2011 Annual Report 1a.Objectives (from AD-416) Objective 1: Determine the impact of a changing cropping landscape on host plant ecology and insect resistance management practices for bollworm using analytical techniques. Objective 2: Determine gene flow and migration patterns by analyzing tobacco budworm and bollworm populations in temporal and spatial scales using genetic and/or empirical/mathematical approaches. Objective 3: Identify possible mechanisms of resistance to Bt toxins by profiling gene expression patterns and develop a marker based genetic linkage map. 1b.Approach (from AD-416) More than 95% of the second generation bollworm within each growing season utilizes field corn as a host. Impact of corn plants expressing multiple Bt toxins on the bollworm populations will be studied by comparing historical pheromone trap data with current and future population estimates influenced by increased acreages of Bt corn expressing multiple Bt toxins. Stable carbon isotope analysis will be used to identify bollworms using corn as a host plant. Influence of local cropping landscape on bollworm populations will be studied using sentential plots of conventional and Bt corn and cotton and early maturing soybeans. Large field cages will be used to evaluate the impact of pyramided-gene Bt corn hybrid/refuge system on resistance management strategies. Expressed genes of tobacco budworm and bollworm will be identified by transcriptome sequencing, and genetic markers developed from polymorphic nucleotide regions will be used in ecological genetic studies of tobacco budworm and bollworm populations. Gene expression profiles will be used to identify biological processes involved in physiological response to ingestion of Bt toxins. Markers developed for candidate loci associated with resistance to Bt toxins will be used to estimate allele frequencies in natural populations. Genetic loci under selection will be identified using statistical methods. A genetic linkage map of the bollworm developed using polymorphic markers will be used to study inheritance of loci of interest to Bt resistance. 3.Progress Report This is the first report for the project 6402-22000-065-00D that started in April 2011. Field and laboratory studies have been initiated, and insect samples are being collected and held for future analyses. Twelve-month milestones will be complete upon the end of the cropping season. Transcriptome of tobacco budworm was assembled using over 20 million nucleotide sequence reads. All nucleotide reads and assemblies were submitted to public databases. Glass slide microarrays containing 44,000 features were developed using curated transcriptome sequences. BW transcriptome sequencing using Roche 454 and Illumina platforms was completed. Assembly and bioinformatic analyses were performed to identify expressed genes in the transcriptome assembly. Nucleotide sequence reads were re-assembled (overlaid) using curated reference gene sequences to identify single nucleotide polymorphisms. Nucleotide reads and assemblies of tobacco budworm and bollworm were submitted to public databases. Primary cell cultures generated from susceptible YDK strain and resistant KCB and CXC strains of tobacco budworm were treated with Cry1Ac and Cry2Ab toxins. Treated and control cells were harvested and total RNA and proteins were extracted. RNA extractions are being used in RNA-Seq based gene expression profiling experiments. Protein fractions were subjected to two-dimensional differential gel electrophoresis to identify differentially expressed proteins. The protein spots identified were excised from the genes and were submitted to a core facility for mass spectrometry based determination of amino acid sequences. Laboratory screening of bollworm larvae collected from transgenic corn plants was initiated to select a bollworm line resistant to Cry1Ac. Bollworm larvae collected from various field sites were used to establish laboratory strains to obtain genetic material for mapping studies. 4.Accomplishments 1. Expression patterns of a membrane-bound alkaline phosphatase (mALP) known to bind Cry1Ac toxin in several lepidopteran species indicated reduced levels of this enzyme in resistant strains. Elevated expression levels of a storage protein transcript was also identified in tobacco budworm resistant to Cry toxins. This protein, a member of storage protein group known as arylphorins commonly found in fatbody tissues, was detected in the midgut tissues of tobacco budworm larvae. The mALP gene and the arylphorin gene were identified as putative biomarkers for Bt resistance. Review Publications Jurat-Fuentes, J., Karumbahiah, L., Jakka, S.K., Ning, C., Liu, C., Wu, K., Jackson, J., Gould, F., Blanco, C., Portilla, M., Perera, O.P., Adang, M.J. 2011. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis. PLoS One. 6:e17606. Hardke, J., Leonard, R., Huang, F., Jackson, R.E. 2011. Damage and survivorship of fall armyworm (Lepidoptera: Noctuidae) on transgenic field corn expressing Bacillus thuringiensis cry proteins. Crop Protection Journal. 30(2):168-172. Blanco, C., Portilla, M., Jurat-Fuentes, J., Sanchez, J.F., Viteri, D., Vega-Aquin, P., Teran-Vargas, A.P., Azuara-Dominguez, A., Lopez, J., Arias De Ares, R.S., Zhu, Y., Barrera, D., Jackson, R.E. 2010. Susceptibility of Spodoptera frugiperda (Lepidoptera: noctuidae) isofamilies to Cry1Ac and Cry1F proteins of Bacillus thuringiensis. Southwestern Entomologist. 35(3):409-415.