2006 Annual Report 4d.Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the University of Maryland to conduct research related to an agreement with the USDA Biotechnology Risk Assessment Grants (BRAG) Program (60-3625-5-0599) entitled "Recommended Protocols for Field Evaluations of Non-Target Organisms in Bt Crops." Additional details of research can be found in the report for the parent project #3625-22000-016-00D, Ecologically-Based Management of Insect Pests of Corn. To clearly define specific accomplishments and future goals, this report is arranged according to the five objectives contained in the original proposal, with each goal restated below. 1. Provide protocols for sampling non-target communities in Bacillus thuringiensis (Bt) corn, including appropriate (a) indicator taxa, (b) sample timing, and (c) replication. Using five multi-year data sets from across the Corn Belt, we have generated recommendations for selecting non-target taxa for field trials. These recommendations are based partly on estimates of statistical power associated with sampling various non-target taxa. The manuscript related to this objective is being reviewed by the collaborators with anticipated submission this fall. Also, a manuscript that specifically addresses sampling methods for ground-dwelling arthropods has been submitted as an additional component to this objective (see Publications section). 2. Develop techniques to identify and rear abundant but currently indistinguishable larvae of non-target soil-dwelling beetles, and create laboratory methods for extended Bt exposure tests. Because of recent advances in rearing methods by other researchers, this Objective has been expanded to include research related to "Linking predator-prey relationships and estimating non-target exposure in rootworm-active transgenic corn (Cry34/35)." Specifically, trophic relationships between beetles and their dietary components in Bt corn is being explored to estimate exposure to plant-incorporated protectants and potential impacts on ecosystem services. A laboratory study examined effects of continuous exposure to Bt-corn pollen by adults of a carabid and staphylinid species, and found no detrimental effects on longevity or behavior. A second study incorporated no-choice tests to estimate diet breadth, and choice tests to estimate the likelihood of Bt-pollen consumption in the presence of a readily accepted prey item. These studies find pollen to be an acceptable and sustaining food item, but one that is not favored in the presence of fly eggs. Currently, enzyme-linked immunosorbent assay (ELISA) tests are being conducted to quantify consumption of Cry34/35. Additionally, laboratory feeding trials and field behavior studies are being conducted to examine the functional role of abundant, minute ground beetles in field corn systems. 3. Assess the suitability of different plot sizes for evaluating effects of cropping practices on non-target communities and specific indicator taxa. This objective has been completed ahead of schedule and the results have been published. In summary, arthropod population estimates differed among plot sizes receiving identical (non-transgenic) management. The effect was seen for arthropods at ground level or in corn foliage, and for taxa sampled visually and using traps. Because non-target taxa show different levels of mobility, an appropriate plot size should be tailored for the specific groups of interest in a study. 4. Test approaches to combine and analyze existing data on the non-target effects of Bt corn to increase statistical power for detecting community and species-level treatment differences. Because Objectives 4 and 1 are related, they will utilize overlapping sets of field data. The key difference is that Objective 4 focuses on making recommendations for optimal use of resources in multiple site (i.e., multiple field) studies, and explores pooling data from multiple studies to increase statistical power. Additional data sets (beyond those used for Objective. 1)are being collected with analysis planned for late 2006. 5. Conduct annual meetings prior to and during research to collect stakeholder input and improve the value of research products from other objectives wherever possible. Relative to 2004, a smaller, more focused meeting was held at the Entomological Society of America’s convention in December 2005. The meeting was intended to facilitate progress between the PIs, but included industry stakeholders as well. Discussion at the meeting allowed the data sets for Objectives 1 and 4 to be fully assembled, resulting in analysis and manuscript preparation for Objective 1. Publications: Prasifka, J.R., R.L. Hellmich, G.P. Dively, L.S. Higgins, P.M. Dixon, and J.J. Duan. Selection of nontarget arthropod taxa for field research on transgenic insecticidal crops: using empirical data and statistical power. Environmental Entomology, in preparation. Prasifka, J.R., M.D. Lopez, R.L. Hellmich, L.C. Lewis, and G.P. Dively. 2006. Comparison of pitfall traps and litter bags for sampling ground-dwelling arthropods. Journal of Applied Entomology, submitted. Prasifka, J.R., R.L. Hellmich, G.P. Dively, and L.C. Lewis. 2005. Assessing the effects of pest management on non-target arthropods: the influence of plot size and isolation. Environmental Entomology 34:1181–1192.