Submitted to: American Chemical Society
Publication Type: Book / Chapter
Publication Acceptance Date: 6/30/2011
Publication Date: 12/31/2011
Citation: Carroll, J.F., Zhang, A., Kramer, M.H. 2011. Using lone star ticks, Amblyomma americanum (Acari: Ixodidae) in in vitro laboratory bioassays of repellents: dimensions, duration, and variability. American Chemical Society. 1090(7):97-120. Interpretive Summary: Tick-borne diseases pose a serious threat to humans throughout much of the habitable world. Repellents provide a critical means of personal protection against tick bite. Behavioral bioassays are an important tool in repellent discovery, development, and registration. A great variety of bioassays are used to test tick repellents. ARS has a memorandum of understanding with the EPA to cooperate in developing standardized methods for testing tick repellents. We evaluated several bioassay methods using two repellents, deet and (-)-isolongifolenone, of similar effectiveness against ticks. We wished to see if the various bioassay methods all gave the same comparative result, that deet and (-)-isolongofloenone were of similar efficacy. We aslo wanted to determine some optimum bioassay conditions. All the methods we used showed that the two repellents were similarly effective. We determined the optimum height for bioassays involving climbing by lone star ticks to be 8 centimeters and that climbing bioassays only need to last 6 minutes. We found that ticks could be tested twice in one day in climbing bioassays, but should be allowed a week or more before being used again. These findings are of interest to the EPA, researchers investigating tick repellents and to manufacturers developing tick repellents.
Technical Abstract: The in vitro bioassay is an important tool in repellent discovery and development, with a variety of bioassays used in recent years. Several factors, such as the dimensions 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 eight different bioassays or configurations. All bioassays used =4 concentrations of repellent and an ethanol control against lone star tick, Amblyomma americanum, nymphs. Climbing bioassays included a 22 X 1 cm vertical filter paper strip and a 4 X 7 cm vertical filter paper strip plus four modifications of that configuration. 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 was used as was a horizontal bioassay in which ticks were confined between two filter paper discs that had one half treated with repellent. For each bioassay, deet and (-)-isolongifolenone were similarly effective. The 22 X 1 cm showed that a height of =8 cm and =6 min duration were optimal for climbing bioassays. When a loop of treated paper was added to the untreated lower portion of the 4 X 7 cm filter paper, as alternative escape for ticks, 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 X 7 cm strip. However, with the ethanol controls more ticks fell from 4 X 7 cm strips with loops than without loops. Several important behaviors associated with host acquisition 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 and allowed detection of a decline in repellency over time. Individual variation among ticks and repeated use of ticks in bioassays were assessed using a vertical paper strip bioassay. The responses of ticks tested twice on one day did not differ between tests. However, continued repeated daily testing compromised results. A hiatus of about a week allowed ticks return to their initial response profiles