Vertical bioassays (Figure 1) were prepared with modifications to a previously described vertical bioassay method [25]. Naïve, unfed, host-seeking (actively questing), I. scapularis ticks were used in all behavioural assays. Uninfected ticks were purchased from the Tick Rearing Facility Laboratory at Oklahoma State University (Stillwater, OK, USA). Ticks were stored on site in plastic containers lined with moistened Kimwipe® at 4 °C in dark conditions.

Vertical bioassay setup: A cotton swap is inserted in a Falcon tube and secured at the base with a polystyrene platform (a). The base of the cotton swap tip is aligned with the 25 mL line (b); the tick is released at the bottom of the cotton tip just below the 20 mL line. The timer starts when the tick crosses the 20 mL line (c); the treatment is applied on top of the swab (d); the observer exposes a finger on the open top of the Falcon tube to stimulate the tick to climb the slender surface (e).

Nymphs were removed from the refrigerator and kept at room temperature for approximately 20 min prior to starting the bioassays. The bottom of 50 mL Falcon tubes (Fisher Scientific, Walthman, MA, USA) were filled with polystyrene foam. Cotton swabs (SolonCare®, Amd-Ritmed Inc., Montréal, QC, Canada) were measured and cut at 7 cm so that the edge cotton tip was at the 25 mL marker on the falcon tube when fully inserted. In addition, 100 mg/mL solutions of ICs were prepared in 1:1 DI H2O:EtOH for use in the assay. Fifty (50) µL of the solution was pipetted to the tip of the cotton swab and left to dry for approximately for 5 min. Active ticks were added with a paintbrush to the wooden stem of the cotton swab just below the 20 mL marker on the tube and positioned so that the tick faced upward. Time was started when the tick crossed the 20 mL marker. Time was stopped when the tick either reached the tip and remained for 10–15 s, reached the tip, and dropped or reached the tip and climbed off the tip. The observer placed their hand on the opening of the tube for the length of the experiment and occasionally removed their hand to breath over the top of the tube.

Statistical analyses were performed using RStudio version 1.1.453 (RStudio Team 2018). Results from the mass of EO extracted from IC and EO volatile emission were analyzed with a linear mixed-effect model (lmer). The significance of the model was assessed by running the anova function. We performed a post-hoc pairwise comparison of interaction using the emmeans function (emmeans package) on the model to determine the differences between means produced by different EOs. Results from different methods and time points were organized in subsets and independently analyzed. Repellency data with not normal distribution were subjected to non-parametric tests (i.e., Kruskal-Wallis), followed by a post-hoc test (i.e., Dunn test) to compare the different treatments. Differences were considered significant at p ≤ 0.05.

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