2007 Annual Report 1a.Objectives (from AD-416) Conduct fundamental research to generate baseline data on population genetic parameters to facilitate research on mechanisms of resistance to Bt proteins at the molecular level in Lepidopteran pests of cotton. Characterize the injury potential of Lepidopterous pests (esp. Helicoverpa zea) to new transgenic cottons to develop accurate action thresholds. Determine the effects that Bt corn (Yieldgard®) has on bollworm biology including: pupal diapause, reproduction, flight potential, physiology, and the ability of progeny to survive and damage transgenic Bt cottons (Bollgard® and Bollgard II®). 1b.Approach (from AD-416) Population genetic studies for characterizing natural populations of H. virescens and H. zea are in need of a comprehensive set of genetic markers. Well defined genetic markers will also facilitate the development of linkage maps which in turn will serve as valuable tools in studying inheritance of traits. Simple sequence repeats (SSR) or microsatellites found in genomes as well as single nucleotide polymorphisms (SNP) found in gut specific genes will be used as genetic markers for H. virescens and H. zea. Genomic DNA libraries enriched for microsatellite sequences will be used to develop SSR markers. Subtraction cDNA libraries enriched for gut specific genes will be used to identify SNP markers. 4.Accomplishments Title: “Production of bollworm and tobacco budworm from cotton and non-cotton crop hosts: Implications for insect resistance management in Bt cotton.” Problem: Since Bollgard cotton was commercially introduced in 1996, structured non-Bt cotton refuges have been required on a percentage of the acreage planted to cotton on a particular farm for the purpose of preventing resistance development to the Bt technology. Description: Contributions of crop hosts, including Bt and non-Bt cotton, field corn, soybeans, grain sorghum and/or peanuts, to the bollworm population was estimated in Arkansas, Georgia, Louisiana, Mississippi and North Carolina in order to determine whether bollworm production from non-cotton crop hosts adequately supplemented that of the structured non-Bt cotton refuge. Impact: In nearly all instances, non-cotton crop hosts collectively produced many more bollworm than non-Bt cotton, which suggested that the structured non-Bt cotton refuge played a minor role in delaying resistance development to Bt cotton in bollworm. In addition, a gossypol detection technique was used to estimate the proportion of the tobacco budworms that developed on cotton or non-cotton hosts in the same five-state area, and results indicated that tobacco budworms from non-cotton hosts made a significant contribution to the general population. Overall conclusions from these studies resulted in the elimination of structured refuge requirements for Bollgard II cottons saving growers up to $70/acre in structured refuge costs. NP304 Crop Protection and Quarantine Action Plan, Insects and Mites Component – Integrated Pest Management Systems and Areawide Suppression Programs. Title: “Development of polymorphic genetic markers for bollworm, Helicoverpa zea, and tobacco budworm, Heliothis virescens.” Problem: Population studies of bollworm and budworm were not possible due to lack of polymorphic co-dominant genetic markers. Description: We developed and evaluated microsatellite markers suitable for population studies as well as genetic mapping studies for these species. The microsatellite markers are being used to study population parameters such as gene flow and migration between different populations of bollworm and budworm. In addition, these markers are also being used in the development of a genetic map of the bollworm. Impact: Genetic markers linked to insecticide resistance alleles will facilitate identification of bollworm/budworm populations with high incidence of insecticide resistance and enable efficient management of field resistance. NP304 Crop Protection and Quarantine Action Plan, Insects and Mites Component – Integrated Pest Management Systems and Areawide Suppression Programs. Title: “Testing the feasibility of an F2 screen for tobacco budworm Bt-resistance monitoring.” Problem: The tobacco budworm is one of the most important cotton pests and it has the potential to develop resistance to Bacillus thuringiensis-producing cotton (Bt cotton) as it has done with other synthetic insecticides. One way to prolong the effectiveness of Bt cotton is early detection of resistance. Description: The second generation (F2) of a known Bt-resistant tobacco budworm colony was screened to document the feasibility of this method. Methodological, logistical and economic aspects of this type of screen were documented and already implemented to tobacco budworm samples obtained from six different regions and two different countries in 2006 and 2007. Impact: This method can be implemented to other resistance detection programs for other pests, but, more importantly, serves as a template for logistical considerations when implementing an F2 screen for Bt-crop resistance monitoring programs. NP304 Crop Protection and Quarantine Action Plan, Insects and Mites Component – Integrated Pest Management Systems and Areawide Suppression Programs. Title: “Interaction between Bt toxins and proteinase inhibitors and other bio-reagents, as potential bio-insecticides to be incorporated into plant genomes to synergize Bt performance.” Problem: Resistance development in a lepidopteran insect is a potential threat to the Bt biotechnology because of the high selection pressure against target insects due to widespread adoption of Bt cotton. Description: Avidin and proteinase inhibitors are potential bio-pesticides for control of a wide range of insects. To prolong the benefit of Bt cotton biotechnology, we explored potential incorporation of avidin and proteinase inhibitors with Bt for expanding control range and for slowing down Bt resistance development. A study was carried out to examine the toxicity of avidin against major cotton insects. We found that avidin significantly synergized Bt toxicity against the bollworm, and the mortality rate was increased by more than 44%. Interaction of Bt and proteinase inhibitors significantly retarded larval growth and resulted in significant delay of larval development and up to 20% mortality. Impact: Determining interaction of Bt with avidin, proteinase inhibitors, and other bio-reagents, could lead to the discovery of potential bio-insecticides that could be incorporated into plant genomes. NP304 Crop Protection and Quarantine Action Plan, Insects and Mites Component – Integrated Pest Management Systems and Areawide Suppression Programs. 5.Significant Activities that Support Special Target Populations NONE 6.Technology Transfer Number of web sites managed 1 Number of non-peer reviewed presentations and proceedings 14 Number of newspaper articles and other presentations for non-science audiences 1 Review Publications Fengyou, J., Maghirang, E.B., Dowell, F.E., Abel, C.A., Sonny, R. 2007. Differentiating Tobacco Budworm and Corn Earworm Using Near-Infrared Spectroscopy. Journal of Economic Entomology. Vol.100(3):759-764. Blanco, C.A., Perera O. P., Boykin D., Abel C.A., Gore J., Matten S. R., Ramirez-Sagahon J. C., Teran-Vargas A. P. 2007. Monitoring Bacillus thuringinesis-susceptibility in insect pests that occur in large geographies: How to get the best information when two countries are involved. J. Inv. Pathol. 95:201-207 Perera, O.P., Blanco C.A., SchefflerB.E., and Abel C.A. 2007. Characteristics of 13 ploymorphic microsatellite makers in the corn earworm, Helicovepa zea (Lepidoptera: Noctuidae). Mol. Ecol. Notes. doi: 10.1111/j.147-8286.2007.01806.x Jackson, R.E., MarcusM.A., Gould F., Bradley, J. R. Jr., and Van Duyn J.W. 2007.cross-resistance responses of cry1Ac-selected Heliothis virescens (Lepidoptera: Noctuidae) to the Bacillus thuringiensis protein Vip3a. J. Econ. Entomol. 100: 180-186. Zhu, Y.C., Abel, C.A., Chen, M.S. 2007. Interaction of Cry1Ac toxin (Bacillus thuringiensis) and proteinase inhibitors on the growth, development, and midgut proteinase activities of the bollworm, Helicoverpa zea. Pesticide Biochem. Physiol. 87:39-46.