B. Wade French

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.

Prairie Grasses as Hosts of the Northern Corn Rootworm (Coleoptera: Chrysomelidae)

Abstract 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.

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.

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

ABSTRACT 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 rootwormactive 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.

Selection for Cry3Bb1 Resistance in a Genetically Diverse Population of Nondiapausing Western Corn Rootworm (Coleoptera: Chrysomelidae)

ABSTRACT 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 &dgr;-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 ≈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.

Capsules containing entomopathogenic nematodes as a Trojan horse approach to control the western corn rootworm

AimsThe use of entomopathogenic nematodes in the biological control of soil insect pests is hampered by the costly and inadequate application techniques. As a possible solution we evaluated a nematode encapsulation approach that offers effective application and may possibly attract the pest by adding attractants to the capsule shell.MethodsHeterorhabditis bacteriophora nematodes, which show high virulence against the maize root pest Diabrotica virgifera virgifera, were encapsulated in a polysaccharide shell derived from the algae Laminaria ssp. Shells of varying thickness and composition were evaluated.ResultsNematodes readily survived the encapsulation process and were able, varying with shell thickness and temperature, to break through the shell and subsequently infect hosts. The added attractants and feeding stimulants to the shell attracted the pest larvae as much as maize roots. In field trials, encapsulated H. bacteriophora nematodes were more effective in controlling D. v. virgifera than those sprayed in water over the soil surface, but in these trials the addition of stimulants did not increase the control efficiency.ConclusionsThe study demonstrates that nematodes can be successfully applied in capsules in the field. Further improvements are needed to make the capsules a cost effective alternative to conventional field application of nematodes.

Adaptation by Western Corn Rootworm (Coleoptera: Chrysomelidae) to Bt Maize: Inheritance, Fitness Costs, and Feeding Preference

ABSTRACT 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.

Genetic Diversity in Laboratory Colonies of Western Corn Rootworm (Coleoptera: Chrysomelidae), Including a Nondiapause Colony

Abstract Laboratory-reared western corn rootworms, Diabrotica virgifera virgifera, from colonies maintained at the North Central Agricultural Research Laboratory (NCARL) in Brookings, SD, are used extensively by many researchers in studies of the biology, ecology, behavior, and genetics of this major insect pest. A nondiapause colony developed through artificial selection in the early 1970s is particularly attractive for many studies because its generation time is much shorter than that of typical diapause colonies. However, the nondiapause colony has been in culture for ≈190 generations without out-crossing. We compared variation at six microsatellite loci among individuals from the NCARL nondiapause colony (≈190 generations), main diapause colony (≈22 generations), four regional diapause colonies (3–8 generations), and four wild populations. Genetic diversity was very similar among the diapause laboratory colonies and wild populations. However, the nondiapause colony showed ≈15–39% loss of diversity depending on the measure. Pairwise estimates of FST were very low, revealing little genetic differentiation among laboratory colonies and natural populations. The nondiapause colony showed the greatest genetic differentiation with an average pairwise FST of 0.153. There was little evidence that the laboratory colonies had undergone genetic bottlenecks except for the nondiapause colony. The nondiapause colony has suffered a moderate loss in genetic diversity and is somewhat differentiated from wild populations. This was not unexpected given its history of artificial selection for the nondiapause trait, and the large number of generations in culture. In contrast, the results indicate that the diapause colonies maintained at NCARL are genetically similar to wild populations.

Effects of Riparian and Grassland Habitats on Ground Beetle (Coleoptera: Carabidae) Assemblages in Adjacent Wheat Fields

Abstract Natural habitats surrounding agricultural fields provide a source of natural enemies to assist in pest control. The boundaries among landscape elements filter some organisms attempting to cross them, resulting in differing communities within the landscape elements. Ground beetles are numerous and generally disperse by walking. These qualities make them excellent organisms for the study of boundary dynamics. Our goal was to determine if natural habitats adjacent to wheat fields affected the species composition of ground beetles within the wheat fields. We captured ground beetles from autumn through spring 1996–1997 at two sites using directional pitfall traps placed in wheat fields and adjacent grasslands and riparian zones. Ground beetle abundance reached two peaks, one in autumn and the other in spring. Species composition was most strongly related to these seasons. Axis 1 of a canonical correspondence analysis separated spring active beetles from autumn active beetles. Axis 2 separated winter active beetles. With the effects of season and sites removed, axes 1 and 2 of a partial canonical correspondence analysis separated beetles with respect to habitat. Axis 1 separated beetles into wheat and natural habitat assemblages. Axis 2 further distinguished assemblages in wheat fields as those adjacent to grasslands and those adjacent to riparian habitats. Axis 2 also separated grassland, grassland edge, and riparian edge assemblages from riparian assemblages. Net dispersal of beetles across the boundaries showed no consistent pattern during autumn, winter, or spring. However, mark–recapture studies showed that several species routinely cross boundaries, which resulted in different community structures and an increase in abundance of beetles in the wheat interiors during spring.

Evidence of Resistance to Cry34/35Ab1 Corn by Western Corn Rootworm (Coleoptera: Chrysomelidae): Root Injury in the Field and Larval Survival in Plant-Based Bioassays

Abstract Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States, and recent management of western corn rootworm has included planting of Bt corn. Beginning in 2009, western corn rootworm populations with resistance to Cry3Bb1 corn and mCry3A corn were found in Iowa and elsewhere. To date, western corn rootworm populations have remained susceptible to corn producing Bt toxin Cry34/35Ab1. In this study, we used single-plant bioassays to test field populations of western corn rootworm for resistance to Cry34/35Ab1 corn, Cry3Bb1 corn, and mCry3A corn. Bioassays included nine rootworm populations collected from fields where severe injury to Bt corn had been observed and six control populations that had never been exposed to Bt corn. We found incomplete resistance to Cry34/35Ab1 corn among field populations collected from fields where severe injury to corn producing Cry34/35Ab1, either singly or as a pyramid, had been observed. Additionally, resistance to Cry3Bb1 corn and mCry3A corn was found among the majority of populations tested. These first cases of resistance to Cry34/35Ab1 corn, and the presence of resistance to multiple Bt toxins by western corn rootworm, highlight the potential vulnerability of Bt corn to the evolution of resistance by western corn rootworm. The use of more diversified management practices, in addition to insect resistance management, likely will be essential to sustain the viability of Bt corn for management of western corn rootworm.