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Using environmental DNA (eDNA) to determine Hellbender distribution : interim report.
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    Environmental DNA (eDNA) methods are non-invasive genetic sampling in which DNA from organisms is detected via sampling of water or soil, typically for the purposes of determining the presence or absence of an organism. In this project, we have evaluated the efficacy of eDNA sampling to detect populations of the eastern hellbender indirectly from their aquatic environments. We developed species-specific primers, validated their specificity and sensitivity, and assessed the utility of our methods. Water samples were collected from 3 creeks and an aquaculture facility at the Columbus Zoo, the latter of which holds animals in captivity for breeding. Salt Creek and Scioto Brush Creek, both in southern-central Ohio were selected for positive control locations upon the recommendation of Greg Lipps, leader of the Hellbender Consortium, part of the Ohio Division of Wildlife. Due to the extremely low population numbers for the animal, coordinates for the release of captive bred animals is protected information, so these coordinates were invaluable in the work. A third waterway (Duck Creek) in the Eastern Cincinnati/Oakley Watershed was chosen as a negative control, due to an abundance of eDNA resulting from municipal wastewater impacting the water and the lack of animal habitat. The negative control sampling location was known to the investigators from previous research, and consists of a concrete raceway adjacent to a Combined-Sewer-Overflow. The structural engineering of the raceway allows for essentially no habitat for aquatic animal life, but the available environmental water contains an abundance of eDNA from sewage overflow, including human and canine mitochondrial DNA. Due to the late start of the project samples were collected in early November, after the typical breeding season Aug-Oct. Thus, detection results represent a minimum detection range for the animal using the protocols detailed in the research section. From the positive detection results at Salt Creek, the animal detection range was found to be approximately 1/3 of a mile downstream of the animal at stream flow rate of approximately 11 ft3 /sec. Comparatively, Scioto Brush Creek had a slightly higher flow rate measured to be around 14 ft3 /sec and significantly wider stream bed width (about 1.5 times larger). The animal at this location was barely detectable at the coordinates provided. It is possible that the animal moved and/or the water volume provided a sufficient dilution to the animal eDNA resulting in only the eDNA detection threshold at the anticipated location. As expected, no animals were detected at the negative control site with the D-loop primers developed in this study. Primers from previous investigations provided little to no animal specific eDNA amplification from the environmental water samples or positive control samples and yielded a variety of amplicons in the negative Duck Creek eDNA samples.
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