Comparison of in vitro and in vivo repellency bioassay methods for Ixodes scapularis nymphs

Abstract

Background Numerous 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. Methods We 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 using Ixodesscapularis nymphs. We compared the results using nymphs from two different tick colonies that were derived from I.scapularis collected 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. Results The 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. Conclusions The 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

Repellent activity of essential oils to the Lone Star tick, Amblyomma americanum

“…americanum nymphs [ 22 , 24 ], yet oil of lemon eucalyptus was more active than DEET, picaridin and IR3535 when tested using the tick carousel assay [ 28 ]. Contrarily, repellent potency of DEET, peppermint oil and rosemary oil against I. scapularis did not differ significantly between in vitro (jar and petri dish) and in vivo assays (fingertip and forearm) [ 30 ]. Thus, the second goal of this study was to assess how the repellent potencies of 16 essential oils, the naturally derived pyrethrin acid, TCA, nootkatone and DEET vary based on two in vitro assays and one in vivo assay.…”

“…Furthermore, the experimental design of in vitro assays is unlikely to account for external stimuli that are relevant to questing behavior [ 33 ] and raises the question of how well in vitro assays correlate to repellent efficacy to questing ticks in the field. Although there have been some efforts to compare repellency bioassay methods for ticks [ 30 , 31 ], there is still no consensus for the most appropriate bioassay to test for tick repellency.…”

Ixodes scapularis Is the Most Susceptible of the Three Canonical Human-Biting Tick Species of North America to Repellent and Acaricidal Effects of the Natural Sesquiterpene, (+)-Nootkatone

“…Multiple variables may impact tick response even when the same species or life stage and repellent formulation are used. These include bioassay design, genetic differences within tick populations, tick age and storage conditions, and the working definition of repellency, which is largely unstandardized [44].…”

“…Personal protection compounds used against mosquitos and other insects that spend mere seconds on a host typically act on odorant receptors, disrupting host cue detection through spatial repellency effects or preventing landing through an irritant effect [53]. The host seeking and feeding behaviors of ticks are very different from that of insects and requires different means of measure [44]. Ticks quest by climbing vegetation or other vertical surfaces to wait for a passing host on which to grab and navigate up the host in search of a feeding site, where they remain attached for several days [54].…”

“…Ticks quest by climbing vegetation or other vertical surfaces to wait for a passing host on which to grab and navigate up the host in search of a feeding site, where they remain attached for several days [54]. The EPA considers this behavior in their recommendations for in vitro testing via the human arm bioassay, where repellency is defined by ticks crossing 3 cm upwards into a treated region after being released to walk from an untreated surface [44]. However, we can speculate that distinct events occur during the initial interaction with a protected human, which may affect the tick response.…”