Development of an Environmental DNA Assay for Eastern Massasauga

Utilizing environmental DNA (eDNA) for the detection of species in the field is a potentially cost-effective and time-saving technology that may be useful in understanding the distribution and abundance of threatened or endangered species such as the Eastern Massasauga (Sistrurus catenatus). I describe the development of an eDNA assay for the species and evaluate its ability to detect eDNA in laboratory and field conditions. In the field samples, I also investigated the potential for abiotic conditions to influence eDNA detection. Species-specific primers and probe were designed to amplify a 152 bp segment of the massasauga COI gene. Target eDNA could be detected in samples containing as little as 100 copies of target DNA/μL. Water samples collected from laboratory housed snakes indicated that eDNA can be detected in water 56 days after massasauga removal. Field samples were taken from crayfish burrows, known overwintering habitat for the species, from four sites that vary in snake use as ascertained by traditional visual surveys. Of the 60 burrows sampled, seven had a positive detection for massasauga eDNA with no difference in detection rate between DNA extracted from burrow water and burrow sediment. Occupancy models fitted to burrow water indicated that larger amounts of total DNA in a sample may increase the probability of detection of a massasauga eDNA. Large confidence intervals in site occupancy (ѱ) and burrow detection (Θ) values suggest that a larger sample size is needed for more reliable occupancy models. Abiotic conditions within crayfish burrows varied among sites but correlation with eDNA detection was not supported. Estimates of qPCR detection within a burrow with eDNA (ρ) suggest that up to 10 qPCR replicates per burrow sample may be necessary. Further studies need to examine eDNA degradation in the field, improve upon the limit of detection, and sample a larger number of sites for eDNA sampling to be a stand-alone survey method for Eastern Massasaugas.