Spatial Variation in Western Corn Rootworm (Coleoptera: Chrysomelidae) Susceptibility to \u3ci\u3eBacillus thuringiensis\u3c/i\u3e Corn Events in Nebraska

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an economically important pest species of field corn (Zea mays L.) in the U.S. Corn Belt. Yield losses and control costs exceed greater than $1 billion annually. Over the past 10+ years, growers have adopted transgenic corn hybrids expressing rootworm-active Bacillus thuringiensis (Bt) proteins as a primary WCR management tactic. Field-evolved resistance to Cry3Bb1 and mCry3A proteins expressed in single-trait Bt hybrids has been confirmed in some areas of Nebraska. Growers have used different tactics as needed to prevent or mitigate resistance, resulting in a mosaic of selection pressure placed on local populations. Currently, research is needed to characterize WCR susceptibility to these Bt toxins on a local spatial scale. Therefore, this study was conducted to characterize spatial variation in WCR susceptibility to Bt corn events in two corn-intensive production areas of Nebraska. Single-plant larval bioassays were conducted in 2016 and 2017 to characterize the susceptibility of populations from pre-established grids in Keith and Buffalo counties to single-trait Cry3Bb1 and mCry3A proteins. Bioassay results confirmed a mosaic of susceptibility to Cry3Bb1 and mCry3A existed within the two landscapes. Patterns at several spatial scales were observed and some gene flow of resistant alleles was indirectly documented in the landscape. A field history index, comprised of additive and weighted WCR management tactics and agronomic practices was developed in an attempt to explain variation in WCR susceptibility. Regression analyses indicated a strong relationship between past management practices and WCR survival on Cry3Bb1, indicating that localized selection pressure is a major contributing factor in determining current susceptibility. These two study areas can be viewed as model systems to draw potential inferences about the relationship of susceptibility to past Bt management histories. These retrospective case histories will inform use of current rootworm-Bt technologies and contribute to the future development of sustainable rootworm management strategies conducted within an IPM framework. Advisor: Lance J. Meink

Reduced susceptibility of western corn rootworm (\u3ci\u3eDiabrotica virgifera virgifera\u3c/i\u3e LeConte) populations to Cry34/35Ab1‑expressing maize in northeast Nebraska

The western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) is a significant pest of maize (Zea mays L.) across the United States Corn Belt. Transgenic maize hybrids expressing insecticidal proteins derived from Bacillus thuringiensis (Bt) have been used to manage WCR since 2003. Widespread resistance to Cry3Bb1 (and associated cross-resistance to mCry3A and eCry3.1Ab) has placed increased selection pressure on Cry34/35Ab1 in single-protein and pyramided transgenic maize hybrids. Data on the susceptibility of Nebraska WCR populations to Cry34/35Ab1 has not been published since 2015 and plant-based bioassays conducted in 2017–2018 confirmed resistance to Cry3Bb1 + Cry34/35Ab1 maize, suggesting resistance to Cry34/35Ab1 has evolved in the Nebraska landscape. Therefore, plant-based bioassays were conducted on F1 progeny of WCR populations collected from northeast Nebraska in 2018 and 2019. Larval survival and development were used to classify resistance to Cry34/35Ab1 in each WCR population. Bioassays confirmed incomplete resistance to Cry34/35Ab1 maize in 21 of 30 WCR populations; 9 of 30 WCR populations remained susceptible to Cry34/35Ab1. Collectively, results indicate that northeast Nebraska WCR populations were in the initial stages of resistance evolution to Cry34/35Ab1 during 2018–2019. Appropriate resistance management strategies are needed to mitigate resistance and preserve efficacy of rootworm-active products containing Cry34/35Ab1

Evidence of western corn rootworm (\u3ci\u3eDiabrotica virgifera virgifera\u3c/i\u3e LeConte) field-evolved resistance to Cry3Bb1 + Cry34/ 35Ab1 maize in Nebraska

BACKGROUND: Western corn rootworm (WCR; Diabrotica virgifera virgifera) field-evolved resistance to transgenic maize expressing the Cry3Bb1 protein derived from Bacillus thuringiensis (Bt) has been confirmed across the United States Corn Belt. Although use of pyramided hybrids expressing Cry3Bb1 + Cry34/35Ab1 has increased in recent years to mitigate existing WCR Bt resistance, susceptibility of Nebraska WCR populations to this rootworm–Bt pyramid has not been assessed. Plant-based bioassays were used to characterize the susceptibility of WCR populations to Cry3Bb1 and Cry3Bb1 + Cry34/35Ab1 maize. Populations were collected from areas of northeastern Nebraska with a history of planting Bt maize that expressed Cry3Bb1 and Cry34/35Ab1. RESULTS: Significant differences in mean corrected survival among populations within Bt hybrids indicated a mosaic of WCR susceptibility to Cry3Bb1 + Cry34/35Ab1 and Cry3Bb1 maize occurred in the landscape. All field populations exhibited some level of resistance to one or both Bt hybrids when compared to susceptible laboratory control populations in bioassays. Most WCR populations exhibited incomplete resistance to Cry3Bb1 + Cry34/35Ab1 maize (92%) and complete resistance to Cry3Bb1 maize (79%). CONCLUSION: The present study confirms the first cases of field-evolved resistance to Cry3Bb1 + Cry34/35Ab1 maize in Nebraska and documents a landscape-wide WCR Cry3Bb1 resistance pattern in areas characterized by long-term continuous maize production and associated planting of Cry3Bb1 hybrids. Use of a multi-tactic integrated pest management approach is needed in areas of continuous maize production to slow or mitigate resistance evolution to Bt maize

Characterizing the sublethal effects of SmartStax PRO dietary exposure on life history traits of the western corn rootworm, \u3ci\u3e Diabrotica virgifera virgifera\u3c/i\u3e LeConte

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an economically important pest of field corn (Zea mays L.) across the United States (U.S.) Corn Belt. Repeated use of transgenic hybrids expressing Bacillus thuringiensis (Bt) proteins has selected for field-evolved resistance to all current rootworm-active Bt proteins. The newest product available for WCR management is SmartStax® PRO, a rootworm-active pyramid containing Cry3Bb1, Cry34/35Ab1 [now reclassified as Gpp34Ab1/Tpp35Ab1] and a new mode of action, DvSnf7 dsRNA. Understanding the fitness of adult WCR after dietary exposure to SmartStax® PRO will identify potential impacts on WCR population dynamics and inform efforts to optimize resistance management strategies. Therefore, the objective of the present study was to characterize the effect of SmartStax® PRO dietary exposure on WCR life history traits. Adult WCR were collected during 2018 and 2019 from emergence tents placed over replicated field plots of SmartStax® PRO or non-rootworm Bt corn at a site with a history of rootworm-Bt trait use and suspected resistance to Cry3Bb1 and Cry34/35Ab1. Adult survival was reduced by 97.1–99.7% in SmartStax® PRO plots relative to the non-rootworm Bt corn plots during the study. Individual male/female pairs were fed different diets of ear tissue to simulate lifetime or adult exposure. Life history parameters measured included adult longevity, adult head capsule width, lifetime female egg production, and egg viability. Results indicate that lifetime or adult exposure to SmartStax® PRO significantly reduced adult longevity and lifetime egg production. Larval exposure to SmartStax® PRO significantly reduced WCR adult size. Results from this study collectively suggest that SmartStax® PRO may negatively impact WCR life history traits, which may lead to reduced population growth when deployed in an area with WCR resistance to Bt traits

Effects of SmartStax\u3csup\u3e®\u3c/sup\u3e and SmartStax\u3csup\u3e®\u3c/sup\u3e PRO maize on western corn rootworm (\u3ci\u3eDiabrotica virgifera virgifera\u3c/i\u3e LeConte) larval feeding injury and adult life history parameters

Field-evolved resistance of the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, to Bacillus thuringiensis Berliner (Bt) proteins Cry3Bb1 and Cry34/35Ab1 (now classified as Gpp34Ab1/Tpp35Ab1) expressed in the pyramid SmartStax® has been documented in areas of the United States (U.S.) Corn Belt. SmartStax® PRO is a recently registered rootworm-active pyramid containing the same Bt proteins expressed in SmartStax® plus DvSnf7 dsRNA. Little to no published data is available comparing efficacy of the technologies or potential effects of dietary exposure on adult WCR fitness. Therefore, experiments were conducted to compare effects of adult WCR dietary exposure to SmartStax® and SmartStax® PRO on life history parameters and efficacy of the technologies in the field with both Bt-susceptible and Bt-resistant WCR populations. WCR life history parameters evaluated included adult longevity, head capsule width, egg production, and egg viability. Results of small-plot field trials indicated that both technologies provided a high level of root protection when a Bt-susceptible WCR population was present. Root protection was reduced on SmartStax® but maintained on SmartStax® PRO when WCR Bt resistance occurred. Lifetime egg production was the key life history parameter that was significantly reduced when either Bt-susceptible or Bt-resistant adult WCR were fed SmartStax® or SmartStax® PRO diet. A potential fitness advantage was apparent as egg production was significantly higher in the Bt-resistant than Bt-susceptible population. The similar response by the Bt-susceptible WCR population to SmartStax® and SmartStax® PRO indicates that results were caused by sublethal dietary exposure to Bt proteins. Adult size (males \u3c females) and egg viability (high: \u3e95%) were not significantly different among treatments but longevity results were inconsistent between years. Collectively, the field efficacy and life history parameter data expand existing knowledge of SmartStax® and SmartStax® PRO technologies, which will inform practical WCR resistance management programs

Spatial variation in western corn rootworm (Coleoptera: Chrysomelidae) susceptibility to Cry3 toxins in Nebraska

Repeated use of field corn (Zea mays L.) hybrids expressing the Cry3Bb1 and mCry3A traits in Nebraska has selected for field-evolved resistance in some western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) populations. Therefore, this study was conducted to characterize spatial variation in local WCR susceptibility to Cry3Bb1 and mCry3A traits in Keith and Buffalo counties, Nebraska, and determine the relationship between past management practices and current WCR susceptibility. Adult WCR populations were collected from sampling grids during 2015 and 2016 and single-plant larval bioassays conducted with F1 progeny documented significant variation in WCR susceptibility to Cry3Bb1 and mCry3A on different spatial scales in both sampling grids. At the local level, results revealed that neighboring cornfields may support WCR populations with very different susceptibility levels, indicating that gene flow of resistant alleles from high trait survival sites is not inundating large areas. A field history index, comprised of additive and weighted variables including past WCR management tactics and agronomic practices, was developed to quantify relative selection pressure in individual fields. The field history index-Cry3 trait survivorship relationship from year 1 data was highly predictive of year 2 Cry3 trait survivorship when year 2 field history indices were inserted into the year 1 base model. Sensitivity analyses indicated years of trait use and associated selection pressure at the local level were the key drivers of WCR susceptibility to Cry3 traits in this system. Retrospective case histories from this study will inform development of optimal resistance management programs and increase understanding of plant-insect interactions that may occur when transgenic corn is deployed in the landscape. Results from this study also support current recommendations to slow or mitigate the evolution of resistance by using a multi-tactic approach to manage WCR densities in individual fields within an integrated pest management framework

Resistance management and integrated pest management insights from deployment of a Cry3Bb1+ Gpp34Ab1/Tpp35Ab1 pyramid in a resistant western corn rootworm landscape

In Nebraska USA, many populations of western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, now exhibit some level of resistance to all corn rootworm-active Bacillus thuringiensis Berliner (Bt) proteins expressed in commercial hybrids. Therefore, a study was conducted in northeast Nebraska from 2020–2022 to reevaluate current corn rootworm management options in continuous maize (consecutive planting for ≥2 years). Results from on-farm experiments to evaluate a standard soil-applied insecticide (Aztec® 4.67G) in combination with non-rootworm Bt or rootworm-active Bt pyramided maize (Cry3Bb1 + Gpp34Ab1/Tpp35Ab1) are reported within the context of WCR Bt resistance levels present. Corrected survival from Bt pyramid single-plant bioassays (\u3c0.3, 0.3–0.49, \u3e0.5) was used to place populations into 3 resistance categories. Variables evaluated included root injury, adult emergence, proportion lodged maize, and grain yield. Key results: A composite analysis of all populations across resistance levels indicated that addition of soil insecticide to Bt pyramid significantly reduced adult emergence and lodging but did not significantly increase root protection or yield. Within and among resistance category analyses of root injury revealed that the Bt pyramid remained highly efficacious at any non-rootworm Bt root injury level when resistance was absent or low. When corrected survival was \u3e0.3, mean Bt pyramid root injury tracked more closely in a positive linear fashion with mean non-rootworm Bt root injury (rootworm density x level of resistance interaction). Similar trends were obtained for adult emergence but not yield. Mean Bt pyramid root injury rating was \u3c0.75 in most populations with Bt resistance, which contributed to no significant yield differences among categories. Results are discussed within the context of IPM:IRM tradeoffs and the need to reduce WCR densities in this system to decrease the impact of the density x resistance interaction to bridge use of current pyramids with new technologies introduced over the next decade

Characterization of Western Corn Rootworm (Coleoptera: Chrysomelidae) susceptibility to foliar insecticides in northeast Nebraska

Foliar-applied insecticides are commonly used for adult Western Corn Rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), control in Nebraska but little efficacy data is available. Anecdotal reports of reduced efficacy in areas of northeast Nebraska led to the conduct of this study. Objectives were to (i) evaluate the efficacy of commercial applications of commonly used formulated insecticides (bifenthrin, lambda-cyhalothrin, chlorpyrifos, or tank mixes) for WCR control in 7 northeast Nebraska counties during 2019 and 2020 and (ii) conduct adult WCR concentration-response vial bioassays with bifenthrin, chlorpyrifos, and dimethoate active ingredients on a subset of field populations. Whole plant counts (WPC) were used to measure WCR densities in insecticide-treated and untreated maize fields before and after insecticide application. Field control was excellent with organophosphate/pyrethroid tank mixes as proportional change in mean WPC of treated fields was significantly reduced (\u3e0.90) versus untreated fields where little change in WPC occurred. The exception was one treated Boone County field where proportional reduction in WPC was ≤0.78. Bioassays revealed LC50s and resistance ratios of most populations exposed to bifenthrin and dimethoate were not significantly different than the susceptible control. Most populations exhibited a low level of chlorpyrifos resistance when compared to the susceptible control. Field and lab data suggest the local onset of practical WCR field-evolved resistance to bifenthrin in Boone County and chlorpyrifos in Boone and Colfax counties. Results of this study will increase our understanding of WCR resistance evolution, serve as a comprehensive baseline for future research, and inform WCR management programs

RNAi for Western Corn Rootworm Management: Lessons Learned, Challenges, and Future Directions

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered one of the most economically important pests of maize (Zea mays L.) in the United States (U.S.) Corn Belt with costs of management and yield losses exceeding USD ~1–2 billion annually. WCR management has proven challenging given the ability of this insect to evolve resistance to multiple management strategies including synthetic insecticides, cultural practices, and plant-incorporated protectants, generating a constant need to develop new management tools. One of the most recent developments is maize expressing double-stranded hairpin RNA structures targeting housekeeping genes, which triggers an RNA interference (RNAi) response and eventually leads to insect death. Following the first description of in planta RNAi in 2007, traits targeting multiple genes have been explored. In June 2017, the U.S. Environmental Protection Agency approved the first in planta RNAi product against insects for commercial use. This product expresses a dsRNA targeting the WCR snf7 gene in combination with Bt proteins (Cry3Bb1 and Cry34Ab1/Cry35Ab1) to improve trait durability and will be introduced for commercial use in 2022

Optimizing Egg Recovery From Wild Northern Corn Rootworm Beetles (Coleoptera: Chrysomelidae)

The northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), is one of the most important insect pests in the U.S. Corn Belt. Efforts to obtain eggs from wild northern corn rootworm populations using techniques developed for other rootworm species have been unsuccessful due to lack of oviposition. In 2016, we evaluated four oviposition media in choice tests within each of three female densities in 30.5 × 30.5 × 30.5 cm BugDorm cages. The number of eggs laid per female was significantly affected by female density and the interaction of female density × oviposition media, but oviposition was relatively poor in all oviposition media (1.2 eggs per female when averaging the three female densities and all oviposition media). Single females were also evaluated in nonchoice assays in 6 cm × 6 cm × 8 cm clear plastic boxes and averaged up to 108 eggs per female depending on the oviposition media. In 2017, the cumulative number of eggs laid per female in boxes with one female was not significantly different from the number of eggs laid per female in boxes with 3 females. In 2018, the cumulative number of eggs laid per female was not significantly different between female densities of 1, 3, 5, or 10 females per box. Total egg production per box therefore increased as female density increased. More than 27,000 wild northern corn rootworm eggs were collected from just 190 females when collected relatively early in the field season. We now have an efficient and robust system for obtaining eggs from wild northern corn rootworm females