To date little is known about mode of

To date, little is known about mode of action or the underlying genetic basis of DEET repellency for human-biting ticks. To begin to unveil these mechanisms, we carried out behavioral assays to confirm the effectiveness of DEET as a deterrent for D. variabilis. RNA sequencing was then used to build the D. variabilis transcriptome and to characterize global changes in gene Imiquimod hydrochloride mass in response to DEET in ticks at 0.25 h, 4 h and 24 h post exposure. Two major patterns emerged from these novel datasets. First, we identified an immediate and substantial reduction in transcript abundance of cytochrome P450 genes, which recovered over time. This finding was corroborated by enzymatic assays, and provides support for the hypothesis that P450 inhibition plays an important part in repellence. Second, we found molecular support for DEET as an acetylcholinesterase inhibitor in ticks. While this work does not fully unravel the repellent’s mode of action per say, it provides novel and valuable insights into the responses of ticks to DEET at the molecular level.
Materials and methods
Results
Discussion Despite the global reliance on DEET to repel a myriad of invertebrate pests and prevent vector-borne diseases, its mode of action and genetic underpinnings remain unresolved. The American dog tick, D. variabilis, was chosen for these studies because of its abundance in the study area, importance as a vector of human pathogens and the fact that virtually nothing is known about the molecular responses of ticks to DEET. Our experiments indicated that ticks are effectively deterred by DEET, and that exposure can induce muscle spasms and uncoordinated movements. Underlying these behaviors was a small genetic signature, characterized by an immediate and dramatic reduction in the accumulation of transcripts encoding cytochrome P450 s and acetylcholinesterases. In addition, relative oxidases enzyme activity of D. variabilis was impeded nearly 30% in the presence of DEET. This is in agreement with recent toxicological and biochemical studies of mosquitoes, which show inhibition of these enzymes, particularly CYP450, in the presence of DEET (Bonnet et al., 2009; Corbel et al., 2009; Ramirez et al., 2012). The transcriptional downregulation was most pronounced within the first 15 min post DEET exposure and gradually recovered over the 24 h time period. Such abrupt changes to only a small number of specific gene groups prompt speculation into their mechanistic roles in DEET avoidance by ticks and provide unique insights into the repellent’s mode of action. The two most commonly accepted theories for DEET’s mode of action – “confusion” and “smell and avoid” – are both fundamentally based on odor perception. Both vertebrates and insects use a variety of families of transmembrane receptor proteins to detect and discriminate odors, though their molecular mechanisms of chemosensory signalling are radically different (Pellegrino and Nakagawa, 2009; Silbering and Benton, 2010; Spehr and Munger, 2009; Touhara and Vosshall, 2009). Insects (and perhaps all arthropods) predominately use ionotropic receptors, which form ion channels that are generally closed until a ligand (e.g., odor molecule) binds. On the other hand, vertebrates use metabotropic receptors, which do not form channels. Instead, binding of the ligand indirectly activates ion channels through secondary messengers. From a functional standpoint, ionotropic receptors respond rapidly, whereas metabotropic receptors tend to have slower but broader effects, depending on the number and types of secondary messengers involved in the signaling cascade. From Drosophila studies, it has been speculated that an odor molecule binding to a specific chemosensory receptor stimulates an immediate ionotropic pathway followed by a slower forming metabotropic response (Hansson et al., 2010; Wicher et al., 2008). Ramirez et al. (2012) proposed that the metabotropic pathway requires cytochrome P450 activity at some point in the signalling cascade. If this were the case, DEET may initially trigger an attraction via ionotropic receptors and subsequent repellency due to an impediment of the P450 s along the metabotropic pathway, as discussed below in the context of D. variabilis and other ticks.