2013 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.
The Pheromone Gland Fatty-Acyl Reductase (pgfar) molecular assay we developed is substantially more accurate than previous E or Z pheromone-baited traps at identifying E and Z races of European corn borer (ECB) and their hybrids. The new assay revealed that hybridization between races is much more common than previously suspected. The surprisingly high frequency at which ECB races hybridize suggests that if Bacillus Thuringiensis (Bt) resistance develops in E-race populations, it will spread quickly to the Z race and spread into the Corn Belt. The pgfar assay also was used to re-examine a population genetics study with ECB collected from nine locations in Pennsylvania. Improved identification accuracy along with use of a new analytical technique called "Population Graphs" provided the precision necessary to detect population differentiation across space and time within each race. Genetic differences are not related simply to distance from one another, indicating that something else is serving as a barrier to dispersal, possibly topology (e.g., mountains, forests) or patterns of alternative host use. Results confirmed preliminary tests that gene flow is lower between E-race populations than Z-race populations, which could lead to faster development of resistance to transgenic Bt corn in the E race. The genetic basis of larval ECB resistance to feeding on Bt corn that express Cry1F toxin was investigated in two backcross families, and DNA and RNA have been collected from resistant and susceptible progeny for Quantitative Trait Loci (QTL) mapping and gene expression analyses. Western corn rootworm Bacterial Artificial Chromosome (BAC) clones (37) were fully sequenced and submitted to public databases after gene coding regions were identified. Candidate Bt-resistance genes were isolated from western corn rootworm, and identification BACs carrying these genes is ongoing. Genome sequencing of pools of western corn rootworm individuals was completed for a laboratory strain and a field population, and subsequent low coverage genome assembly allowed isolation of full mitochondrial genome sequences, the genome of the Wolbochia endosymbiont and novel transposons not discovered by prior BAC sequencing. DNA was extracted from two Northern corn rootworm F1 crosses that show one year and extended diapause traits, and QTL analysis is ongoing. Confirmation of resistance to transgenic Bt corn in many western corn rootworm populations has emphasized the urgent need for better understanding of this pest's dispersal behavior, which strongly influences rate of resistance development and the rate at which resistance spreads after it develops. Thorough, systematic sampling of adult population numbers were taken in 50 fields across five locations in the species native home range of eastern Colorado and western Kansas. In combination with follow-up analyses of genetic variation at 500 single-nucleotide polymorphisms markers, we will be able to estimate how far typical individuals fly per generation before reproducing. One more year of such sampling will be conducted to allow the necessary analysis of genetic changes over time.
Quick development of genetic markers for new insect pests. The western bean cutworm, once confined to the Great Plains, is undergoing a dramatic range expansion in North America. It is now a serious pest of corn throughout the Midwest. The reason for its spread is unknown, and other important aspects of its ecology and population dynamics are poorly understood. Population genetics research could provide important information to improve management of this species, but has been hindered by a lack of DNA-Based genetic markers. ARS researchers in Ames, Iowa used a novel way to economically develop 71 genetic markers that are suitable for population studies. This work not only provides significant genetic resources for future research on western bean cutworm, but demonstrates the power of this new tool. This information will be used by university, government, and industry scientists interested in managing western bean cutworm and other new insect pest.
Genetic markers differentiate Z- and E-races of European corn borer. European corn borers, an important pest in the U.S., have two races whose females produce different Z and E pheromones. The Z race is commonly found in corn and the E race is often found in weeds. There is concern that the E race may adapt to Bacillus Thuringiensis (Bt) corn first and be a conduit for resistance to the Z race. ARS scientists from Ames, Iowa developed DNA markers that target the gene associated with pheromone production. These markers rapidly and accurately identify the pheromone race of field-collected European corn borers, using any growth stage and either sex. This is an important tool for studies that focus on the population genetics of European corn borers. This information will help scientists delay or prevent these insects from becoming resistant to Bt corn.
Kuester, A.P., Jones, R.W., Sappington, T.W., Kim, K., Barr, N.B., Roehrdanz, R.L., Senechal, P., Nason, J.D. 2012. Population structure and genetic diversity of the boll weevil, Anthonomus grandis (Coleoptera: Curculionidae), on Gossypium in North America. Annals of the Entomological Society of America. 105(6):902-916.
Cheng, Y., Luo, L., Jiang, X., Sappington, T.W. 2012. Synchronized oviposition triggered by migratory flight intensifies larval outbreaks of beet webworm. PLoS One. 7(2):e31562.
Coates, B.S., Johnson, H., Kim, K., Hellmich II, R.L., Abel, C.A., Mason, C., Sappington, T.W. 2013. Frequency of hybridization between Ostrinia nubilalis E-and Z-pheromone races in regions of sympatry within the United States. Ecology and Evolution. 3(8):2459-2470.
Coates, B.S., Alves, A., Wang, H., Walden, K., French, B.W., Miller, N.M., Abel, C.A., Robertson, H.M., Sappington, T.W., Siegfried, B.D. 2012. Distribution of genes and repetitive elements in the Diabrotica virgifera virgifera genome estimated using BAC sequencing. Journal of Biomedicine and Biotechnology. 2012:1.
French, B.W., Coates, B.S., Sappington, T.W. 2012. Inheritance of an extended diapause trait in the Northern corn rootworm, Diabrotica barberi (Coleoptera: Chrysomelidae). Journal of Applied Entomology. DOI: 10.1111/j.1439-0418.2012.01751.x.
Jiang, X., Jian, C., Zhang, L., Sappington, T.W., Luo, L. 2013. Increased long-flight activity triggered in beet armyworm by larval feeding on diet containing Cry1Ac protoxin. PLoS One. 8(5):e63554.
Kim, K., Sappington, T.W. 2013. Population genetics strategies to characterize long-distance dispersal of insects. Journal of Asia-Pacific Entomology. 16(1):87-97.
Kong, H., Luo, L., Sappington, T.W., Jiang, X., Zhang, L., Cheng, Y. 2013. Density-dependent prophylaxis in crowded beet webworm, Loxostege sticticalis (Lepidoptera: Pyralidae) larvae to a parasitoid and a fungal pathogen. International Journal of Pest Management. DOI:10.1080/09670874.2013.807957.
Miller, N.J., Sun, J., Sappington, T.W. 2012. High-throughput transcriptome sequencing for SNP and gene discovery in a moth. Environmental Entomology. 41(4):997-1007.
Shelton, A., Naranjo, S.E., Romeis, J., Hellmich II, R.L. 2012. Errors in logic and statistics plague a meta-analysis. Environmental Entomology. 41:1047-1049.
Tian, J., Collins, H.L., Romeis, J., Naranjo, S.E., Hellmich II, R.L., Shelton, A.M. 2012. Using field-evolved resistance to Cry1F maize in a lepidopteran pest to demonstrate no adverse effects of Cry1F on one of its major predators. Transgenic Research. 21(6):1303-1310.
Tian, J.C., Wang, X.P., Long, L.P., Romeis, J., Naranjo, S.E., Hellmich II, R.L., Wang, P., Earle, E.D., Shelton, A.M. 2013. Bt crops producing Cry1Ac, Cry2Ab and Cry1F do not harm the green lacewing, chrysoperla rufilabris. PLoS One. 8(3):e60125.
Wang, L., Jiang, X., Luo, L., Sappington, T.W., Stanley, D.W., Zhang, L. 2013. A cadherin-like protein influences Bacillus thuringiensis Cry1Ab toxicity in the oriental armyworm, Mythimna separata. Environmental Microbiology Reports. 5(3):438-443.
Kim, K., Sappington, T.W. 2013. Microsatellite data analysis for population genetics. In: Kantartzi, S.K., editor. Microsatellites: Methods and Protocols. Methods in Molecular Biology. New York, NY: Humana Press, Springer Science+Business Media, LLC. p. 271-295.
Romeis, J., Raybould, A., Bigler, F., Candolfi, M., Hellmich II, R.L., Huesing, J.E., Shelton, A. 2012. Deriving criteria to select arthropod species for laboratory tests to assess the ecological risks from cultivating transgenic crops. Chemosphere. 90(3):901-909.
Xie, D., Luo, L., Sappington, T.W., Jiang, X., Zhang, L. 2012. Comparison of reproductive and flight capacity of beet webworm, Loxostege sticticalis (Lepidoptera: Pyralidae), developing from diapause and non-diapause larvae. Environmental Entomology. 41(5):1199-1207.
Bowers, E., Hellmich II, R.L., Munkvold, G.P. 2013. Vip3Aa and Cry1Ab proteins in maize reduce Fusarium ear rot and fumonisins by deterring kernel injury from multiple Lepidopteran pests. World Mycotoxin Journal. 6(2):127-135.