A new paradigm for personal protection against ticks: efficacy of spatial repellents to reduce host seeking activities in three major tick species of

2 Abstract 32 Addressing the prevalence of tick-borne disease requires robust chemical options as an integral 33 component of Integrative Vector Management (IVM) program. Spatial repellency is a novel 34 concept in tick bite prevention. To date, there is no standard for the evaluation of spatial 35 repellency against ticks, despite the speculated value of volatilized chemicals in control systems. 36 This study reports a novel vertical climb assay that was specifically created for the quantitative 37 evaluation of spatial repellency in ticks. Controlled release devices (CRDs) were used to control 38 the dispersion of multiple Active Ingredients (AIs) transfluthrin, metofluthrin, nootkatone, and 39 DEET against adult females of three medically important tick species: Dermacentor variabilis, 40 Amblyomma americanum, and Ixodes scapulari s . Results of our study indicate significant 41 associations between AI exposure and changing in tick climbing behavior when compared 42 controls in the absence of the AI, from several perspectives, including changes in tick movement 43 velocity, displacement, detachment, and rate of successful vertical climbing. Metofluthrin and 44 transfluthrin caused strong reductions in host seeking activities against D. variabilis and A. 45 americanum , while both demonstrated slightly weaker effects against I. scapularis . Further work 46 is planned to evaluate spatial repellency in ticks in more natural environments and assess their 47 potential in future tick control programs. 48 49

165 Repellency bioassay 166 The CRD containing a particular test repellent solution was placed at the upper end of the 167 chamber ( Figure 2). An induction time of 20 minutes after each initial device activation was set 168 prior to the introduction of the ticks for each trial. Each trial included 3 female ticks of the same 169 species. One tick was placed at the base of one of the 3 vertical sticks. The array of three 170 climbing sticks in the chamber allowed the assessment of effect of the AI diffusion in the . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made     237 Control trials established baseline behaviors that were compared with ticks exposed to each 238 active ingredient. When the ticks were placed in the assay box, they climbed to the top of their 239 stick. They rarely moved down the stick or detached. A. Americanum ticks took slightly longer to 240 orient than the other species at the bottom of their stick but climbed to the top with a mean 241 velocity much greater than that of any D. variabilis or I. scapularis ticks. They also showed more . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (Table I). The ticks of the same species move at a similar velocity that does not vary much based 247 on time or height. All ticks survived both control and AI trials.
248 Table 1. Comparison of tick detachments among three tick species in response to the control and 249 AI trials.   was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Comparisons of tick activity parameters
298 AIs showed variable influence on the integrative activity parameters measured (Figures 6-8).
299 Most tick activity parameters measured in responses to DEET and Nootkatone were not 300 statistically significant, and effect sizes of those that were significant tended to be lower than 301 what were observed with transfluthrin and metofluthrin. Metofluthrin and transfluthrin showed 302 more significant and large effects from all perspectives against D. variabilis and A. americanum 303 but were generally less effective against I. scapularis (Table 3).
304 Figure 6. Climbing success of ticks in each control group is compared with ticks in AI trials as a 305 measure of inhibition. A tick that is considered "successful" reaches the 27 cm-30 cm height mark 306 of its stick without detaching and maintains a meaningful presence here, as measured by presence 307 at this point at trial end (t = 600 sec). Difference of proportion show significant differences between 308 AI and controls. was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Figure 7. Mean velocity of ticks while moving is compared between AI and control groups, 316 measured in cm/sec. Mann-Whitney U tests showed large, significant differences in D. variabilis 317 with metofluthrin (r = .79, p < .001) and transfluthrin (r = .82, p < .001), and much smaller, non-318 significant differences with nootkatone (r = .14, p = .443) and DEET (r = .08, p = .638). A. 319 americanum showed similar results: metofluthrin r = .66 (p < .001), transfluthrin r = .71 (p < .001), 320 nootkatone r = .25 (p = .140), DEET r = .25 (p = .158). I. scapularis did not show significant effects 321 with metofluthrin (r = 31, p = .067) or transfluthrin (r = .26, p = 113). Significant differences, 322 though smaller in magnitude, were observed with nootkatone (r = .23, p = .044) and DEET (r = 323 .47, p = .006). Significant probability values are considered in tiers: * p < .05, ** p < .01, *** p < 324 .001.  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 17, 2022. . There is no current standard for assessing spatial repellency in ticks, however 362 the two targets of repellents are defined by the prevention of movement across a "protected" 363 surface and preventing attachment for subsequent feeding and disease transmission. The VTA-364 ESR assay considers these in analyses of behaviors that are integral to a tick's successful . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 17, 2022. ; https://doi.org/10.1101/2022.05.17.492268 doi: bioRxiv preprint 365 navigation around these measures, revolving around successful climbing, which is required of a 366 tick for host-seeking and feeding.

367
Ticks have a finite amount of energy and moisture available to fuel host-seeking. Thus, 368 they must use this supply wisely [22]. In conditions conducive to host-seeking, they climb 378 however these metrics are unable to make this distinction from other stages of host-seeking and 379 feeding as performed.

380
In addition to observing gross behaviors as a simulation of host-seeking, an activity 381 analysis of velocity and displacement was performed to visualize any specific effects that AI-382 exposure may have had on their capability or desire to move, translating to a physical ability to 383 carry out host-seeking and on-host movement. There were several occurrences of large changes 384 in the distance ticks traveled. The greatest of which were with metofluthrin and transfluthrin, 385 which reduced the displacement of all three species. DEET showed a meaningful reduction in I.
386 scapularis and an increase in A. americanum but did not result in a change in D. variabilis.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  475 is unable to distinguish which of the two are being simulated, however the behavior changes that 476 are considered here may be applicable to each. Tick response to volatilized compounds, as 477 opposed to tactile chemoreception, has been speculated in the past but has yet to be effectively 478 demonstrated. The VTA-ESR is therefore useful for the evaluation of several behavior factors . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 17, 2022. ; https://doi.org/10.1101/2022.05.17.492268 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 17, 2022. ; https://doi.org/10.1101/2022.05.17.492268 doi: bioRxiv preprint