BackgroundNumerous bioassay methods have been used to test the efficacy of repellents for ticks, but the comparability of results across different methods has only been evaluated in a single study. Of particular interest are comparisons between bioassays that use artificial containers (in vitro) with those conducted on a human subject (in vivo) for efficacy testing of new potential unregistered active ingredients, which most commonly use in vitro methods.MethodsWe compared four different bioassay methods and evaluated three ingredients (DEET [N,N-Diethyl-meta-toluamide], peppermint oil and rosemary oil) and a negative control (ethanol) over a 6-h period. Two of the methods tested were in vivo bioassay methods in which the active ingredient was applied to human skin (finger and forearm bioassays), and the other two methods were in vitro methods using artificial containers (jar and petri dish bioassays). All four bioassays were conducted usingIxodesscapularisnymphs. We compared the results using nymphs from two different tick colonies that were derived fromI.scapulariscollected in the US states of Connecticut and Rhode Island (northern origin) and Oklahoma (southern origin), expecting that ticks of different origin would display differences in host-seeking behavior.ResultsThe results between bioassay methods did not differ significantly, even when comparing those that provide the stimulus of human skin with those that do not. We also found that tick colony source can impact the outcome of repellency bioassays due to differences in movement speed; behavioral differences were incorporated into the assay screening. DEET effectively repelled nymphs for the full 6-h duration of the study. Peppermint oil showed a similar repellent efficacy to DEET during the first hour, but it decreased sharply afterwards. Rosemary oil did not effectively repel nymphs across any of the time points.ConclusionsThe repellency results did not differ significantly between the four bioassay methods tested. The results also highlight the need to consider the geographic origin of ticks used in repellency bioassays in addition to species and life stage. Finally, our results indicate a limited repellent efficacy of the two essential oils tested, which highlights the need for further studies on the duration of repellency for similar botanically derived active ingredients and for evaluation of formulated products.Graphical Abstract