2010 Annual Report 1a.Objectives (from AD-416) 1) Define genetic and ecological factors impacting management of insect pests of corn;. 2)Define the impact of pest management programs on corn agroecosystems;. 3)Define relationships between insect pests of corn, indigenous biological control organisms, corn plants, and the environment; and. 4)Develop methodology to allow prescription planting of insect resistant corn. 1b.Approach (from AD-416) A combination of methods including classical genetics and molecular techniques will determine what regions of the European corn borer (ECB) genome are associated with resistance to Bacillus thuringiensis (Bt) proteins. Three resistant colonies will be crossed to a susceptible colony; subsequent F2 segregating populations will be analyzed for significant linkage relationships between phenotypes and genetic markers/candidate genes. A major unknown with resistance monitoring methods that rely on the mass mating of field-collected individuals is whether eggs collected from these mating arenas are a representative genetic sample of the collected populations. Best strategy to sample from mass mating arenas will be investigated to better ensure the adequate representation of the genetic variation present in the original field-collected individuals. ECB moths (>100 per location) will be sampled at 80 km intervals along two perpendicular 720-km transects centered in Iowa. In addition to the 80-km scale sampling, an additional eight sites will be distributed along two different parts of the transects, breaking each of two 80-km intervals into five 16-km intervals. This hierarchical design will provide a wide range of distances for sampling the first year. Genetic variation in microsatellite and RFLP molecular markers will be used to determine genetic differentiation and estimate gene flow among locations. Western corn rootworm (WCR) have been sampled from 10 widely separated locations. At least 50 individuals from each location will be genotyped at 10 microsatellite loci. A mark-release-recapture strategy will be used to examine spatial fidelity of newly emerged ECB adults to their natal fields when presented with adjacent areas of grass (cereal crop) suitable as action sites. Careful selection of representative taxa for non-target field studies is a fundamental need. Goals of selection are to choose taxa that effectively represent the invertebrate community while also providing the best chance of detecting any unanticipated effects of Bt crops. Diversity, abundance, functional importance, and exposure or susceptibility to Cry toxins will influence the choice of representative taxa. Bioassays will be conducted using both N. pyrausta-infected and non-infected neonatal larvae susceptible to Bt, and N. pyrausta infected and non-infected neonatal larvae with partial resistance to Bt. The assay regime will expose these insects to laboratory diets containing discriminating amounts of Bt-corn leaf tissue. Controlled experiments in replicated Bt/nonBt strip plots will be used to correlate the level of damage in a nonBt strip of corn to strength of the signal in the yield map data. Our data also will be used to quantify spatial scaling effects on levels of statistical noise and signal strength, and guide design of computational methodology for mapping natural damage in large-scale field experiments. 3.Progress Report In the past year, ecological and genetic studies were conducted on the corn insects, European corn borer (Ostrinia nubilalis), western bean cutworm (Striacosta albicosta), and western corn rootworm (Diabrotica virgifera virgifera). Genetic markers were used to differentiate between two populations of European corn borer within each of two races that were separated by possible physical barriers to movement in Pennsylvania. Results indicate that gene flow is high between sites, over time, and between and within races, suggesting that resistance to transgenic corn may be slow to develop, even in populations partially isolated in mountain valleys. A molecular genetics approach was taken to isolate two genes (slowmo and shaker) from European corn borer that are known behavior-linked genes from the fruit fly (Drosophila melanogaster). The intention is to learn if these genes are associated with European corn borer movement, particularly larval movement. The manner in which Lepidoptera larvae move in the field can have widespread impacts on the methods used to manage them, and on the sustainability of these methods. A laboratory colony of European corn borer was selected for increased tolerance (greater than 12,000 fold) to the Bacillus thuringiensis (Bt)-corn toxin, Cry1F, compared to a susceptible colony. Genomic research demonstrated the Cry1F resistance trait is controlled at a specific region of chromosomal linkage group 12. Laboratory flight mill experiments showed that infection of European corn borer with the microorganism, Nosema pyrausta, has a negative effect on moth flight. Analyses indicate that the negative effects are due to competition by the pathogen for adult energy reserves. A genetic map of the Z sex chromosome for European corn borer was developed that incorporates and compares a genetic map, gene expression data, and sequence data gathered for a panel of 50 sex-linked genes identified in the silkworm genome. Genes located on the Z chromosome in Lepidoptera (butterflies and moths) play important roles in establishing the numbers of insects produced per season as well as the ability of opposite sexes to locate mates in the field. High throughput DNA sequencing technology was used to develop 22 genetic markers for the western bean cutworm, a growing pest of corn in the Midwest. The genetic markers will allow detailed studies of insect population ecology and genetics. Gene sequences from a pathogen were found in the DNA, which will allow development of a test to determine the presence of this pathogen and level of infection in individual moths. Over 1,500 possible new genetic markers were identified from western corn rootworm and submitted for sequencing and verification. Western corn rootworm samples were collected from 10 locations in its original range in Colorado and western Kansas, including the species type-location (Wallace County, Kansas), for genetic diversity and gene flow analyses. Samples from this area are important for obtaining estimates of long-range movement, and the DNA has been isolated in preparation for determining genetic differences between locations, which will allow such estimates to be made. 4.Accomplishments 1. Flight behavior of European corn borer infected with Nosema pyrausta. ARS researchers in Ames, Iowa demonstrated the importance of long-distance movement of European corn borer to its population dynamics and to the effectiveness of current strategies being used to slow its resistance to transgenic Bacillus thuringiensis (Bt)-corn. A pathogen, Nosema pyrausta, is a common disease of European corn borers that reduces longevity and the number of lifetime eggs laid by the moths. In laboratory flight mill experiments, we found that Nosema infection has a negative effect on measures of flight distance, duration, and speed, especially in males, with effects in females detected only when infection was at a moderate to heavy level. Elucidation of the relationship between Nosema infection, wing length, body weight, and flight performance indicates that the negative effects of Nosema infection on flight activity are due to reduction in fuel reserves needed to support flight rather than to effects on wing length and body size. Our results will be used by university and government scientists trying to understand and model European corn borer population dynamics, as well as potential development and spread of resistance to transgenic Bt-corn in the presence of natural Nosema infections. 2. The potential of wild hosts to serve as unstructured refuge for European corn borer in managing resistance to Bacillus thuringiensis (Bt)-corn. To prevent or delay European corn borer developing resistance to Bt-corn, growers are required to plant a certain percentage of their corn acreage to non-Bt corn as a nursery "to grow" susceptible borers that will mate with any resistant borers that might emerge from the Bt corn. Because the European corn borer can feed on other kinds of plants besides corn, it has been suggested that larvae developing and moths emerging from these plants ("unstructured" refuge) might be enough to offset the "structured" non-Bt corn refuge requirement. By using a technique that measures the form of carbon in the larval diet typical of corn or non-corn plants the larvae fed on, ARS researchers in Ames, Iowa, in collaboration with Cornell University found that one race of European corn borer (called "E race," where females produce a particular form of male-attractant pheromone, "E pheromone") does produce a substantial proportion of adults from non-corn host plants; however, the other kind of European corn borer (the Z race) is produced almost entirely on corn. Thus, even though unstructured refuge might be possible for the E race, it is not possible for the Z race, and because we found that in every county where the E race occurs, the Z race also occurs, our study does not support a relaxation of the current structured refuge requirements for European corn borer. The results of this study will be used by regulatory agencies, biotech seed industry scientists, and other government and university scientists in the U.S., Europe, and Asia who conduct research on insect resistance management, and who are involved in developing, recommending, or implementing improved refuge requirements for Bt corn. 3. Z sex chromosome of European corn borer. Genes located on the Z sex chromosome in Lepidoptera (butterflies and moths) play important roles in establishing the numbers of insects produced per season (voltinism) as well as the ability of opposite sexes to locate mates in the field (male pheromone responses). These traits can be extremely important for pest species such as the European corn borer whose inherent ability to find mates, abundance, and generation times in the field directly impacts whether established economic injury levels are reached. This study provides an enhanced resolution of the Z chromosome architecture in the European corn borer, incorporating and comparing a genetic map, gene expression data, and sequence data gathered for a panel of 50 sex-linked genes identified in the silkworm genome. Together, these data provide new and valuable insights into Lepidopteran genetic structures that can be used to identify and evaluate important genes responsible for influencing the field status of pest populations. The type and degree of change in a pest’s phenotype can directly influence its potential for developing both physiological and behavioral resistance against modern control tactics such as genetically-engineered crops. Understanding where and when these genetic changes occur in pest populations enhances our ability to trace and predict their agricultural impacts, and to establish new effective control strategies. 4. The discovery of mobile sections of European corn borer DNA. Microsatellite DNA are small, repeating sequences of DNA that can aid in understanding insect populations and interpreting the inheritance of traits that may affect the longevity of Bacillus thuringiensis (Bt) toxin based insecticides. In the process of developing and using these microsatellite genetic markers for research, we discovered mobile sections of DNA that autonomously move among different positions within the genome of the European corn borer. These mobile DNAs are a common feature within the genomes of other Lepidoptera, and appear to carry microsatellite DNA. The consequence of this finding is that microsatellite genetic markers for European corn borer may no longer be useful for population studies of this insect. This discovery has a major implication on the future genetic studies of lepidopteran crop pests and may lend insight into how their genomes have evolved. 5. Bacillus thuringiensis (Bt) resistance alleles influence behavior of European corn borer larvae following exposure to the Bt-corn toxin, Cry1Ab. Behaviors of insects, including movement and mating, have been recognized as important factors that can influence how fast resistance to Bt crops is likely to develop in the field. However, it is often unknown whether insects with genes that contribute to resistance behave differently than insects without such resistance genes. When European corn borer larvae from a colony selected for Cry1Ab resistance were exposed to diet with Cry1Ab toxin, they did not show a greater ability to avoid toxins (called "behavioral resistance") compared with susceptible corn borers. This information will be incorporated into a model and used to guide recommendations for new types of refuges for managing European corn borer resistance to Bt corn. Review Publications Carroll, M.W., Glaser, J.A., Hellmich II, R.L., Hunt, T.E., Calvin, D., Sappington, T.W., Copenhaver, K., Fridgen, J. 2009. Detection of European Corn Borer Infestation in Iowa Corn Plots using Spectral Vegetation Indices Derived from Airborne Hyperspectral Imagery. Journal of Economic Entomology. 101(5):1614-1623. O'Rourke, M.E., Sappington, T.W., Fleischer, S.J. 2010. Managing Resistance to Bt Crops in a Genetically Variable Insect Herbivore, Ostrinia nubilalis. Ecological Applications. 20(5):1228-1236. Dorhout, D.L., Sappington, T.W., Lewis, L.C., Rice, M.E. 2011. Flight behavior of European corn borer infected with Nosema pyrausta. Journal of Applied Entomology. 135(1-2):25-37. Kim, K.S., Bagley, M.J., Coates, B.S., Hellmich II, R.L., Sappington, T.W. 2009. Spatial and Temporal Genetic Analyses Show High Gene Flow Among European Corn Borer (Lepidoptera: Crambidae) Populations Across the Central U.S. Corn Belt. Environmental Entomology. 38(4):1312-1323. Coates, B.S., Miller, N.J., Sumerford, D.V., Sappington, T.W., Siegfried, B.D., Lewis, L.C. 2009. Comparative Performance of Single Nucleotide Polymorphism (SNP) and Microsatellite Markers for the Detection of Population Differentiation in Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Journal of Heredity. 100(5):556-564. Miller, N.J., Richards, S., Sappington, T.W. 2010. The Prospects for Sequencing the Western Corn Rootworm Genome. Journal of Applied Entomology. 134(5):420-428. Greenberg, S.M., Adamczyk Jr, J.J., Sappington, T.W., Jones, W.A., Liu, T. 2008. Interrelationship between plants and insects as the basis for IPM systems. Egyptian Journal of Agricultural Research. 86(1):133-148. Greenberg, S.M., Sappington, T.W., Adamczyk Jr, J.J., Liu, T., Setamou, M. 2008. Effects of photoperiod on boll weevil (Coleoptera: Curculionidae) development, survival, and reproduction. Environmental Entomology. 37(6):1396-1402. Spurgeon, D.W., Sappington, T.W., and Rummel, D.R. 2008. Host-free Survival of Boll Weevils (Coleoptera: Curculionidae) from Two Regions of Texas. Southwest Entomol. 33:151-152. Coates, B.S., Sumerford, D.V., Hellmich II, R.L., Lewis, L.C. 2008. Mining an Ostrinia nubilalis Midgut Expressed Sequence Tag (EST) Library for Candidate Genes and Single Nucleotide Polymorphisms (SNPs). Insect Molecular Biology. 17(6):607-620. Coates, B.S., Sumerford, D.V., Hellmich II, R.L., Lewis, L.C. 2009. Repetitive Genomic Elements in a European Corn Borer, Ostrinia nubilalis, Bacterial Artificial Chromosome Library were Indicated by Bacterial Artificial Chromosome End Sequencing and Development of Sequence Tag Site Markers: Implications for Lepidopteran Genomic Research. Genome. 52(1):57-67. Hellmich II, R.L., Gorecka, J. 2008. Non-target Risk Assessment for Crops Engineered for Insect Resistance. Annals of Warsaw University of Life Sciences. 29:8-17. Prasifka, J.R., Hellmich II, R.L., Dively, G.P., Higgins, L.S., Dixon, P.M., Duan, J.J. 2008. Selection of Nontarget Arthropod Taxa for Field Research on Transgenic Insecticidal Crops: Using Empirical Data and Statistical Power. Environmental Entomology. 37(1):1-10. Romeis, J., Bartsch, D., Bigler, F., Candolfi, M., Gielkens, M.C., Hartley, S.E., Hellmich II, R.L., Huesing, J.E., Jepson, P.C., Layton, R., Quemada, H., Raybould, A., Rose, R., Schiemann, J., Sears, M., Shelton, A., Sweet, J., Vaituzis, Z., Wolt, J. 2008. Assessment of Risk of Insect-resistant Transgenic Crops to Nontarget Arthropods. Nature Biotechnology. 26(2):203-208. Kim, K.S., Jones, G.D., Westbrook, J.K., Sappington, T.W. 2010. Multidisciplinary Fingerprints: Forensic Reconstruction of an Insect Reinvasion. Journal of the Royal Society Interface. 7(45):677-686.