Effect of Cry3Bb1-expressing transgenic corn on plant-to-plant movement by western corn rootworm larvae (Coleoptera: Chrysomelidae)

Dispersal of larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, in specific combinations of transgenic corn expressing the Cry3Bb1 protein and nontransgenic, isoline corn was evaluated in a 2-yr field study. In total, 1,500 viable western corn rootworm eggs were infested in each subplot. Each year, plant damage and larval recovery were evaluated among four pedigree combinations (straight transgenic; straight nontransgenic corn; nontransgenic corn with a transgenic central, infested plant; and transgenic corn with a nontransgenic central, infested plant) on six sample dates between egg hatch and pupation. For each subplot, the infested plant, three successive plants down the row (P1, P2, and P3), the closest plant in the adjacent row of the plot, and a control plant were sampled. The number of western corn rootworm larvae recovered from transgenic rootworm-resistant plants adjacent to infested nontransgenic plants was low and not statistically significant in either 2001 or 2002. In 2001, significantly fewer larvae were recovered from transgenic rootworm-resistant plants than from nontransgenic plants when both were adjacent to infested, nontransgenic plants. In 2002, significantly more neonate western corn rootworm larvae were recovered from nontransgenic plants adjacent to infested, transgenic rootworm-resistant plants than nontransgenic plants adjacent to infested, nontransgenic plants on the second sample date. Together, these data imply that both neonate and later instar western corn rootworm larvae prefer nontransgenic roots to transgenic rootworm-resistant roots when a choice is possible. However, when damage to the infested, nontransgenic plant was high, western corn rootworm larvae apparently moved to neighboring transgenic rootworm-resistant plants and caused statistically significant, although only marginally economic, damage on the last sample date in 2001. Implications of these data toward resistance management plan are discussed.     

Post-establishment movement of western corn rootworm larvae (Coleoptera: Chrysomelidae) in Central Missouri corn

If registered, transgenic corn, Zea mays L., with corn rootworm resistance will offer a viable alternative to insecticides for managing Diabrotica spp. corn rootworms. Resistance management to maintain susceptibility is in the interest of growers, the Environmental Protection Agency, and industry, but little is known about many aspects of corn rootworm biology required for an effective resistance management program. The extent of larval movement by the western corn rootworm, Diabrotica virgifera virgifera LeConte, that occurs from plant-to-plant or row-to-row after initial establishment was evaluated in 1998 and 1999 in a Central Missouri cornfield. Post-establishment movement by western corn rootworm larvae was clearly documented in two of four treatment combinations in 1999 where larvae moved up to three plants down the row and across a 0.46-m row. Larvae did not significantly cross a 0.91-m row after initial host establishment in 1998 or 1999, whether or not the soil had been compacted by a tractor and planter. In the current experiment, western corn rootworm larvae moved from highly damaged, infested plants to nearby plants with little to no previous root damage. Our data do not provide significant insight into how larvae might disperse after initial establishment when all plants in an area are heavily damaged or when only moderate damage occurs on an infested plant. A similar situation might also occur if a seed mixture of transgenic and isoline plants were used and if transgenic plants with rootworm resistance are not repellent to corn rootworm larvae.     

The impact of Bt maize as a natal host on adult head capsule width in field populations of western corn rootworm

The success of the current resistance management plan for transgenic maize, Zea mays L. (Poaceae), targeting the rootworm complex hinges upon high rates of mating between resistant and susceptible beetles. However, differences in the fitness of adult beetles could result in assortative mating, which could, in turn, change the rate of resistance evolution. Adult head capsule widths of naturally occurring populations of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), were examined in a variety of refuge configurations. Beetles were classified into treatments based on the hybrid – non‐Bt refuge or Bt maize targeting larval rootworms (hereafter Bt‐RW maize) – and location – proximity to other Bt‐RW or refuge plants – of the natal host plant. Treatments included the following: a refuge plant surrounded by other refuge plants, a refuge plant located near a Bt‐RW plant, a Bt‐RW plant surrounded by Bt‐RW plants, and a Bt‐RW plant located near a refuge plant. The mean head capsule width of males emerging from Bt‐RW plants was significantly smaller than the mean head capsule width of males emerging from refuge plants. These results indicate that males emerging from Bt‐RW maize plants may be exposed to sublethal doses of the Bt toxin as larvae. No differences were detected between females emerging from refuge plants compared with Bt‐RW plants. Overall mean head capsule width decreased as the season progressed, regardless of treatment. The diminished head capsule width of western corn rootworm males emerging from Bt‐RW maize may act to enhance resistance management, particularly in a seed mix refuge system.     

Quantifying rates of random mating in western corn rootworm emerging from Cry3Bb1‐expressing and refuge maize in field cages

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is the most significant pest of field maize, Zea mays L. (Poaceae), in the USA. Maize plants expressing Bt toxins targeting the corn rootworm complex have been widely adopted and are the primary insecticidal control measure for this pest in North America. Insect resistance management tactics using various refuge structures have been adopted to ensure Bt products will retain durability. An assumption of the refuge strategy is that males and females emerging from Bt and refuge plantings mate randomly; this has not been tested in the field. We conducted cage studies using field populations of WCR in Indiana, USA, to generate empirical field data on mating rates between beetles emerging from Cry3Bb1‐expressing Bt and refuge maize plants. Two refuge configurations were tested; all refuge plants were labeled using the stable isotope ¹⁵N. This mark persists in adult beetles after eclosion, allowing for collection and analysis of isotopic ratios of all beetles. Additional data collected included adult emergence rates, timing and sex ratios for each of the treatments, and head capsule size and dry weights of beetles collected. Treatment had a significant effect on dry weight; mean dry weight decreased in Bt‐only treatments. Fisher's exact test of proportions of mating pairs of refuge and Bt insects indicated that mating was not random in 20% strip refuges and 5% seed blend treatments. We found high percentages of beetles that fed on Bt‐expressing plants as larvae, suggesting that mating between resistant beetles may not be rare even if random mating did occur.     

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.     

Mortality of western corn rootworm larvae on MIR604 transgenic maize roots: field survivorship has no significant impact on survivorship of F1 progeny on MIR604

Mortality of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae due to MIR604 transgenic corn, Zea mays L., expressing the modified Cry3A (mCry3A) protein relative to survivorship on corn with the same genetic background without the gene (isoline corn) was evaluated at three Missouri sites in both 2005 and 2006. We made these comparisons by using wild-type western corn rootworm at three different egg densities (6,000, 3,000, and 1,500 eggs per m) so that the role of density-dependent mortality would be known. The mortality due to the mCry3A protein was 94.88% when averaged across all environments and both years. Fifty percent emergence of beetles was delayed approximately 5.5 d. Beetles were kept alive and their progeny evaluated on MIR604 and isoline corn in the greenhouse to determine whether survivorship on MIR604 in the field for one generation increased survivorship on MIR604 in the greenhouse in the subsequent generation. There was no significant difference in survivorship on MIR604 in greenhouse assays between larvae whose parents survived isoline and larvae whose parents survived MIR604 in the field the previous generation, indicating that many susceptible beetles survived MIR604 in the field the previous season along with any potentially resistant beetles. The data are discussed in terms of rootworm insect resistance management.     

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.     

Greenhouse-Selected Resistance to Cry3Bb1-Producing Corn in Three Western Corn Rootworm Populations

Transgenic corn producing the Bacillus thuringiensis (Bt) toxin Cry3Bb1 has been useful for controlling western corn rootworm, Diabrotica virgifera virgifera LeConte, one of the most economically important crop pests in the United States. However, rapid evolution of resistance by this beetle to Bt corn producing Cry3Bb1 has been reported previously from the laboratory, greenhouse, and field. Here we selected in the greenhouse for resistance to Cry3Bb1 corn in three colonies of WCR derived from Kansas, Minnesota, and Wisconsin, respectively. Three generations of rearing on Cry3Bb1 corn significantly increased larval survival on Cry3Bb1 corn, resulting in similar survival in the greenhouse for selected colonies on Cry3Bb1 corn and isoline corn that does not produce Bt toxin. After four to seven generations of rearing on Cry3Bb1 corn, survival in the field on Cry3Bb1 corn relative to isoline corn more than doubled for selected colonies (72%) compared with control colonies (33%). For both selected and control colonies, survival in the field was significantly lower on Cry3Bb1 corn than on isoline corn. On isoline corn, most fitness components were similar for selected colonies and control colonies. However, fecundity was significantly lower for selected colonies than control colonies, indicating a fitness cost associated with resistance. The rapid evolution of resistance by western corn rootworm to Bt corn reported here and previously underlines the importance of effective resistance management for this pest.     

Interactions of alternate hosts, postemergence grass control, and rootworm-resistant transgenic corn on western corn rootworm (Coleoptera: Chrysomelidae) damage and adult emergence

Field studies were conducted in 2003 and 2004 to determine the effects of grassy weeds, postemergence grass control, transgenic rootworm-resistant corn, Zea mays L., expressing the Cry3Bb1 endotoxin and glyphosate herbicide tolerance (Bt corn), and the interactions of these factors on western corn rootworm, Diabrotica virgifera virgifera LeConte, damage and adult emergence. Three insect management tactics (Bt corn, its nontransgenic isoline, and isoline plus tefluthrin) were evaluated with two weed species (giant foxtail, Setaria faberi Herrm, and large crabgrass, Digitaria sanquinalis L. Scop), and four weed management regimes (weed free, no weed management, early [V3-4] weed management and late [V5-6] weed management) in a factorial arrangement of a randomized split split-plot design. In each case, the isoline was also tolerant to glyphosate. Root damage was significantly affected by insect management tactics in both years, but weed species and weed management did not significantly affect damage to Bt corn in either year. Adult emergence was significantly affected by insect management tactics in both years and by weed species in 2003, but weed management and the interaction of all three factors was not significant in either year. The sex ratio of female beetles produced on Bt corn without weeds was generally greater than when weeds were present and this difference was significant for several treatments each year. Average dry weight of male and female beetles emerging from Bt corn was greater than the weights of beetles emerging from isoline or isoline plus tefluthrin in 2003, but there was no difference for females in 2004 and males weighed significantly less than other treatments in 2004. The implications of these results in insect resistance management are discussed.     

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.     

Role of egg density on establishment and plant-to-plant movement by western corn rootworm larvae (Coleoptera: Chrysomelidae)

The effect of egg density on establishment and dispersal of larvae of the western corn rootworm, Diabrotica virgifera virgifera LeConte, was evaluated in a 3-yr field study. Implications of these data for resistance management plans for Bt crops are discussed. Viable egg levels of 100, 200, 400, 800, and 1600 eggs per infested plant were evaluated in 2000, 2001, and 2002. A 3200 viable egg level was also tested in 2001 and 2002. All eggs were infested on one plant per subplot in a field that was planted to soybean, Glycine max (L.), in the previous year. For each subplot, the infested plant, three plants down the row, the closest plant in the adjacent row of the plot, and a control plant at least 1.5 m from any infested plant (six plants total) were sampled. In 2000, there were five sample dates between egg hatch and pupation, and in 2001 and 2002, there were six sample dates. On each sample date, four replications of each egg density were sampled for both larval recovery and plant damage. Initial establishment on a corn plant seemed to not be density-dependent because a similar percentage of larvae was recovered from all infestation rates. Plant damage and, secondarily, subsequent postestablishment larval movement were density-dependent. Very little damage and postestablishment movement occurred at lower infestation levels, but significant damage and movement occurred at higher infestation rates. Movement generally occurred at a similar time as significant plant damage and not at initial establishment, so timing of movement seemed to be motivated by available food resources rather than crowding. At the highest infestation level in 2001, significant movement three plants down the row and across the 0.76 m row was detected, perhaps impacting refuge strategies for transgenic corn.     

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.     

Western corn rootworm (Coleoptera: Chrysomelidae) beetle emergence from weedy Cry3Bbl rootworm-resistant transgenic corn

Three greenhouse experiments were conducted to evaluate western corn rootworm, Diabrotica virgifera virgifera LeConte, beetle emergence from individual pots containing glyphosate-tolerant transgenic corn, Zea mays L., expressing the Cry3Bbl endotoxin from the soil bacterium Bacillus thuringiensis Berliner (MON863), nontransgenic glyphosate-tolerant isoline corn, grassy weeds (giant foxtail, Setariafaberi R.A.W. Herrm; and large crabgrass, Digitaria sanguinalis (L.) Scop.), and combinations thereof infested with 40 neonate larvae. In the first two experiments, pots with corn and weed combinations were treated with glyphosate 5 d after larval infestation to kill the weeds. The third experiment was similar to the first two except that untreated corn-weed combinations were added. In all three experiments beetle emergence varied significantly. Beetle recovery generally did not vary between the nontransgenic, nontransgenic + weeds, and MON863 + weeds. Significantly more beetles were recovered from MON863 + weeds than MON863 alone or weeds alone. Beetle emergence from MON863 + weeds was likely enhanced by larvae that initially survived on weeds before application of glyphosate. Preliminary data indicated that fecundity was highest from beetles reared on nontransgenic isoline corn and fewer eggs were laid by beetles reared on MON863 alone. Egg viability was generally lowest from beetles reared on MON863. The implications of these results in relation to insect resistant management are discussed.     

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.     

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.     

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.     

Evaluating multiple approaches for managing western corn rootworm larvae with seed blends

Corn rootworm, Diabrotica spp., larvae represent a significant and widespread economic threat to corn, Zea mays (L.), production in the United States, where control costs and yield losses associated with these insect pests exceed $1 billion annually. Preventing root injury and associated yield loss caused by corn rootworm larvae may be accomplished by the independent use of planting time soil insecticides or transgenic Bt hybrids. However, recent reports of both confirmed and suspected Bt resistance in corn rootworm populations throughout the Corn Belt have led to significant interest in the use of these two management tactics simultaneously. Although this approach has been investigated to some extent previously, information is lacking on how the use of a soil insecticide in tandem with a Bt seed blend—Bt and refuge (non‐Bt) seed mixed into a single product—may affect root protection and yield. We describe an experiment including six trial sites conducted over a three‐year period where various seed blends and soil insecticide/seed blend combinations were evaluated. The predominant species contributing to root injury across all sites was the western corn rootworm (Diabrotica virgifera virgifera LeConte). A weighted technique is presented for evaluating root injury for seed blends that offers a reliable estimate of product performance. The addition of a soil insecticide to the seed blend treatments never resulted in significantly improved root protection and failed to provide a consistent yield benefit. Our results suggest that a soil insecticide/seed blend combination approach is not warranted. Additionally, a subanalysis performed on individual refuge and nearby Bt root systems for seed blend treatments provides insight into the spatial characteristics of root injury in seed blend scenarios.     

Mortality impact of Bt transgenic maize roots expressing eCry3.1Ab, mCry3A, and eCry3.1Ab plus mCry3A on western corn rootworm larvae in the field

Mortality of the western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae due to feeding on maize, Zea mays L., expressing Bacillus thuringiensis Berliner (Bt) was evaluated in five Missouri sites in 2007, 2008, and 2009. Specifically, eCry3.1Ab (5307), mCry3A (MIR604), and eCry3.1Ab plus mCry3A proteins relative to survivorship on maize with the same genetic background without these genes (isoline maize) was evaluated. An average of 890.8 +/- 152.3 beetles emerged from isoline plots, whereas average beetle emergence from 5307, MIR604, and 5307 x MIR604 was 1.9 +/- 0.6, 19.3 +/- 6.3, and 0.8 +/- 0.3, respectively, when averaged across 22 replications in five environments. Overall, 66, 50, 61, and 51% of beetles recovered from 5307, MIR604, 5307 x MIR604, and isoline maize, respectively, were female, and there was no significant difference between the number of male and female beetles that emerged from any of these treatments. Mortality due to 5307, MIR604, and 5307 x MIR604 was 99.79, 97.83, and 99.91%, respectively. There was an 8.0-d delay in time to 50% beetle emergence from 5307 compared with isoline maize, which was significantly later than to the other three maize lines. The average delay to 50% emergence from MIR604 and 5307 x MIR604 averaged 4.1 and 4.6 d, respectively later than 50% emergence from isoline maize. Female beetles had a significant delay in time to 50% emergence compared with male beetles from all treatments with the exception of 5307 x MIR604. Data are discussed in terms of insect resistance management in relation to other control measures for western corn rootworm.     

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.     

Field measures of western corn rootworm (Coleoptera: Chrysomelidae) mortality caused by Cry34/35Ab1 proteins expressed in maize event 59122 and implications for trait durability

Maize, Zea mays L., has been transformed to express the Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis strain PS149B1. These two proteins act together as a binary insecticidal protein that is effective against corn rootworm (Coleoptera: Chrysomelidae) species. The design of the resistance management plan to preserve the long-term durability of this trait largely depends on the level of rootworm mortality induced by Cry34/35Ab1 corn rootworm-protected maize (frequently referred to as "dose" in this context). Here, we report on studies that showed Cry34/35Ab1-expressing maize event 59122 caused 99.1 to 99.98% mortality of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae, after adjusting adult emergence numbers for density-dependent mortality. In two of three studies, there was a short delay in time to 50% adult emergence from 59122 maize plots compared with control plots, although emergence was completed at approximately the same time from both types of maize. These data support an expectation that alleles conferring resistance to the Cry34/35Ab1 proteins in western corn rootworm will be functionally nearly completely to completely recessive on 59122 maize and that there is unlikely to be assortative mating of Cry34/35Ab1-resistant and susceptible rootworms. When incorporated into simulation models of rootworm adaptation to transgenic maize, these findings suggest that a 20% refuge is likely to be highly effective at prolonging the durability of 59122 maize.