NERL Research Abstract EPA's National Exposure Research Laboratory GPRA Goal 8 - Sound Science APM # 628 Significant Research Findings Draft Report on the Feasibility of Genetic and Molecular Biomarkers as an Indirect Measure of Pesticide, Toxics and Exposure to Fish in the Eastern Cornbelt Plains Ecoregion Purpose This report describes studies of the genetic diversity of a selected stream fish throughout the Eastern Cornbelt Plains ecoregion (as classfied by Omernik) in EPA Region 5. Genetic diversity is one component of overall biodiversity, along with species diversity and habitat diversity. It represents the sum total of genetically-based variations among individuals within populations and among populations within species. Genetic diversity within species of aquatic organisms is an indicator of the condition of stream ecosystems since it is responsive to changes in stream habitat. It is also an indicator of the sustainability of populations since populations with low genetic diversity are less able to adapt to novel environmental stressors. This report also addresses the feasibility of conducting a large-scale regional genetic study since regional evaluations of species genetic diversity have not been reported previously. Eventual technology transfer of advanced methods requires an evaluation of the feasibility of new field applications of biological indicators. Issues of scale are addressed for field sample collection, laboratory molecular biological analyses, and data management. Measures of population genetic diversity have the potential to be excellent integrative indicators of exposure. These measures will contribute uniquely to the exposure component of regional ecological risk assessments since they can be evaluated and interpreted over large scales, geographically and temporally. The genetic diversity data will be correlated with those generated from biomarker measurements (indicators of toxic chemical stressors), species diversity metrics, and abiotic measurements to provide data on exposure patterns at different scales. Since levels of genetic diversity influence local extinction probabilities following exposure, regional vulnerabilities to exposure can begin to be mapped for populations of interest. National Exposure Research Laboratory - September 2000 ------- Research Fish tissue samples were collected from over 400 stream sites during a 1995 Approach Region 5 REMAP study of the Eastern Cornbelt Plains ecoregion. A total of 110 sites contained populations of the central stoneroller (Campostoma anomalum). Genetic markers were developed using a molecular biological DNA fingerprinting method referred to as randomly amplified polymorphic DNA (RAPD) analysis. Samples from all 110 field sites have been assayed for the presence or absence of each of these genetic markers. Population genetic analyses are being conducted on the output of the RAPD fingerprint assays. A "similarity index" will be calculated for each site that measures the average relatedness of individual organisms within that population. Additional analyses will measure the average relatedness between fish in different populations. The data will be used to produce an ecoregional map of genetic diversity (a "genetic landscape" atlas). A relational database will be constructed that contains chemical stressor and biological index data for many of these sites. The database will allow us to correlate species genetic diversity with the stressor data. A subset of data were analyzed and have been reported previously (Silbiger et al., 1998). Similarity indices were calculated for 20 of the 110 sites. Significant variation was observed in genetic diversity within and among these 20 sites. Initial evaluations revealed a significant correlation of within- population genetic diversity with ecosystem bioassessment indicators (index of biological integrity) but not with levels of chemical stressors poly aromatic hydrocarbons (PAHs). At this time, technical barriers for a regional genetic study have been overcome. The final report will detail the success of the study of field collections after scaling up laboratory analyses from small bench operations to major laboratory throughput. The report will also describe the development of new quality assurance procedures and provide recommendations for cradle to grave data management for regional genetic studies. Preliminary data were presented to academic scientists at an Ecological Indicators STAR grant review meeting in May 2000. Discussions of the roles of academic and federal government researchers confirmed the importance of Offic of Research and Development's NERL Ecological Exposure Research Division research on genetic diversity at large regional scales and suggested avenues for future collaboration. Examples of publications resulting from this study follow. Silbiger, R.N., Christ, S.A., Leonard, A.C., Garg, M., Dawes, S., Dimsoski, P., McCormick, F., Wessendarp, T., Gordon, D.A., Roth, A.C., Smith, M.K., Toth, G.P. Preliminary studies on the population genetics of the central stoneroller (Campostoma anomalum) from the Great Miami River Basin, Ohio. Environmental Monitoring and Assessment 51: 481-495, 1998. Major Findings and Significance Research Collaboration and Publications National Exposure Research Laboratory - September 2000 ------- Leonard, A.C., Franson, S.E., Hertzberg, V.S., Smith, M.K., Toth, G.P. Hypothesis testing with the similarity index. Molecular Ecology 8: 2105-2114, 1999. Christ, S.A., Silbiger, R.N., Garg, M., Franson, S.E., Toth, G.P. Quality assurance considerations for use of the Fluorimager SI and FragmeNT analysis software. Electrophoresis 21: 874-888, 2000. Future The present design provides unparalleled information on the regional genetic Research structure of a native fish species and on the environmental forces that influence this structure. However, since each species "sees" the environment differently, the ideal ecosystem indicator will incorporate genetic information gathered from several species with varying life-history attributes. Towards this end, pilot studies have been initiated on the genetic diversity of a second stream fish, the creek chub Semotilus atromaticulatus, with the aim of eventually incorporating it into the regional genetic profile. Genetic analyses of both central stoneroller and creek chubs are also being expanded to the Mid-Atlantic Integrated Assessment (MAIA) region (encompassing a region ranging from southern New York state to northern North Carolina) in order to expand the geographic coverage and assess possible differences in the genetic indicator among regions. The new analyses incorporate recent advances in molecular detection of genetic diversity, such as microsatellite and amplified fragment length polymorphism analyses, in order to provide more resolution of genetic structure and to facilitate development of assays that are best suited for technology transfer. Questions and inquiries concerning this research abstract can be directed to: Mark Bagley, Ph.D. U.S. Environmental Protection Agency National Exposure Research Laboratory (MD-642) Cincinnati, OH 45268 Phone: (513)569-7455 E-mail: bagley.mark@epa.gov Greg Toth, Ph.D. U.S. Environmental Protection Agency National Exposure Research Laboratory (MD-642) Cincinnati, OH 45268 Phone: (513)569-7242 E-mail: toth.greg@epa.gov National Exposure Research Laboratory - September 2000 ------- |