Chapter 7 Using Lone Star Ticks, Amblyomma americanum (Acari: Ixodidae), in in Vitro Laboratory Bioassays of Repellents: Dimensions, Duration, and Variability

摘要

The in vitro laboratory bioassay is an important tool in tick repellent discovery and development, with a variety of bioassays used in recent years. Several factors, such as size and configuration of test surfaces and duration of tick exposure, can influence the outcome of bioassays. We tested two tick repellents, N,N-diethyl-3-methyl benzamide (deet) and (-)-isolongifolenone, in seven different bioassays or configurations. All bioassays used ≥4 concentrations of repellent and an ethanol control applied to filter paper against lone star tick nymphs, Amblyomma americanum (L.). Climbing bioassays included a 22 × 1 cm vertical filter paper strip and a 4 × 7 cm vertical filter paper strip plus four modifications of the basic 4 × 7 cm configuration. We used a moving object bioassay (MOB), in which a strip of filter paper treated with test solution was affixed to a rotating heated brass drum and ticks allowed to transfer to the paper. A horizontal bioassay in which ticks were confined between two filter paper discs that had one half treated with repellent was also used. For each bioassay, deet and (-)-isolongifolenone were similarly effective, but in some bioassays ticks were repelled by lower concentrations of both repellents than in other bioassays. The 22 × 1 cm strip proved impractical for regular bioassay use, but showed that a height of 8-9 cm and ~6 min duration were optimal for climbing bioassays. When a loop of treated paper was added to untreated lower portion of the 4 × 7 cm filter paper, as alternative escape for ticks responding to repellents, more ticks were on the loop and lower untreated area of the strip at 10 min (end of the test) than were on the lower untreated area of the basic 4 × 7 cm strip. However, with the ethanol controls more ticks fell from 4 × 7 cm strips with loops than those without loops. Several important behaviors associated with host acquisition (contacting, transferring to and remaining on a moving surface) were recorded in the MOB, but we only found significant differences between treatment and control for the proportion of ticks that transferred to the filter paper and the length of time the ticks remained on paper. The petri dish bioassays lasted longer than other bioassays (2h compared to 10 min for the vertical filter bioassays) and allowed detection of a decline in repellency over time. Individual variation among ticks and fatigue (change in response) in repeatedly tested ticks were assessed in a vertical paper strip bioassay using deet. The responses of ticks tested twice on one day (morning and afternoon) did not differ between tests. However, continued repeated daily testing compromised results. A hiatus of about a week between tests allowed ticks to return to their initial response profiles.