Utilizing Environmental DNA for the Detection of Invasive Goldfish (Carassius Auratus) and Koi (Cyprinus rubrofuscus) in Southeastern Massachusetts Water System 

By: Mia Oliviera

I am happy to report that this semester’s research has been a huge, fun, learning opportunity, and I am very appreciative of the OUR’s grant to assist me in my work this Spring. In March, I spent my spring break taking water samples and negative controls at all ten sites across Southeastern Massachusetts. These samples were then brought to the UMD campus to be filtered over the following few weeks within Dr. Drew’s genetics lab. Samples were filtered using a vacuum pump setup under a sterile hood, and bleach was used to ensure no cross-contamination between samples.

Starting in early April. I utilized the DNeasy Blood and Tissue Kit and the Powersoil PowerLyzer kit to optimize an eDNA extraction method that best suits my extraction needs. My two positive control samples (goldfish and koi ponds) were extracted with both kits to determine which would produce the best product. It was found that the Powersoil PowerLyzer kit allowed the filter and cells to break down best and produce a clearly visible PCR product. I determined the best extraction method was to utilize the DNEasy Powersoil PowerLyzer kit for the remainder of my samples, with a few modifications to the listed protocol.

Once all samples and negative controls were extracted, I ran a PCR utilizing Dr. Drew’s genetics lab resources and using universal fish DNA primers that amplify all fish species. I chose to use fish primers instead of my species-specific primers to ensure the filtering, extraction, and PCR methods captured fish eDNA. The PCR products were run in an electrophoresis gel and imaged. The first PCR I ran produced a lot of DNA bands but showed signs of potential contamination within the PCR step. I chose to do a second PCR while under a sterile hood, which produced a cleaner product and a few samples containing DNA, but did show some amplification of fish DNA within my negative control samples. Dr. Drew and I worked through troubleshooting methods and discussed ways we could optimize protocols for the Fall semester and ways to use different primers, as well as my species-specific primers. I decided to do more research on eDNA sampling, extraction, and PCR methods over the summer to find the best protocols for my research in the Fall.

I have learned so much about the world of eDNA over the last few months. I find the troubleshooting aspect not to be a roadblock, but a huge opportunity to problem-solve and optimize my own protocols. eDNA research is still in its early stages, but it has so much potential in its application. I am very excited to continue my research on this in the Fall, and once again thank the OUR for this funding opportunity.

(Sampling at Snows Pond, Rochester, MA)