What state is environmental DNA found and does that affect detection rates in the North American river otter? Sydney Waloven and Dr. Nicholas Sard State University of New York at Oswego Department of Biological Sciences Background Threatened and endangered species are challenging to manage due to difficulties associated with sampling small populations. Results in uncertainty when attempting to conserve populations 1 . Animals in all populations release trace amounts of DNA known as environmental DNA (eDNA) 2 . eDNA sampling methods can be used to improve conservation management efforts by improving sampling efficiency 2 . eDNA is used to detect mammals, fishes, and amphibians 3 . For example, eDNA has been used to characterize spatial trends in keynote species like the sea otters ( Enhydra lutris ) that are vital to the health of kelp forest ecosystems 4 . and transport) of eDNA is poorly understood 5 . Relative concentrations between intracellular and extracellular DNA (i.e., the state of eDNA) are currently unknown. Based on the microbial community literature, the state of eDNA may vary by several orders of magnitude depending on sampling location 6,7 . Species detections may be affected by the state of eDNA. Studying the ecology of eDNA can be used to optimize sampling efforts. North American river otters ( Lontra canadensis ) currently have a stable population (Figure 1). Exhibits many of the same behaviors and uses similar habitats as the endangered species (e.g., sea otter). eDNA assay has already been developed 8 and was studied as part of Ms. Capstone research project in the Fall of 2019. Could be used as a model to study the ecology of eDNA of aquatic mammals to improve detection rates and sampling methodology. General Methods Collected 15 mL samples (n = 25) from each of the following locations at Rice Creek Field Station (Figure 2) Locations consist of sampling upstream, downstream, and near a known river otter den. Centrifuged samples at 5,000 g for 10 minutes to pellet animal cells. Decanted supernatant (containing extracellular DNA) into another 15 mL tube, precipitated external DNA, and centrifuged samples at 14,000 g for 20 minutes . DNA concentration ([DNA]) and quality quantified with NanoDrop . Double stranded [DNA] quantified with PicoGreen assay. Converted published qPCR assay 8 to end point PCR. Final volumes for PCRs was 20 Âµl and consisted of 1X Gold PCR Buffer, 2 mM MgCl 2 , 0.24 mM dNTPs, 1 Âµg/Âµl BSA, 0.3 mM each primer, and 1.25 U of Dream TAQ polymerase. Test PCR conductions using known river otter, fisher ( Martes pennanti ), and marten ( Martes americana ) DNA samples. Preliminary Results No eDNA differential centrifugation protocol currently published. We created a protocol to separate intracellular and extracellular DNA components of water samples. Used eDNA from a captive spotted turtle ( Clemmys guttata ) named Jerry to develop protocol. Used 15 mL water samples to test variations of the protocol now described in general methods. Method is successful at extracting high quality eDNA (Table 1). Future Work Ms. Waloven submitted a proposal to the Rice Creek Small Grants Association to continue this project this summer (Figure 4). Intends to expand sampling to capture summer eDNA variation. To reduce non specific amplification, PCR conditions will be altered. References 1) Elith , J. et al. Ecography 29(2). 2) Goldberg, C. et al . (2016) Critical considerations for the application of environmental DNA methods to detect aquatic species. Methods Ecol Evol 7(11): 1299 1307. 3) Rees, H. et al . (2014) The detection of aquatic animal species using environmental DNA a review of eDNA as a survey tool in ecology. J Appl Ecol 51(5): 1450 1459. 4) Port, J. et al . (2016) Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA. Mol Ecol 25(2): 527 541. 5) Barnes, M. and Turner, C. (2016) The ecology of environmental DNA and implications for conservation genetics. Conserv Genet 17(1): 1 17. 6) Corinaldesi , C. et al . (2005) Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments. Appl Environ Microbiol 71: 46 50. 7) , A. and Corinaldesi , C. et al . (2004) Degradation and turnover of extracellular DNA in marine sediments: ecological and methodological considerations. Appl Environ Microb 70: 4384 4386. 8) Padgett Stewart, T. et al . (2016) An eDNA assay for river otter detection: a tool for surveying a semi aquatic mammal. Conserv Genet Resour 8(1): 5 7. 9) Winnepenninckx , B. et al. (1993) DNA Extraction CTAB Method. TIG 9(12): 407. Objectives Determine the state (intracellular or extracellular) at which eDNA is collected in water samples . Quantify the relative concentration of intracellular to extracellular DNA extracted from water DNA samples collected . Determine whether eDNA collection state affects detection rates . Compare the detection rate (via end point and quantitative Polymerase Chain Reaction (PCR) assays for the river otter using intracellular and extracellular DNA extracts per sample) . Learn more at sardlab.com/ Figure 3. eDNA assay shows evidence of non specific amplification. Figure 2. Study Map of sampling locations throughout Rice Creek Pond and Stream. Figure 1. North American river otter https://www.animalsportal.net/wp content/uploads/2016/09/North American river otter.jpg Winter Intracellular DNA Extracellular DNA Extracellular DNA Intracellular DNA r Summer River Otter detections River Otter detections River Otter detections River Otter detections Endpoint PCR qPCR Endpoint PCR qPCR Endpoint PCR qPCR Endpoint PCR qPCR Figure 4. Schematic depiction of summer plan and the statistical comparisons we would be calculating. Acknowledgments Special thanks to Dr. Bendinskas for the helpful comments on developing the differential centrifugation protocol and Dr. Sample [DNA] A260/A280 Jerry 1 78.2 2.18 Jerry 2 173.1 2.09 Table 1: DNA concentration ([DNA] ng/Âµl) results The in silico analysis suggested published assay would produce a 147 base pairs PCR product with river otter eDNA. Also, suggested that fishers eDNA would not amplify, but marten eDNA may amplify. The in vitro analysis confirmed amplification of non target species (Figure 3).