A draft Diabrotica virgifera virgifera genome: insights into control and host plant adaption by a major maize pest insect.

BACKGROUND: Adaptations by arthropod pests to host plant defenses of crops determine their impacts on agricultural production. The larval host range of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is restricted to maize and a few grasses. Resistance of D. v. virgifera to crop rotation practices and multiple insecticides contributes to its status as the most damaging pest of cultivated maize in North America and Europe. The extent to which adaptations by this pest contributes to host plant specialization remains unknown. RESULTS: A 2.42 Gb draft D. v. virgifera genome, Dvir_v2.0, was assembled from short shotgun reads and scaffolded using long-insert mate-pair, transcriptome and linked read data. K-mer analysis predicted a repeat content of ≥ 61.5%. Ortholog assignments for Dvir_2.0 RefSeq models predict a greater number of species-specific gene duplications, including expansions in ATP binding cassette transporter and chemosensory gene families, than in other Coleoptera. A majority of annotated D. v. virgifera cytochrome P450s belong to CYP4, 6, and 9 clades. A total of 5,404 transcripts were differentially-expressed between D. v. virgifera larvae fed maize roots compared to alternative host (Miscanthus), a marginal host (Panicum virgatum), a poor host (Sorghum bicolor) and starvation treatments; Among differentially-expressed transcripts, 1,908 were shared across treatments and the least number were between Miscanthus compared to maize. Differentially-expressed transcripts were enriched for putative spliceosome, proteosome, and intracellular transport functions. General stress pathway functions were unique and enriched among up-regulated transcripts in marginal host, poor host, and starvation responses compared to responses on primary (maize) and alternate hosts. CONCLUSIONS: Manual annotation of D. v. virgifera Dvir_2.0 RefSeq models predicted expansion of paralogs with gene families putatively involved in insecticide resistance and chemosensory perception. Our study also suggests that adaptations of D. v. virgifera larvae to feeding on an alternate host plant invoke fewer transcriptional changes compared to marginal or poor hosts. The shared up-regulation of stress response pathways between marginal host and poor host, and starvation treatments may reflect nutrient deprivation. This study provides insight into transcriptomic responses of larval feeding on different host plants and resources for genomic research on this economically significant pest of maize.

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

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

A Physiological and Behavioral Mechanism for Leaf Herbivore-Induced Systemic Root Resistance.

Indirect plant-mediated interactions between herbivores are important drivers of community composition in terrestrial ecosystems. Among the most striking examples are the strong indirect interactions between spatially separated leaf- and root-feeding insects sharing a host plant. Although leaf feeders generally reduce the performance of root herbivores, little is known about the underlying systemic changes in root physiology and the associated behavioral responses of the root feeders. We investigated the consequences of maize (Zea mays) leaf infestation by Spodoptera littoralis caterpillars for the root-feeding larvae of the beetle Diabrotica virgifera virgifera, a major pest of maize. D. virgifera strongly avoided leaf-infested plants by recognizing systemic changes in soluble root components. The avoidance response occurred within 12 h and was induced by real and mimicked herbivory, but not wounding alone. Roots of leaf-infested plants showed altered patterns in soluble free and soluble conjugated phenolic acids. Biochemical inhibition and genetic manipulation of phenolic acid biosynthesis led to a complete disappearance of the avoidance response of D. virgifera. Furthermore, bioactivity-guided fractionation revealed a direct link between the avoidance response of D. virgifera and changes in soluble conjugated phenolic acids in the roots of leaf-attacked plants. Our study provides a physiological mechanism for a behavioral pattern that explains the negative effect of leaf attack on a root-feeding insect. Furthermore, it opens up the possibility to control D. virgifera in the field by genetically mimicking leaf herbivore-induced changes in root phenylpropanoid patterns.

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

Transgenic plants have been widely adopted by growers to manage the western corn rootworm, Diabrotica virgifera virgifera LeConte, in field corn. Because of reduced efficacy in some Nebraska fields after repeated use of Cry3Bb1-expressing hybrids, single plant bioassays were conducted in 2012 and 2013 to characterize the susceptibility of western corn rootworm populations to the rootworm-active proteins Cry3Bb1, mCry3A, and Cry34/35Ab1. Results demonstrate that there are heritable differences in susceptibility of Nebraska western corn rootworm populations to rootworm-active Bt traits. Proportional survival and corrected survival data coupled with field histories collectively support the conclusion that a level of field resistance to Cry3Bb1 has evolved in some Nebraska populations in response to selection pressure and that cross-resistance exists between Cry3Bb1 and mCry3A. There was no apparent cross-resistance between Cry34/35Ab1 and either Cry3Bb1 or mCry3A. The potential implications of these results on current and future corn rootworm management strategies are discussed.

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

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

Distribution of genes and repetitive elements in the Diabrotica virgifera virgifera genome estimated using BAC sequencing.

Feeding damage caused by the western corn rootworm, Diabrotica virgifera virgifera, is destructive to corn plants in North America and Europe where control remains challenging due to evolution of resistance to chemical and transgenic toxins. A BAC library, DvvBAC1, containing 109,486 clones with 104 ± 34.5 kb inserts was created, which has an ~4.56X genome coverage based upon a 2.58 Gb (2.80 pg) flow cytometry-estimated haploid genome size. Paired end sequencing of 1037 BAC inserts produced 1.17 Mb of data (~0.05% genome coverage) and indicated ~9.4 and 16.0% of reads encode, respectively, endogenous genes and transposable elements (TEs). Sequencing genes within BAC full inserts demonstrated that TE densities are high within intergenic and intron regions and contribute to the increased gene size. Comparison of homologous genome regions cloned within different BAC clones indicated that TE movement may cause haplotype variation within the inbred strain. The data presented here indicate that the D. virgifera virgifera genome is large in size and contains a high proportion of repetitive sequence. These BAC sequencing methods that are applicable for characterization of genomes prior to sequencing may likely be valuable resources for genome annotation as well as scaffolding.

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.

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.

Effect of MIR604 transgenic maize at different stages of development on western corn rootworm (Coleoptera: Chrysomelidae) in a central Missouri field environment.

The establishment and survival of western corn rootworm, Diabrotica virgifera virgifera LeConte, was evaluated on transgenic Bacillus thuringiensis Berliner maize, Zea mays L., expressing the mCry3A protein (MIR604) and non-Bt maize with the same genetic background (isoline maize) at different stages of development in 2007 and 2008. Overall, western corn rootworm larval recovery, root damage, and adult emergence were significantly higher on isoline maize compared with MIR604. The number of larvae and adults collected from MIR604 did not significantly differ among egg hatch dates from each maize developmental stage evaluated in either year. In 2007, damage to isoline maize roots was lower than expected and never exceeded 0.24 nodes of damage. In 2008, over 0.60 nodes of damage occurred on isoline maize roots. The mean weight and head capsule width of larvae and adults recovered from MIR604 and isoline maize were generally not significantly different. Results are discussed in relation to insect resistance management of western corn rootworm.

Characterizing the Relationship Between Western Corn Rootworm (Coleoptera: Chrysomelidae) Larval Survival on Cry3Bb1-Expressing Corn and Larval Development Metrics.

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a significant pest of field corn, Zea mays L. (Poales: Poaceae), across the United States Corn Belt. Widespread adoption and continuous use of corn hybrids expressing the Cry3Bb1 protein to manage the western corn rootworm has resulted in greater than expected injury to Cry3Bb1-expressing hybrids in multiple areas of Nebraska. Single-plant bioassays were conducted on larval western corn rootworm populations to determine the level of resistance present in various Nebraska counties. The results confirmed a mosaic of susceptibility to Cry3Bb1 across Nebraska. Larval development metrics, including head capsule width and fresh weight, were measured to quantify the relationship between the level of resistance to Cry3Bb1 and larval developmental rate. Regression and correlation analyses indicate a significant positive relationship between Cry3Bb1 corrected survival and both larval development metrics. Results indicate that as the level of resistance to Cry3Bb1 within field populations increases, mean head capsule width and larval fresh weight also increase. This increases our understanding of western corn rootworm population dynamics and age structure variability present in the transgenic landscape that is part of the complex interaction of factors that drives resistance evolution. This collective variability and complexity within the landscape reinforces the importance of making corn rootworm management decisions based on information collected at the local level.

Author Correction: Multidimensional approach to formulating a specialized diet for northern corn rootworm larvae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Native resistance to western corn rootworm (Coleoptera: Chrysomelidae) larval feeding: characterization and mechanisms.

Seven maize, Zea mays L., genotypes selected for native resistance to western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), larval feeding damage (SUM2068, SUM2162, CRW3(S1)C6, NSS1 x CRW3(S1)C6, PI583927, CRW2(C5), and AR17056-16) were evaluated along with three control maize genotypes in the field for plant damage, larval recovery, adult emergence, root size, and root regrowth. Larvae recovered were further analyzed for head capsule width and dry weight and adults for dry weight. All factors evaluated with the exception of adult dry weight varied significantly among maize genotypes. Control genotypes included a highly susceptible hybrid, B37 x H84, a transgenic rootworm-resistant hybrid expressing the modified Cry3A protein (MIR604), and the untransformed modern hybrid with the same genetic background as the MIR604 we used (isoline) as a second susceptible control. In general, the genotypes previously selected for resistance to western corn rootworm larval feeding had less damage, fewer larvae recovered, smaller larvae recovered, and fewer adults recovered than the susceptible controls. SUM2162 was significantly less damaged than all other native sources of resistance. Western corn rootworm larvae recovered from SUM2162 and SUM2068 were significantly smaller in terms of head capsule width and average weight than larvae recovered from all other maize genotypes, indicating that antibiosis is a mechanism of resistance for these two hybrids.

Field-Evolved Resistance of Northern and Western Corn Rootworm (Coleoptera: Chrysomelidae) Populations to Corn Hybrids Expressing Single and Pyramided Cry3Bb1 and Cry34/35Ab1 Bt Proteins in North Dakota.

Northern, Diabrotica barberi Smith & Lawrence, and western, D. virgifera virgifera LeConte, corn rootworms (Coleoptera: Chrysomelidae) are major economic pests of corn, Zea mays L., in North America. Corn hybrids expressing Bacillus thuringiensis Berliner (Bt) toxins are commonly used by growers to manage these pests. Several cases of field-evolved resistance to insecticidal proteins expressed by Bt corn hybrids have been documented in many corn-producing areas of North America, but only for D. v. virgifera. In 2016, beetles of both species were collected from five eastern North Dakota corn fields and reared in a growth chamber. In 2017, larvae reared from those populations were subjected to single-plant bioassays to screen for potential resistance to Cry3Bb1, Cry34/35Ab1, and pyramided Cry3Bb1 + Cry34/35Ab1 Bt toxins. Our results provide the first documented report of field-evolved resistance in D. barberi to corn hybrids expressing Cry3Bb1 (Arthur problem population) and Cry34/35Ab1 (Arthur and Page problem populations, and the Ransom and Sargent populations) proteins in North America. Resistance to Cry3Bb1 was also observed in the Ransom population of D. v. virgifera. Increased larval survival on the pyramided Cry3Bb1 + Cry34/35Ab1 hybrid was observed in both species. No cross-resistance was evident between Cry3Bb1 and Cry34/35Ab1 in any of the D. barberi populations tested. Our experiments identified field-evolved resistance to Bt toxins in some North Dakota populations of D. barberi and D. v. virgifera. Thus, more effective control tools and improved resistance management strategies are needed to prolong the durability of this technology for managing these important pests.

Comparative Susceptibility of Western Corn Rootworm (Coleoptera: Chrysomelidae) Neonates to Selected Insecticides and Bt Proteins in the Presence and Absence of Feeding Stimulants.

The susceptibility of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae to nine insecticides from five different classes and to Bt proteins eCry3.1Ab and mCry3A in the presence or absence of feeding stimulants, was estimated in filter paper and diet toxicity assays, respectively. The use of a synthetic feeding stimulant blend of the sugars glucose, sucrose, and fructose plus linoleic acid at a ratio of 30:4:4:0.3 mg/ml of distilled water was evaluated to determine whether they increase the efficacy of insecticides and Bt proteins. The efficacy of thiamethoxam diluted in solutions with feeding stimulants was significantly increased when compared to thiamethoxam dilutions in water (>60-fold). Differences in the efficacy of the other insecticide classes when diluted in feeding stimulant solutions were no greater than fivefold when compared to the insecticides diluted in water. The presence of corn root juice as a natural feeding stimulant diminished toxicity of the insecticides, except for thiamethoxam, even though larval fresh weight was higher when fed on root juice compared to feeding stimulant or water. The use of feeding stimulants in diet toxicity assays did not enhance efficacy of eCry3.1Ab nor mCry3A proteins. Feeding stimulants can be recommended in combination with thiamethoxam to increase larval mortality. These results are discussed in terms of applicability of feeding stimulants to improve susceptibility of western corn rootworm larvae to pesticides in general.

Multidimensional approach to formulating a specialized diet for northern corn rootworm larvae.

The northern corn rootworm (NCR), Diabrotica barberi Smith & Lawrence, is a major pest of maize (Zea mays L.). This pest has developed resistance to insecticides and adapted to crop rotation and may already be in the early stages of adaptation to toxins produced by Bacillus thuringiensis (Bt). Toxicity bioassays using artificial diet have proven to be valuable for monitoring resistance in many species, but no artificial diet has been developed specifically for NCR larvae. Toward this end, we first evaluated known Diabrotica diets to identify a starting media. We then developed a specialized diet for NCR using an iterative approach. Screening designs including 8 diet components were performed to identify the principal nutritional components contributing to multiple developmental parameters (survival, weight, and molting). We then applied mixture designs coupled with response surface modeling to optimize a blend of those components. Finally, we validated an improved NCR diet formulation that supports approximately 97% survival and molting, and a 150% increase in larval weight after 10 days of feeding compared with the best previously published artificial diet. This formulation appears suitable for use in diet bioassays as a tool for evaluating the resistance of NCR populations to insecticides.

Utility of EST-derived SSRs as population genetics markers in a beetle.

Microsatellite, or simple sequence repeat (SSR), loci can be identified by mining expressed sequence tag (EST) databases, and where these are available, marker development time and expense can be decreased considerably over conventional strategies of probing the entire genome. However, it is unclear whether they provide information on population structure similar to that generated by anonymous genomic SSRs. We performed comparative population genetic analyses between EST-derived SSRs (EST-SSRs) and anonymous SSRs developed from genomic DNA for the same set of populations of the insect Diabrotica virgifera, a beetle in the family Chrysomelidae. Compared with noncoding, nontranscribed regions, EST-SSRs were generally less polymorphic but had reduced occurrence of null alleles and greater cross-species amplification. Neutrality tests suggested the loci were not under positive selection. Across all populations and all loci, the genomic and EST-SSRs performed similarly in estimating genetic diversity, F(IS), F(ST), population assignment and exclusion tests, and detection of distinct populations. These findings, therefore, indicate that the EST-SSRs examined can be used with confidence in future genetic studies of Diabrotica populations and suggest that EST libraries can be added as a valuable source of markers for population genetics studies in insects and other animals.

Prairie grasses as hosts of the northern corn rootworm (Coleoptera: Chrysomelidae).

We evaluated 27 prairie grass species thought to be among those dominant 200 yr ago in the northern midwest as larval hosts of the northern corn rootworm, Diabrotica barberi Smith and Lawrence. Maize (Zea mays L.), spring wheat (Triticum aestivum L.), and grain sorghum (Sorghum bicolor L.) were included as controls for a total of 30 species. Twenty pots of each species were planted in a randomized complete block design. Each pot was infested 5 wk later with 20 neonate northern corn rootworm larvae. Two pots within each species and block were assigned an extraction date of 7 or 14 d after infestation. The remaining two pots from each block were used to monitor adult emergence. The percentage of larvae recovered, change in larval head capsule width, and change in average dry weights varied significantly among the grass species. The highest percentage of larvae was recovered from slender wheatgrass, Elymus trachycaulus (Link), and this was significantly greater than the percentage recovered from all other species including maize for the 14-d sample date. Several additional species were also relatively good hosts, in that the percentage of larvae recovered from these species was not significantly different from maize. The average dry weight of larvae recovered was significantly greater for larvae recovered from maize than for larvae recovered from all other species except slender wheatgrass, when the two samples dates were combined. Overall, adults were produced from only 6 of the 28 species evaluated, and no analysis was performed because of the low numbers. The results of this study are discussed in relation to the potential of alternate hosts of northern corn rootworm to serve as a bridge to survival on transgenic maize.

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.

Author Correction: A new artificial diet for western corn rootworm larvae is compatible with and detects resistance to all current Bt toxins.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Transgenic Bt Corn, Soil Insecticide, and Insecticidal Seed Treatment Effects on Corn Rootworm (Coleoptera: Chrysomelidae) Beetle Emergence, Larval Feeding Injury, and Corn Yield in North Dakota.

Northern, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), and western, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), corn rootworms are economic pests of corn, Zea mays L. in North America. We measured the impacts of corn hybrids incorporated with Cry3Bb1, Cry34/35Ab1, and pyramided (Cry3Bb1 + Cry34/35Ab1) Bacillus thuringiensis Berliner (Bt) proteins, tefluthrin soil insecticide, and clothianidin insecticidal seed treatment on beetle emergence, larval feeding injury, and corn yield at five locations from 2013 to 2015 in eastern North Dakota. In most cases, emergence was significantly lower in Bt-protected corn than in non-Bt corn hybrids. Exceptions included Wyndmere, ND (2013), where D. barberi emergence from Cry34/35Ab1 plots was not different from that in the non-Bt hybrid, and Arthur, ND (2013), where D. v. virgifera emergence from Cry3Bb1 plots did not differ from that in the non-Bt hybrid. Bt hybrids generally produced increased grain yield compared with non-Bt corn where rootworm densities were high, and larval root-feeding injury was consistently lower in Bt-protected plots than in non-Bt corn. The lowest overall feeding injury and emergence levels occurred in plots planted with the Cry3Bb1 + Cry34/35Ab1 hybrid. Time to 50% cumulative emergence of both species was 5-7 d later in Bt-protected than in non-Bt hybrids. Tefluthrin and clothianidin were mostly inconsequential in relation to beetle emergence and larval root injury. Our findings could suggest that some North Dakota populations could be in early stages of increased tolerance to some Bt toxins; however, Bt corn hybrids currently provide effective protection against rootworm injury in eastern North Dakota.