2005 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 #3625-22000-015-00D, Ecologically-Based Management of Selected Insects Associated with 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 Bt corn, including appropriate (a) indicator taxa, (b) sample timing, and (c) replication. Currently we have access to three multi-year data sets and have met with additional potential collaborators (November 2004) to evaluate their interest. Several researchers indicated their willingness to share data. To provide data in a standard format (with supplemental information that would also allow data to be leveraged for other related projects), we are developing a simple Microsoft Excel form that collaborators can complete, attaching their data. Analysis should begin in fall 2005. 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. This objective has been modified because of recent advances in rearing methods by other researchers. A tentative title for the new Objective is “Linking predator-prey relationships and estimating non-target exposure in rootworm-active transgenic corn (Cry34/35).” Specifically, trophic relationships between beetle species and their dietary components in Bacillus thuringiensis (Bt) corn will be explored to estimate exposure to plant-incorporated protectants and potential impacts on ecosystem services. Carabids and staphylinids (larvae and adults) are currently being collected for analysis of the presence of DNA corresponding to prey species. Using generic primer pairs, predator and prey barcode genes will be sequenced; subsequently, unique primers will be designed to conduct multiplex PCR (multiple primer pair reactions), allowing for detection of multiple prey items in the gut of a single predator. Also enzyme-linked immunosorbent assay (ELISA) tests will be used to quantify consumption of Cry34/35. Later, laboratory feeding trials will be conducted with meridic diets (incorporating various sources of Cry34/35 protein) to assess possible effects on larval and adult mortality and development at environmentally-relevant exposures. 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 are currently in press. 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 will utilize overlapping sets of field data, current progress on these objectives is similar. 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 the feasibility of pooling data from multiple studies to increase statistical power. Analysis should begin during late 2005. 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. A meeting was held in conjunction with the Entomological Society of America’s convention in November 2004. Stakeholder attendance was dominated by researchers from academia and industry. Productive outcomes were the identification of contributors of data sets for use in Objectives (1) and (4), and the discussion of novel projects (possibly led by collaborators not identified in the agreement) using the collection of many non-target data sets in a standard format.