qPCR results from design and partial validation of three novel eDNA qPCR assays for several 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 three novel eDNA qPCR assays for three common North American tick species (Dermacentor variabilis, Amblyomma americanum, and Ixodes scapularis) and tested them on samples of grasses collected from grasslands and forests in 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 that are 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.

Methods
Plant matter samples containing grasses and leaf litter were collected in the fall of 2020, and from May through October of 2021 from various sites throughout central and southern Illinois.  Three 250 meter linear transects were established at each site and sampled for ticks and leaf litter via random selection (without replacement) to reduce bias (i.e. one of the three transects was selected at random for each site per visit, for a total of three visits on average).  50mL Falcon tubes were filled halfway with unpacked plant matter collected at approximately 0m, 150m, and 250m along the transect to ensure adequate coverage. The leaf litter sample tube and a control (blank) 50 mL tube were filled with enough CTAB extraction buffer to cover all plant matter, and tightly capped. Both tubes were inverted five-ten times (as necessary) to ensure all litter was in contact with the buffer.

All field samples were extracted 30 days post-collection via a modified chloroform: isoamyl alcohol method (Renshaw et al., 2015).  All samples and field blanks were tested for tick presence via qPCR. All reactions contained 12.5 µl of TaqMan Environmental Master Mix 2.0, 1 µl of forward and reverse primers, 1 µl of probe, 6.5 µl of molecular grade water, and 3 µl of DNA.  Three replicates of seven gBlock standards containing target DNA from each species were run alongside samples from each plate in order to estimate copy number. Concentration of the seven standards ranged from 1 copy/µl to 1,000,000 copies/µl by a factor of 10.  Thermocycler conditions included an initial denaturation step at 95°C for 5 minutes followed by 45 cycles of 95°C for 30 s, annealing (56.5, 57, and 57.5 depending on species) for 30 s, and extension at 72°C for 1 min.