1. The expression of Hsp 70 in Hexagenia limbata: A molecular biomarker of environmental stress M. Peplinski, J. Provazza, B. O’Donnell Department of Biological Science, Plymouth State University, Plymouth, NH Abstract Studying organism level responses to environmental stress can be used to monitor disturbance in ecological systems. In this study, the expression of a heat shock protein in the mayfly Hexagenia limbata is used as an indicator of environmental stress. The regulation of heat shock protein 70 ( Hsp 70) in Hexagenia can be used as a molecular biomarker of the health of the surrounding ecosystem. We are using the relative expression of Hsp 70 in baseline experiments versus osmotic shock experiments. With further studies there is great promise for the development and implementation of new molecular approaches to biomonitoring as well as reducing mitigation measures. There is also potential for broad application as an educational resource. Introduction/Background Mayflies are one of the classic insect groups used in biomonitoring and there is a long history in using these insects as indicators of the integrity of ecological systems, (Resh and Jackson, 1993). The mayfly Hexagenia limbata is a particularly good biomonitor for this study because it is common and very widespread throughout North America. The heat shock protein that we are targeting, Hsp 70 plays an important role in resistance of environmental stress, ( Fu, et. Al., 2013). In a study done by De Jong et. Al., in France, another mayfly species was used to study heat shock protein expression under environmental perturbation, (2005). In this study, increased expression of heath shock protein in the gills of the mayflies was correlated with annual increase in conductivity following road salting. The combination of an osmotic shock biomarker with this biomonitor species presents a helpful example of a molecular tool used to monitor freshwater ecological systems. Monitoring water quality in the long term improves understanding of the importance of environmental health. Also, water quality testing is an effective and low cost way of analyzing the health of an ecosystem. With potential for studying Hexagenia in a number of watersheds, there is the capacity for a useful molecular approach to biomonitoring. Methods Specimen collected from Sky pond in New Hampton, NH All Black wing pad stage of development Dissected at various time intervals after collection; Head, Thorax, Abdomen, Gills Protein Extraction (Trizol) Protein Quantification using QuBit SDS- Gel Electrophoresis Western Blotting Western Blot Detection and Imaging Preliminary Results Discussion Western Blot image 1: Primary Antibody- Hsp 70 (mouse anti-Hsp 70), Secondary Antibody- Alexa Fluor 555 (goat anti-mouse), Biorad Ladder Precision plus dual standard Osmotic Shock specimen used Protein only in wells, 3,5,7,9 Non-specific binding Optimization needed What we expected: Intense bands around 70 kDa range in lanes 3,5,7,9 Conclusion/Summary The relative expression of Hsp 70 in Hexagenia limbata can be used as an indicator of environmental stress. By performing Western Blots, we were able to create a baseline level of protein expression within this species. By comparing the relative intensities of bands within these Blots, we are able to correlate it with the amount of stress being applied to the organism. Though ideal results have not been achieved, we are currently working to address possible issues with our protocols and materials. Goals: Short Term: Optimization of protocols IHC Long Term: Collection of Specimen in Spring from different watersheds Education Climate Change Correlation, Temperature Literature Cited De Jong, L., Moreau, X., Jean, S., Scher, O., & Thiéry, A. (2006). Expression of the heat shock protein Hsp70 in chloride target cells of mayfly larvae from motorway retention pond: A biomarker of osmotic shock. Science of the total environment, 366(1), 164-173. Fu, W., Zhang, F., Liao, M., Liu, M., Zheng, B., Yang, H., & Zhong, M. (2013). Molecular cloning and expression analysis of a cytosolic heat shock protein 70 gene from mud crab Scylla serrata. Fish & Shellfish Immunology, 34(5), 1306-1314. Rosenberg, David M., and Vincent H. Resh. Freshwater biomonitoring and benthic macroinvertebrates. Chapman & Hall, 1993. Acknowledgements This research was funded through the NSF NH EPSCoR program, grant number #1101245.