Date Approved

2014

Date Posted

4-22-2014

Degree Type

Campus Only Senior Honors Thesis

Department

Biology

First Advisor

Katherine Greenwald

Second Advisor

Kristin E. Judd

Abstract

Environmental DNA (eDNA) analysis is a technology of emerging importance in ecology. It enables the detection of specific species within an environment through the isolation of extracellular DNA shed by the target species into surrounding soil or water. The goal of this study was to establish effective eDNA protocols in a seasonal freshwater wetland (the Edwin S. George Reserve of Pinckney, MI), by comparing eDNA results for the detection of Blue Spotted Salamanders (Ambystoma laterale) to field observations collected at the same sites. The protocols used combined sampling and DNA isolation methods that have been successful in past experiments. Water samples were collected in Nalgene Polypropylene 1000 mL bottles from four vernal pools, at two to three sites per pool. Sites were determined based on suspected larvae and juvenile locations based on corresponding organismal sampling. Collections were repeated at three different dates (May 24'h, 2013; July 19'h, 2013; September 9'h, 2013). These dates, based on corresponding field data, ranged from the beginning of A. laterale larvae phase to the end of the migration of A. laterale juveniles from ponds to land. All samples were then vacuum filtered using 0.45 11m cellulose nitrate filters. Two filter storage methods were tested, as well as two primer sets for PCR amplification. Results from UV gel images of the samples were inconclusive, with no eDNA definitively detectable, even in the positive control samples. This may be unrelated to the success of the protocols used; the lack of a clear positive control is indicative of an issue with the PTC-1 00 Progranunable Thermal Controller used. Future testing and adjusting of the PRC equipment and settings may yield much different results for the samples already collected, when retested with the eDNA protocols used in this experiment.

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