Project Number: 6402-22000-047-38
Project Type: Specific Cooperative Agreement

Start Date: Aug 31, 2006
End Date: Mar 31, 2011

Objective:
(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.

Approach:
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).