This post was contributed by Allison Walker, Mika Kirkhus, Rielle Hoeg, and Dave Shutler, authors of a recent paper in The Wilson Journal of Ornithology about using eDNA to identify raptor pellets.

In the field, bird researchers regularly encounter raptor pellets, which are regurgitated clumps of indigestible animal material such as fur, feather, and bones. However, where multiple species of similar-sized raptors occur, it can be hard to tell which species produced a pellet based on the pellet’s appearance alone. After field seasons spent on two Nova Scotian islands collecting raptor pellets as part of a study on predation on seabirds, one sample of pellets raised our eyebrows. The pellets we collected from an island known to host Great Horned Owls were small, about half the size of a computer mouse; those we collected from another island were huge ⁠— easily four times the size. We speculated about what could have disgorged such a monstrosity. Could it have been a Snowy Owl? Probably not, given the similar size of the two owls. An eagle? Seemed more likely, but how could we know for sure? Thus, we became interested in developing a way to identify bird species producing pellets based on residual environmental DNA (eDNA). We suspected DNA in pellets could come from a bird’s digestive tract cells, leaving behind a genetic signature in the field telling us “Who puked?”

To test this, we used a process called DNA barcoding, where DNA is extracted, a specific region is sequenced, and that sequence is then compared to a library of known DNA sequences. In this case, we used a region of the mitochondrial gene cytochrome c oxidase 1 subunit (CO1), which is typically used to identify animal species. Enterprising undergraduate biology student, Mika Kirkhus, took this on as part of a fourth-year Molecular Markers course at Acadia University with Allison Walker. The pellets they studied came from Country Island, Nova Scotia, as well as from a wildlife rehabilitation facility and a local zoo; the latter two each house several raptor species.

Mika and Allison extracted DNA from each pellet and amplified it using Polymerase Chain Reaction (PCR). The PCR, using avian primers, allowed us to make millions of copies of our gene region of interest, enough so that we could study the DNA in detail. After troubleshooting different reaction temperatures in our procedure and applying multiple PCR primer sets to the same eDNA sample, we were successful in visualizing PCR products using gel electrophoresis. From there, we sent successfully amplified samples to the Genome Québec Innovation Centre for sequencing, and we were able to develop DNA barcodes that successfully distinguished between Red-tailed Hawk and Great Horned Owl pellets. Because eDNA degrades, this protocol will only work on pellets that have been in the environment for a limited time, probably less than 6 months. Nonetheless, we believe it will be a useful eDNA-based protocol for future raptor studies, such as for untangling predator-prey interactions in remote locations.

This collaborative multidisciplinary undergrad research experience catapulted Mika into her current master’s degree research project. During this project, she wanted to take barcoding to the next level in a study using metabarcoding, which allows for simultaneous identification of many taxa from the same eDNA sample. Using lichens from all over Norway and applying metabarcoding, she found hidden fungal diversity within the genus Pertusaria. Mika’s fascination for molecularly identifying species with DNA has only increased, and she is currently exploring PhD opportunities involving metabarcoding of either birds or fungi. She believes the opportunity to do research as an undergrad and produce a publication is highly encouraging for students to continue down the path of the science. Mika is therefore very happy she participated in finding out “who puked,” because in the process, she also discovered her passion for research.