LB 19-175 - Lessons learned from field environmental DNA sampling methods for terrestrial crayfish (Creaserinus fodiens and Lacunicambarus diogenes) in Ontario, Canada.

Environmental DNA (eDNA) has become commercially available to industry and is being used to support a wide range of ecological assessments within the private sector. eDNA is effective in detecting species from soil or water samples, making it efficient, non-invasive, and cost-effective. As an emerging ecological survey tool, eDNA has provided unprecedented results for rare species detection and biodiversity monitoring. However, it is also known to produce false positive and negative results from DNA cross-contamination, degradation, production, and transport.In Ontario Canada, two of only three native terrestrial crayfish species are rare and protected as Significant Wildlife Habitat. These fossorial crayfish Creaserinus fodiens and Lacunicambarus diogenes) are difficult to study, and no standardized sampling protocols exist. The goal of this study was to develop an innovative eDNA field collection methodology, use metabarcoding analysis to confirm presence or absence of Significant Wildlife Habitat and develop an agency accepted field collection protocol for terrestrial crayfish in Ontario.Our approach involved the collection of eDNA from crayfish burrow soils in October. Burrow site selection was determined through random sub-sampling and determination of occupied burrows. A total of 10 eDNA soil samples were collected. Sterilization protocols were developed to minimize eDNA site cross-contamination. Soil samples were taken from burrow cap structures or interior walls and sent to a laboratory for metabarcoding analysis using invertebrate and macroinvertebrate assays. Visual surveys were conducted to support data interpretation of eDNA false negatives or false positives.The metabarcoding results did not detect terrestrial crayfish eDNA in any of the 10 samples. However, high-quality macroinvertebrate sequence data was obtained from 20% of the samples. Visual assessments verified the presence of Creaserinus fodiens, a protected species and Significant Wildlife Habitat. Therefore, the eDNA field sampling methodology resulted in a false-negative for terrestrial crayfish. In conclusion, additional research is required to support the development of a standardized crayfish field protocol to include eDNA. Future research and protocol development should consider sampling in the spring when crayfish have higher actively rates and DNA shed rates during reproduction. In addition, where burrows have an accessible water table, eDNA water samples should be collected to determine if eDNA detection rates vary between water and soil samples.