Design and partial validation of novel eDNA qPCR assays for three common North American tick (Arachnida: Ixodida) species

The range expansion of ticks to higher latitudes poses a severe threat to human health exposing human populations who had no prior contact with ticks to several harmful tick-borne diseases. Early detection of ticks in new areas is critical to help inform the public and medical professionals of the dangers associated with tick encounters. Environmental DNA represents a novel survey method that could provide reliable records of tick occurrences and timely warnings of their range expansions. In this study, we designed novel eDNA qPCR assays for three common North American tick species (Dermacentor variabilis, Amblyomma americanum, and Ixodes scapularis) and tested them on 51 samples of grasses and leaf litter collected from 12 grassland and forest sites in central and southern Illinois. We provide in silico and in vitro validation of all three assays; however, we were unable to generate any positive detections from field samples. Our lack of eDNA detections likely stems from low eDNA deposition rates coupled with rapid degradation in grasslands and forests, a problem exacerbated by terrestrial eDNA sampling methods limited by volume of substrate. We provide recommendations for improving sample collection methods to increase detection probability in future efforts. Continued research should focus on the viability of eDNA to detect small terrestrial invertebrates, like ticks, and it potential as early warning indicator of the spread of vector-borne diseases.