2007 Annual Report 1a.Objectives (from AD-416) (1) Annually monitor the susceptibility of H. zea populations from thirty to fifty field populations across the U.S. Cotton Belt. Ideally the populations studied will represent at least 10 different geographic locations with samples repeated three times within the growing season (i.e. early-, mid-, and late-season). (2) Further characterize and study reduced susceptibility to Bt proteins observed in the monitoring activities. 1b.Approach (from AD-416) Our approach will be the procedures described by Ali et al. (2006a). Progeny from field collections will be exposed to a series of toxin concentrations in a diet-incorporation assay. The number of toxins assayed will vary from year-to-year, but our intent would be to include all commercial and soon-to-be-commercialized toxins in the study. Potential toxins in 2006 would include Cry1Ac, Cry2Ab, Cry1F, and Vip3A. Concentrations to be tested would include the critical diagnostic doses chosen from initial baseline data. At a minimum this will include two concentrations approximating a LC50 and a LC90 and an appropriate experimental control with no toxin. Additional concentrations will be added as numbers of insects are available in an attempt to generate concentration-mortality regressions for each protein and each collection. The critical diagnostic doses will be determined in consultation with the EPA, the USDA-ARS, and the cooperating industry group. Consideration of historical data and evolving resistance levels will be considered. Blanco et al. (2005) suggested the following diagnostic concentrations for monitoring Bt resistance in H. zea: (1) 50, 100 and 250 ug/ml for Cry1Ac, (2) 50, 100 and 200 ug/ml for Cry2Ab2, and (3) 1000 and 2000 ug/ml for Cry1Fa. Neonate larvae will be exposed to the treated diet in CD International assay trays containing ~ one ml of the toxin incorporated diet. Each replicate of the key diagnostic doses will include 32 larvae. Replicates of additional concentration added to generate concentration-response regression lines will include 16 larvae. Each concentration will be replicated a minimum of four times given sufficient larval numbers. After 7 d of exposure, observations will made, and the number of dead larvae, the number of larvae alive but remaining as first instars, and the number of larvae alive but remaining as second instars will be recorded as response variables. Paired assays will be run with the designated laboratory colony as an experiment control of variable assay conditions. Response data at the diagnostic doses will be studied by analysis of variance and descriptive statistics with results compared to historical benchmark information and paired results from the laboratory susceptible colony. When sufficient concentrations are included to develop probit regression lines, susceptibilities of the different populations will be compared by the methods described by Ali et al. (2006a). 3.Progress Report This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the University of Arkansas. Additional details of research can be found in the report for the in-house project 6402-22000-047-00D, "Resistance Management and Injury Potential of Lepidopterous Pests to Transgenic Cottons." In cooperation with the USDA-ARS Southern Insect Management Research Unit (SIMRU), the University of Arkansas proposes an annual monitoring of bollworm (corn earworm) populations from across the U.S. Cotton Belt for relative susceptibility to the range of insecticidal proteins expressed in transgenic Bt cottons. Bollworm field populations from six U.S. Cotton Belt states collected on an array of host plants were exposed to Cry1Ac and Cry2Ab Bt-toxins in diet incorporated assays at the University of Arkansas in 2006. Assays were not conducted with Cry1F because of the lack of preliminary data and access to protein. Corrected dose-mortality responses including estimates for lethal concentrations of mortality (LC50) and mortality plus those larvae failed to molt to second instars (MIC50) were measured for 39 and 31 field populations exposed to Cry1Ac and Cry2Ab, respectively. Considerable dose-response variation was observed in field populations of H. zea to Cry1Ac and Cry2Ab toxins. Thirty-two field populations had mortalities at 250 µg of Cry1Ac significantly lower than the lower 99.9% confidence interval for a University Arkansas susceptible laboratory H. zea colony (UALab). Twenty-eight field populations had higher LC50s than UALab and eight populations had LC50 estimates higher than the highest dose tested (250 µg/ml). The MIC50 estimates of these populations varied up to 106-fold and followed a similar trend to the LC50s. At the highest tested dose of Cry2Ab (150 µg/ml), 26 populations had less mortality than the lower confidence interval of UALab. Twenty field populations had higher LC50s than the UALab, of these six LC50 estimates were larger than the highest dose tested. MIC50 estimates for 18 field populations were higher than the UALab and varied up to 69-fold. No association of the observed response variability in field populations of H. zea to Cry1Ac or Cry2Ab with host plants, geographic locations or seasonal time of collection was calculated for this preliminary report. This agreement was monitored by holding a meeting at the Cotton Beltwide Conference.