Results from these assays suggest that this population is resistant to Cry3Bb1 and is incompletely resistant to Cry34/35Ab1. In diet toxicity assays, larvae of the Minnesota (MN) population had resistance ratios of 4.71 and >13.22 for Cry34/35Ab1 and Cry3Bb1 proteins, respectively, compared with the control colonies. In all on‐plant assays, the relative survival of the MN population on the DAS‐59122‐7 and MON88017 hybrids was significantly greater than the control colonies. Larvae of the MN population had inhibited development when reared on DAS‐59122‐7 compared with larvae reared on the non‐Bt hybrid, indicating resistance was incomplete. Overall, these results document resistance to Cry3Bb1 and an incomplete resistance to Cry34/35Ab1 in a population of WCR from a SmartStax^® performance problem field.     

Effects of adult emergence timing on susceptibility and fitness of Cry3Bb1‐resistant western corn rootworms

Field‐evolved resistance by the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte to the Cry3Bb1 trait expressed in maize, has been documented in areas of Nebraska USA. Currently, only limited information is available on life‐history traits of Cry3Bb1‐resistant field populations. Therefore, the Gassmann on‐plant bioassay was used to investigate the potential variability among four Cry3Bb1‐resistant WCR field collections made in 2011–2012 by focusing on the key parameters: larval survival, developmental stage and weight with specific emphasis on the impact of adult emergence timing on these parameters in subsequent progeny. Key results: In three of four collections, the susceptibility of larval progeny from adults that emerged early or late within a generation from Cry3Bb1 plants was similar. Each of the three collections exhibited complete resistance; that is, survival on Cry3Bb1 plants was greater or equal to survival on non‐Bt isoline plants. Bioassays from an additional field collection from one site 2 years (2013) after the original collection (2011) (both from Cry3Bb1 maize) indicated that resistance to Cry3Bb1 was maintained over time at the site despite Bt trait rotation in 2012. In general, comparative WCR life‐history parameter data from Cry3Bb1 and isoline maize indicate that fitness of field collections exhibiting complete resistance was similar on each hybrid. The mean proportion of larvae in third instar and mean weight of larvae recovered in bioassays from progeny of early‐ and late‐emerged adults was not significantly affected by emergence period. This suggests that delays in development and associated mean adult emergence commonly observed in populations that are susceptible to Cry3Bb1 may become smaller as populations become resistant to Cry3Bb1. Results from this article will inform Cry3Bb1 resistance mitigation efforts and contribute to the development of sustainable WCR management programmes.     

Selection for resistance to mCry3A-expressing transgenic corn in western corn rootworm

To investigate the development of resistance to mCry3A, a laboratory colony of the western corn rootworm, Diabrotica virgifera virgifera LeConte, was established from field survivors of mCry3A-expressing (MIR604) corn, Zea mays L. Feral adults emerging from MIR604 (selected) and isoline (control) field plots were collected and returned to the laboratory. Progeny of each colony was reared one generation on isoline corn and then crossed reciprocally with a nondiapausing colony. The resulting nondiapausing progeny were then reared on greenhouse corn in accordance with the wild type parent's origin (on MIR604 or isoline corn). After four, seven, and 10 total generations of selection, the resistance ratio of the selected colony was 0.5, 4.3, and 15.4 in terms of lethal concentration (LC)50 values in toxicity assays, with the latter two LC50 values being significant. After seven generations of selection in total, selected and control colonies were screened on MIR604 and isoline corn under field conditions. There was a significant colony x corn pedigree interaction in terms of plant damage. There was no significant difference in damage between MIR604 and isoline corn, whereas this difference was significant for the control colony. After 14 generations of selection, a seedling bioassay was performed. Again, there was a significant colony x corn pedigree interaction, this time in terms of the number of larvae recovered. There was no significant difference in the number of larvae recovered from MIR604 and isoline corn for the selected colony, whereas this difference was significant for the control colony, although larval size was greater on isoline corn for both colonies. Resistance has developed in western corn rootworm laboratory colonies to all Bt proteins currently registered for corn rootworm management, which emphasizes the importance of adhering to resistance management plans for maintaining product efficacy.     

Western corn rootworm larval movement in SmartStax seed blend scenarios

Insect resistance management (IRM) can extend the lifetime of management options, but depends on extensive knowledge of the biology of the pest species involved for an optimal plan. Recently, the Environmental Protection Agency (EPA) registered seed blends refuge for two of the transgenic Bacillus thuringiensis (Bt) corn products targeting the western corn rootworm, Diabrotica virgifera virgifera LeConte. Larval movement between Bt and isoline plants can be detrimental to resistance management for high dose Bt products because the larger larvae can be more tolerant of the Bt toxins. We assessed movement of western corn rootworm larvae among four spatial arrangements of SmartStax corn (expressing both the Cry34/35Ab1 and Cry3Bb1 proteins) and isoline plants by infesting specific plants with wild type western corn rootworm eggs. Significantly fewer western corn rootworm larvae, on average, were recovered from infested SmartStax plants than infested isoline plants, and the SmartStax plants were significantly less damaged than corresponding isoline plants. However, when two infested isoline plants surrounded a SmartStax plant, a significant number of larvae moved onto the SmartStax plant late in the season. These larvae caused significant damage both years and produced significantly more beetles than any other plant configuration in the study (including isoline plants) in the first year of the study. This plant configuration would occur rarely in a 5% seed blend refuge and may produce beetles of a susceptible genotype because much of their initial larval development was on isoline plants. Results are discussed in terms of their potential effects on resistance management.     

Baseline susceptibility of western corn rootworm (Coleoptera: Crysomelidae) to Cry3Bb1 Bacillus thuringiensis toxin

Susceptibility to Cry3Bb1 toxin from Bacillus thuringiensis (Bt) was determined for western corn rootworm, Diabrotica virgifera virgifera LeConte, neonates from both laboratory and field populations collected from across the Corn Belt. Rootworm larvae were exposed to artificial diet treated with increasing Cry3Bb1 concentrations, and mortality and growth inhibition were evaluated after 4-7 d. The range of variation in Bt susceptibility indicated by growth inhibition was similar to that indicated by mortality. Although interpopulation variation in susceptibility was observed, the magnitude of the differences was comparable with the variability observed between generations of the same population. In general, the toxin was not highly toxic to larvae and estimated LC50 and EC50 values were several times higher than those reported for lepidopteran-specific Cry toxins by using similar bioassay techniques. These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among rootworm populations and provide a baseline for estimating potential shifts in susceptibility that might result from selection and exposure to Cry3Bb1-expressing corn hybrids.     

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.     

Effect of Bacillus thuringiensis cry3Bb1 protein on the feeding behavior and longevity of adult western corn rootworms (Coleoptera: Chrysomelidae)

The first transgenic corn hybrids expressing the Bacillus thuringiensis (Bt) Cry3Bb1 protein to control corn rootworm (Diabrotica spp.) larvae were registered for commercial use in 2003. This study was conducted to investigate the effect of Cry3Bb1 protein in combination with a cucurbitacin bait on adult feeding and longevity of both organophosphate-resistant and -susceptible western corn rootworms, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). In choice and no-choice tests, possible repellency to the Bt protein was quantified by comparing beetle consumption of cellulose disks treated with three concentrations of Bt in combination with a feeding stimulant (Invite EC) to disks treated with stimulant alone. A lethal-time assay also was conducted to examine survival of beetles exposed to Bt protein in their diet. Results from these assays indicate that adult rootworms are not significantly deterred by the presence of Cry3Bb1 on the treated discs and that ingestion of toxin does not adversely affect adult longevity.     

Evaluation of adult emergence and larval root injury for Cry3Bb1‐resistant populations of the western corn rootworm

The transgenic maize (Zea mays L.) event MON 88017 produces the Bacillus thuringiensis Berliner (Bt) toxin Cry3Bb1 to provide protection from western corn rootworm (Diabrotica virgifera virgifera LeConte) larval feeding. In response to reports of reduced performance of Cry3Bb1‐expressing maize at two locations in Illinois, we conducted a two‐year experiment at these sites to characterize suspected resistance, as well as to evaluate root injury and adult emergence. Single‐plant bioassays were performed on larvae from each population that was suspected to be resistant. Results indicate that these populations had reduced mortality on Cry3Bb1‐expressing maize relative to susceptible control populations. No evidence of cross‐resistance between Cry3Bb1 and Cry34/35Ab1 was documented for the Cry3Bb1‐resistant populations. Field studies were conducted that included treatments with commercially available rootworm Bt hybrids and their corresponding non‐Bt near‐isolines. When compared with their near‐isolines, larval root injury and adult emergence were typically reduced for hybrids expressing Cry34/35Ab1 either alone or in a pyramid. In many instances, larval root injury and adult emergence were not significantly different for hybrids expressing mCry3A or Cry3Bb1 alone when compared with their non‐Bt near‐isolines. These findings suggest that Cry34/35Ab1‐expressing Bt maize may represent a valuable option for maize growers where Cry3Bb1 resistance is either confirmed or suspected. Consistent trends in adult size (head capsule width and dry mass) for individuals recovered from emergence cages were not detected during either year of this experiment. Because of the global importance of transgenic crops for managing insect pests, these results suggest that improved decision‐making for insect resistance management is needed to ensure the durability of Bt maize.     

Influence of calcareous soil on Cry3Bb1 expression and efficacy in the field

Greater than expected injury by western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) to Cry3Bb1 expressing maize hybrids (Zea mays L.) has been reported in southwestern Nebraska. Affected areas of some fields are often associated with high pH calcareous soils where maize growth is poor and iron chlorosis is common. As part of a comprehensive study to understand potential causes of unexpected injury, experiments were conducted during 2013 and 2014 to ascertain whether the calcareous soil conditions and associated poor maize growth negatively affect the expression of Cry3Bb1. Quantitative determination of Cry3Bb1 protein expression levels in root tissues was carried out on plants at V5–V6 growth stage using the enzyme-linked immunosorbent assay. Cry3Bb1 and non-Bt near isoline maize hybrids were artificially infested with Cry3Bb1-susceptible WCR eggs to measure survival and efficacy of Cry3Bb1 maize in calcareous and non-calcareous soils. Results showed that there was not a significant difference in expression of Cry3Bb1 protein between plants from calcareous and non-calcareous soils (18.9–21.2 µg/g fresh weight). Western corn rootworm survival was about sevenfold greater from the non-Bt isoline than Cry3Bb1 maize indicating that Cry3Bb1 performed as expected when infested with a Cry3Bb1 susceptible rootworm population. When survival from calcareous and non-calcareous soils was compared, no significant differences were observed in each soil. A significant positive correlation between soil pH and expression of Cry3Bb1 protein in roots was detected from samples collected in 2014 but not in 2013. No such correlation was found between soil pH and survival of WCR. Results suggest that Cry3Bb1 expression levels were sufficient to provide adequate root protection against WCR regardless of soil environment, indicating that lowered Cry3Bb1 expression is not a contributing factor to the greater than expected WCR injury observed in some southwestern Nebraska maize fields.     

Mortality impact of MON863 transgenic maize roots on western corn rootworm larvae in the field

Mortality of western corn rootworm (Diabrotica virgifera virgifera LeConte) due to feeding on MON863 transgenic maize (Zea mays L.) expressing the Cry3Bb1 protein was evaluated at three Missouri sites in both 2003 and 2004 and at one site each in South Dakota, Nebraska and Iowa in 2004. To do this, survivorship relative to survivorship on isoline maize (i.e. the same genetic background, but without Cry3Bb1) was evaluated. Comparisons were made using low (1650–2500 eggs/m) and high (3300–3500 eggs/m) western corn rootworm egg densities. Significantly fewer beetles were recovered from MON863 than from isoline maize. Emergence from MON863 as a percentage of viable eggs ranged from 0.02% to 0.10%, whereas percentage emergence from isoline maize ranged from 1.09% to 7.14%. Survivorship on MON863 relative to survivorship on isoline averaged 1.51% when averaged across all environments and both years, so mortality because of the Cry3Bb1 protein averaged 98.49%. The average time delay to 50% cumulative beetle emergence from MON863 was 18.3 days later than from isoline maize. Females comprised 56% and 71% of total beetles recovered from MON863 in 2003 and 2004, respectively. Results are discussed in relation to insect resistance management (IRM) of western corn rootworm.     

Bacillus thuringiensis Cry34Ab1/Cry35Ab1 Interactions with Western Corn Rootworm Midgut Membrane Binding Sites

Background

Bacillus thuringiensis (Bt) Cry34Ab1/Cry35Ab1 are binary insecticidal proteins that are co-expressed in transgenic corn hybrids for control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Bt crystal (Cry) proteins with limited potential for field-relevant cross-resistance are used in combination, along with non-transgenic corn refuges, as a strategy to delay development of resistant rootworm populations. Differences in insect midgut membrane binding site interactions are one line of evidence that Bt protein mechanisms of action differ and that the probability of receptor-mediated cross-resistance is low.

Methodology/Principal Findings

Binding site interactions were investigated between Cry34Ab1/Cry35Ab1 and coleopteran active insecticidal proteins Cry3Aa, Cry6Aa, and Cry8Ba on western corn rootworm midgut brush border membrane vesicles (BBMV). Competitive binding of radio-labeled proteins to western corn rootworm BBMV was used as a measure of shared binding sites. Our work shows that ¹²⁵I-Cry35Ab1 binds to rootworm BBMV, Cry34Ab1 enhances ¹²⁵I-Cry35Ab1 specific binding, and that ¹²⁵I-Cry35Ab1 with or without unlabeled Cry34Ab1 does not share binding sites with Cry3Aa, Cry6Aa, or Cry8Ba. Two primary lines of evidence presented here support the lack of shared binding sites between Cry34Ab1/Cry35Ab1 and the aforementioned proteins: 1) No competitive binding to rootworm BBMV was observed for competitor proteins when used in excess with ¹²⁵I-Cry35Ab1 alone or combined with unlabeled Cry34Ab1, and 2) No competitive binding to rootworm BBMV was observed for unlabeled Cry34Ab1 and Cry35Ab1, or a combination of the two, when used in excess with ¹²⁵I-Cry3Aa, or ¹²⁵I-Cry8Ba.

Conclusions/Significance

Combining two or more insecticidal proteins active against the same target pest is one tactic to delay the onset of resistance to either protein. We conclude that Cry34Ab1/Cry35Ab1 are compatible with Cry3Aa, Cry6Aa, or Cry8Ba for deployment as insect resistance management pyramids for in-plant control of western corn rootworm.     

Independent Action between DvSnf7 RNA and Cry3Bb1 Protein in Southern Corn Rootworm,Diabrotica undecimpunctata howardi and Colorado Potato Beetle,Leptinotarsa decemlineata

In recent years, corn rootworm (CRW)-resistant maize events producing two or more CRW-active Bt proteins have been commercialized to enhance efficacy against the target pest(s) by providing multiple modes of action (MoA). The maize hybrid MON 87411 has been developed that produces the CRW-active Cry3Bb1 Bt protein (hereafter Cry3Bb1) and expresses a RNAi-mediated MoA that also targets CRW. As part of an environmental risk assessment for MON 87411, the potential for an interaction between the CRW-active DvSnf7 RNA (hereafter DvSnf7) and Cry3Bb1 was assessed in 12-day diet incorporation bioassays with the southern corn rootworm (SCR, Diabrotica undecimpunctata howardi). The potential for an interaction between DvSnf7 and Cry3Bb1 was evaluated with two established experimental approaches. The first approach evaluated each substance alone and in combination over three different response levels. For all three response levels, observed responses were shown to be additive and not significantly different from predicted responses under the assumption of independent action. The second approach evaluated the potential for a fixed sub-lethal concentration of Cry3Bb1 to decrease the median lethal concentration (LC₅₀) of DvSnf7 and vice-versa. With this approach, the LC50 value of DvSnf7 was not altered by a sub-lethal concentration of Cry3Bb1 and vice-versa. In addition, the potential for an interaction between the Cry3Bb1 and DvSnf7 was tested with Colorado potato beetle (CPB, Leptinotarsa decemlineata), which is sensitive to Cry3Bb1 but not DvSnf7. CPB assays also demonstrated that DvSnf7 does not alter the activity of Cry3Bb1. The results from this study provide multiple lines of evidence that DvSnf7 and Cry3Bb1 produced in MON 87411 have independent action.     

Selection for Cry3Bb1 resistance in a genetically diverse population of nondiapausing western corn rootworm (Coleoptera: Chrysomelidae)

Five short-diapause laboratory lines of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), were selected for resistance to MON863, a variety of corn genetically modified with the Bacillus thuringiensis Berliner (Bt) transgene that expresses the Cry3Bb1 delta-endotoxin. Three of the selected lines were developed by incremental increase in the duration of exposure to MON863 over 11 generations (moderate selected lines). Two selected lines were developed from a control group by constant exposure to MON863 for at least 14 d posthatch over seven generations (intense selected lines). At the end of the experiment, survivorship, as measured by adult emergence, was approximately 4 times higher in each of the selected lines reared on MON863 compared with control lines. Estimates of realized heritabilities (h2) were 0.16 and 0.15 for the moderate and intense selected lines, respectively, and are consistent with h2 estimates reported previously from a variety of pest insects. These lines provide data necessary for evaluating the potential for Bt resistance within diabroticite beetles and will be useful for developing improved insect resistance management strategies.     

Enhancement of Bacillus thuringiensis Cry3Aa and Cry3Bb Toxicities to Coleopteran Larvae by a Toxin-Binding Fragment of an Insect Cadherin

The Cry3Aa and Cry3Bb insecticidal proteins of Bacillus thuringiensis are used in biopesticides and transgenic crops to control larvae of leaf-feeding beetles and rootworms. Cadherins localized in the midgut epithelium are identified as receptors for Cry toxins in lepidopteran and dipteran larvae. Previously, we discovered that a peptide of a toxin-binding cadherin expressed in Escherichia coli functions as a synergist for Cry1A toxicity against lepidopteran larvae and Cry4 toxicity against dipteran larvae. Here we report that the fragment containing the three most C-terminal cadherin repeats (CR) from the cadherin of the western corn rootworm binds toxin and enhances Cry3 toxicity to larvae of naturally susceptible species. The cadherin fragment (CR8 to CR10 [CR8-10]) of western corn rootworm Diabrotica virgifera virgifera was expressed in E. coli as an inclusion body. By an enzyme-linked immunosorbent microplate assay, we demonstrated that the CR8-10 peptide binds α-chymotrypsin-treated Cry3Aa and Cry3Bb toxins at high affinity (11.8 nM and 1.4 nM, respectively). Coleopteran larvae ingesting CR8-10 inclusions had increased susceptibility to Cry3Aa or Cry3Bb toxin. The Cry3 toxin-enhancing effect of CR8-10 was demonstrated for Colorado potato beetle Leptinotarsa decemlineata, southern corn rootworm Diabrotica undecimpunctata howardi, and western corn rootworm. The extent of Cry3 toxin enhancement, which ranged from 3- to 13-fold, may have practical applications for insect control. Cry3-containing biopesticides that include a cadherin fragment could be more efficacious. And Bt corn (i.e., corn treated with B. thuringiensis to make it resistant to pests) coexpressing Cry3Bb and CR8-10 could increase the functional dose level of the insect toxic activity, reducing the overall resistance risk.The Cry3 class of Bacillus thuringiensis Cry proteins is known for toxicity to coleopteran larvae in the family Chrysomelidae. Cry3Aa and Cry3Bb proteins are highly toxic to Colorado potato beetle (CPB) Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), and both were used for the development of Bt crops (crops treated with B. thuringiensis to make them resistant to pests) and Bt biopesticides. Due to the limited efficacy of Cry3-based biopesticides/plants and the success of competing chemical pesticides, these biopesticides have had limited usage and sales (12). Cry3Bb is toxic to corn rootworms (8, 17), and a modified version is expressed in commercialized MON863 corn hybrids (26).Cry3 toxins have a mode of action that is similar to, yet distinct from, the action of lepidopteran-active Cry1 toxins. The Cry3A protoxin (73 kDa) lacks the large C-terminal region of the 130-kDa Cry1 protoxins, which is removed by proteases during activation to toxin. The Cry3A protoxin is activated to a 55-kDa toxin and then further cleaved within the toxin molecule (5, 18). Activated Cry3A toxin binds to brush border membrane vesicles with a K_d (dissociation constant) of ∼37 nM (19) and recognizes a 144-kDa binding protein in brush border membrane vesicles prepared from the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (2). Recently, Ochoa-Campuzano et al. (20) identified an ADAM metalloprotease as a receptor for Cry3Aa toxin in CPB larvae.Structural differences between Cry3Bb and Cry3Aa toxins must underlie the unique rootworm activities of Cry3Bb toxin. As noted by Galitsky et al. (11), differences in toxin solubility, oligomerization, and binding are reported for these Cry3 toxins. Recently, Cry3Aa was modified to have activity against western corn rootworm (WCRW) Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) (27). Those authors introduced a chymotrypsin/cathepsin G cleavage site into domain 1 of Cry3Aa that allowed the processing of the 65-kDa form to a 55-kDa toxin that bound rootworm midgut.Cadherins function as receptors for Cry toxins in lepidopteran and dipteran larvae. A critical Cry1 toxin binding site is localized within the final cadherin repeat (CR), CR12, of cadherins from tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) and tobacco budworm Heliothis virescens (Lepidoptera: Noctuidae) (14, 28). Unexpectedly, a fragment of B. thuringiensis R₁ cadherin, the Cry1A receptor from M. sexta, not only bound toxin but enhanced Cry1A toxicity against lepidopteran larvae (6). If the binding residues within CR12 were removed, the resulting peptide lost the ability to bind toxin and lost its function as a toxin synergist. Recently, we identified a cadherin from mosquito Anopheles gambiae (Diptera: Culicidae) that binds Cry4Ba toxin and probably functions as a receptor. We discovered a similar effect where a fragment of a cadherin from A. gambiae enhanced the toxicity of the mosquitocidal toxin Cry4Ba to mosquito larvae (15). Sayed et al. (22) identified a novel cadherin-like gene in WCRW and proposed this protein as a candidate Bt toxin receptor. The cadherin-like gene is highly expressed in the midgut tissue of larval stages. The encoded protein is conserved in structure relative to that of other insect midgut cadherins.In this study, we hypothesized that a fragment from a beetle cadherin that contains a putative Bt toxin binding region might enhance the insecticidal toxicities of Cry3Aa and Cry3Bb toxins. The region spanning CR8 to CR10 (CR8-10) of the WCRW cadherin (22) was cloned and expressed in E. coli. This cadherin fragment significantly enhanced the toxicities of Cry3Aa and Cry3Bb toxins to CPB and rootworms.