2011 Annual Report
1a.Objectives (from AD-416)
1) Improve knowledge of the ecology, genetics, and behavior of corn pests such as corn borer, corn rootworm, and western bean cutworm in relation to pest abundance and insect resistance to transgenic corn;.
2)Determine the nature and degree of potential impacts of transgenic corn (e.g., Bt corn) on non-target organisms; and.
3)Develop genetic and molecular methods to investigate corn insect biology and plant-insect interactions.
1b.Approach (from AD-416)
Laboratory trials will examine how resistance alleles from European corn borer (ECB) colonies impact dispersal and survival of neonates after exposure to various Bt toxins. Movement and avoidance behaviors will be evaluated using video-tracking and bioassays with lyophilized leaf tissue incorporated into artificial diet. Results of the laboratory trials will guide selection of appropriate treatments for subsequent semi-field experiments. The marker development and screening strategies used for identifying Bt resistance linkage groups in ECB will be adapted for developing molecular markers associated with behavioral adaptations in ECB. We will characterize gene flow and genetic structuring among ECB populations in Pennsylvania (PA). We will use a panel of 74 SNP markers, previously identified and verified from EST sequences, in population genetics analyses to determine the effects of potential geographic barriers on ECB gene flow separately for the pheromone races, and determine if genetic structuring is associated with host plant association in the E race. The development of laboratory tests to evaluate possible effects of stressors from GE crops involves two phases. The first phase is development of a standardized protocol for testing an orally administered test substance on a specific insect and life stage. The second phase is using these protocols to parallel test (a.k.a. ring test) the selected insect at three or more laboratories. We will take a mass-screening approach to validate EST-derived SNPs for WCR using the Illumina Golden Gate SNP assay platform. We anticipate that about 75% of the 4,111 candidate SNPs we have identified will produce a designable assay, which would translate into nearly 3,100 assays. These candidate SNPs, for which an assay can be designed, will be tested for polymorphism against samples of rotation-resistant WCR populations from Illinois and wild-type populations from Iowa. Information from the Illumina GenCall and GTS Reports software will be used to remove bad SNPs and bad samples. Complementing the genomics and bioinformatics groundwork, we will investigate Bt resistance traits by integrating structural and functional genome information. SNP markers will be used for mapping QTL studies of ECB, and population studies of ECB and WBC. Our experiments will investigate separate native resistance (NR) sources leading to differential feeding or survival of WCR larvae on roots, of WCR adults on silks, and of corn earworm (CEW) larvae on silks. For both lepidopteran and coleopteran pests, we will narrowly define chromosomal segments of the corn genome that harbor NR alleles. We will also create large sets of inbred and hybrid isolines to enable future mechanistic analysis of the effects of the NR alleles on both target and non-target insects.
Several ecological and genetic studies related to objective 1 were conducted. Movement of European corn borer later instars (older larvae) is being evaluated under both high and low density conditions. Experiments evaluated pre-dispersal mortality of neonates and third instars using Bacillus thuringiensis (Bt) and non-Bt tissues. Diapause and adult emergence experiments with Cry1Ab-resistant larvae and Cry1F-resistant larvae were completed. Results were incorporated by a collaborator into a simulation model of European corn borer infesting Bt corn to compare insect resistance management (IRM) strategies using seed mixtures and block refuges. Studies are underway to profile the expression of candidate behavior-linked genes in the European corn borer. Selection has produced larvae that rapidly leave (rovers) or stay (sitters) on corn plants. These studies provide a basis for understanding the molecular biology that governs movement behavior in European corn borer larvae and how it may be impacted by pesticide exposure and transgenic corn. European corn borer samples from Europe and the U.S. East Coast were collected, and the DNA extracted, quantified, and exchanged with collaborators. Additionally, a combined single nucleotide polymorphism (SNP) and microsatellite-based genotyping approach was devised with French collaborators to estimate population genetic parameters. 339,438 sequences from Lepidoptera were collected from the GenBank Expressed Sequence Tags database (dbEST) and from collaborators. Each sequence was functionally annotated, and the data displayed on Lepdb.org, an ARS database and web interface developed for use by the research community. A single interval of the European corn borer genome was identified as being linked to Cry1F toxin resistance in larvae. 417 segregating markers were integrated into a merged amplified fragment length polymorphism (AFLP)- and SNP-marker based genetic linkage map, and identification of markers linked to the quantitative trait loci (QTL) responsible for resistance is ongoing. Western corn rootworm SNP validation assays were conducted, and DNA was isolated from five mapping families. An ARS database called WCRbase has been prepared, and is being modified to ensure compatibility with databases being generated by French colleagues. Related to objective 2, testing protocols were developed with collaborators to test potential non-target effects of genetically engineered (GE) crops on the lady beetle, Coleomegilla maculata. Related to objective 3, corn germplasm bred for native resistance to western corn rootworm was used to derive a population of lines that will permit detailed genetic analysis of resistance mechanisms. By leveraging haploid induction and chromosome doubling methods, eight generations of conventional breeding were achieved in eight months. Studies show native corn earworm resistance developed by the lab is as effective at limiting pest growth as transgenic Cry1Ab protein. Genetic analysis of this mechanism has commenced.
New European corn borer migration information improves understanding of Bacillus thuringiensis (Bt) resistance management. The dispersal of adult European corn borer moths affects our understanding of how this pest may become resistant to Bt corn, as well as how effective mitigation efforts are to stop the spread of resistance once it develops. However, good estimates of insect migration rates are very difficult to obtain. ARS scientists from Ames, Iowa used DNA markers and independent estimates of population density to calculate migration rate of corn borer adults. The results indicated that 15% of these moths disperse greater than or equal to 12 km per insect generation. This provides the first useful estimate of typical dispersal distances in this pest, which will help officials in regulatory agencies make informed decisions regarding preventing or delaying the evolution and spread of insect resistance to Bt crops, which will help preserve the value of these crops. It will also be useful to scientists trying to understand and predict rates of resistance development in European corn borer to Bt corn.
Bacillus thuringiensis (Bt) corn suppresses European corn borer populations, which benefits all farmers. Planting corn that is genetically engineered to make insect-killing proteins suppresses areawide populations of European corn borers, a devastating pest of corn. Bt corn is so-named for a protein from the soil bacterium Bacillus thuringiensis that the corn plant produces as a built-in defense against the larval stages of European corn borers and other insect pests. A team of scientists, including ARS researchers in Ames, Iowa, showed this suppression of European corn borers provides significant economic benefits to farmers that use Bt corn and even to neighboring farmers who grow non-Bt corn. From 1996-2009, the researchers estimate that farmers in Iowa, Illinois, Minnesota, Nebraska, and Wisconsin received cumulative economic benefits of nearly $7 billion, with benefits of more than $4 billion for non-Bt corn farmers alone.
Romeis, J., Hellmich II, R.L., Candolfi, M., Carstens, K., De Schrijver, A., Gatehouse, A.M., Herman, R.A., Huesing, J.E., McLean, M.A., Raybould, A., Shelton, A., Waggoner, A. 2011. Recommendations for the design of laboratory studies on non-target arthropods for risk assessment of genetically engineered plants. Transgenic Research. 20(1):1-22.
Coates, B.S., Bayles, D.O., Wanner, K.W., Robertson, H.M., Hellmich II, R.L., Sappington, T.W. 2011. The application and performance of single nucleotide polymorphism markers for population genetic analyses of Lepidoptera. Frontiers in Genetics. 2(38):1-10.
Coates, B.S., Kroemer, J.A., Sumerford, D.V., Hellmich II, R.L. 2011. A novel class of miniature inverted repeat transposable elements (MITEs) that contain hitchhiking (GTCY)n microsatellites. Insect Molecular Biology. 20(1):15-27.
Coates, B.S., Sumerford, D.V., Hellmich II, R.L., Lewis, L.C. 2010. A Helitron-like Transposon Superfamily from Lepidoptera Disrupts (GAAA)n Microsatellites and is Responsible for Flanking Sequence Similarity within a Microsatellite Family. Journal of Molecular Evolution. 70(3):278-288.
Hutchison, W.D., Burkness, E.C., Mitchell, P.D., Moon, R.D., Leslie, T.W., Fleischer, S.J., Abrahamson, M., Hamilton, K.L., Steffey, K.L., Gray, M.E., Hellmich II, R.L., Kaster, V., Hunt, T.E., Wright, R.J., Pecinovsky, K.T., Rabaey, T.L., Flood, B.R., Raun, E.S. 2010. Areawide suppression of European corn borer with Bt maize reaps savings to non-Bt maize growers. Science. 330:222-225.
Jiang, X.F., Luo, L.Z., Sappington, T.W. 2010. Relationship of Flight and Reproduction in Beet Armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), a Migrant Lacking the Oogenesis-flight Syndrome. Journal of Insect Physiology. 56(11):1631-1637.
Jiang, X., Luo, L., Zhang, L., Sappington, T.W., Hu, Y. 2011. Regulation of migration in Mythimna separata (Walker) in China: A review integrating environmental, physiological, hormonal, genetic, and molecular factors. Environmental Entomology. 40(3):516-533.
Kim, K., Coates, B.S., Bagley, M.J., Hellmich II, R.L., Sappington, T.W. 2011. Genetic structure and gene flow among European corn borer populations from the Great Plains to the Appalachians of North America. Agricultural and Forest Entomology. 13(4):383-393.
Kroemer, J.A., Coates, B.S., Nusawardani, T., Rider Jr., S., Fraser, L.M., Hellmich II, R.L. 2011. A rearrangement of the Z chromosome topology influences the sex-linked gene display in the European corn borer, Ostrinia nubilalis. Molecular Genetics and Genomics. 286(1):37-56.
Onstad, D.W., Mitchell, P.D., Hurley, T.M., Lundgren, J.G., Porter, R.P., Krupke, C.H., Spencer, J.L., Difonzo, C.D., Baute, T.S., Hellmich Ii, R.L., Buschman, L., Hutchinson, W., Tooker, J.F. 2011. Seeds of change: corn seed mixtures for resistance management and IPM. Journal of Economic Entomology. 104(2):343-352.
Prasifka, J.R., Buhay, J.E., Sappington, T.W., Heaton, E.A., Bradshaw, J.D., Gray, M.E. 2011. Stem-boring caterpillars of switchgrass in the midwestern United States. Annals of the Entomological Society of America. 104(3):507-514.
Margam, V.M., Coates, B.S., Hellmich II, R.L., Agunbiade, T., Seufferheld, M.J., Sun, W., Ba, M.N., Sanon, A., Binso-Dabire, C.L., Baoua, I., Ishiyaku, M.F., Covas, F.G., Srinivasan, R., Armstrong, J., Murdock, L.L., Pittendrigh, B.R. 2011. Mitochondrial genome sequence and expression profiling for the legume pod borer Maruca vitrata (Lepidoptera: Crambidae). PLoS One. 6(2):e16444.
Margam, V.M., Coates, B.S., Bayles, D.O., Hellmich II, R.L., Agunbiade, T., Seufferheld, M.J., Sun, W., Kroemer, J.A., Ba, M.N., Binso-Dabire, C.L., Baoua, I., Ishiyaku, M.F., Covas, F.G., Srinivasan, R., Armstrong, J., Murdock, L.L., Pittendrigh, B.R. 2011. Transcriptome sequencing, and rapid development and application of SNP markers for the legume pod borer Maruca vitrata (Lepidoptera: Crambidae). PLoS One. 6(7):e21388.
Margam, V.M., Coates, B.S., Ba, M.N., Sun, W., Binso-Dabire, C.L., Baoua, I., Ishiyaku, M.F., Shukle, J.H., Hellmich II, R.L., Covas, F.G., Ramasamy, S., Armstrong, J., Pittendrigh, B.R., Murdock, L.L. 2011. Geographic distribution of phylogenetically-distinct legume pod borer, Maruca vitrata (Lepidoptera: Pyraloidea: Crambidae). Molecular Biology Reports. 38(1):893-903.
Wu, F., Bhatnagar, D., Bui-Klimke, T., Carbone, I., Hellmich II, R.L., Munkvold, G., Paul, P., Payne, G., Takle, E. 2011. Climate change impacts on mycotoxin risks in US maize. World Mycotoxin Journal. 4(1):79-93.
Li, Y., Ostrem, J., Romeis, J., Chen, M., Liu, X., Hellmich II, R.L., Shelton, A., Peng, Y. 2011. Development of a Tier-1 assay for assessing the toxicity of insecticidal substances against Coleomegilla maculata. Environmental Entomology. 40(2):496-502.