Collection of Nitellopsis obtusa and Determination of eDNA Signal Detection Limit

Abstract:

Nitellopsis obtusa is a macro-algae native to Eurasia, brought to the Great Lakes through ballast water of commercial ships. This experiment’s objective was to collect and identify N. obtusa in the PA waters of Lake Erie and determine the detection limit of N. obtusa eDNA. N. obtusa was identified by its white star-shaped bulbils, orange antheridia, and identification by Sanger Sequencing. Two experiments were completed, one with N. obtusa samples of 1.0 g, 2.0 g, and 5.0 g, the second with 0.08 g, 0.4 g, and 0.8 g placed in jar with 800 mL of lake water in triplicates. Samples were taken once a week for three weeks using flat filter housings, a Masterflex pump, and tubing. Filters were subjected to a DNA extraction protocol known to successfully obtain eDNA signal from water samples and eDNA signal was examined using a qPCR protocol. qPCR analysis indicated that N. obtusa samples of 1.0 g, 2.0 g, and 5.0 g in 800 mL of water had insignificant differences in PCR signal (CQ values ranging 26-30), and N. obtusa samples of 0.8 g and less had limited detection (CQ values from 34 to not detectable). This experiment demonstrated that the detection limit for N. obtusa eDNA is 1.0 g per 800 mL of water, much higher than similar trials using aquatic invertebrates. The results are consistent with preliminary field trials in which N. obtusa eDNA was not recovered from water samples taken from locations known to have live populations of the plant.