Located on the fore tarsals of the front pair
Located on the fore-tarsals of the front pair of legs, the Haller’s organ is presumed central to tick chemosensation. Recent genomic, transcriptomic and proteomic investigations have identified several families of transmembrane proteins in the forelegs of ticks that are likely involved in olfactory processes, including numerous ionotropic receptors, odorant-binding (like) proteins and other chemoreception-related proteins (Josek et al., 2018; Carr et al., 2017; Gulia-Nuss et al., 2016; Renthal et al., 2017). Cytochrome P450 s were also well documented in these tissues, as these glycerophosphate mg are involved in a variety of functions including the metabolism of odor ligands (Feyereisen, 1999, 2012). Of particular interest, Carr et al. (2017) identified a possible olfactory G-protein coupled receptor (GPCR) signal cascade in D. variabilis that is seemingly exclusive to the Haller’s organ. Since GPCRs can serve as secondary messengers in the metabotropic pathway, it lends support to a coupling of the ionotropic and metabotropic cascade for tick olfaction and highlights a possible mechanistic basis for tick repellence by DEET through P450 inhibition. A suite of over 120 putative GPCRs were identified in our whole-body D. variabilis transcriptome, though tissue specificity to the Haller’s organ could not be inferred. In terms of mode of action, inhibition of cytochrome P450 s by DEET could provide a viable explanation for the confusion hypothesis (Ramirez et al., 2012). Carr et al. (2017) identified putatively Haller’s organ-specific D. variabilis P450 transcripts, suggesting that they function as odorant degrading enzymes (ODEs) within the olfactory sensilla. ODEs are not well understood (Durand et al., 2011; Leal, 2013) but are proposed to maintain olfactory system sensitivity by rapidly removing the receptor-bound odor molecules (Leal, 2013; Vogt, 2005; Younus et al., 2017). In lieu of functional ODEs, the odorant would continue to induce neural activity leading to sensory adaptation, thereby impairing the tick’s ability to quickly respond to changes in volatiles in its environment (Vogt and Riddiford, 1981; Younus et al., 2014). Therefore if DEET acts as a P450 inhibitor, as our molecular data indicates, the tick’s inability to effectively breakdown odor ligands could cause “confusion” and sensory overload, which would impact host-seeking capabilities (Ramirez et al., 2012). The second major finding of our experiments was a reduction in the expression of transcripts encoding acetylcholinesterase (AChE), which akin to P450 s, was immediate and tapered over time. AChE is a type-B carboxylesterase enzyme involved in the hydrolysis of acetylcholine into acetic acid and choline (Taylor et al., 2009). Acetylcholine is an essential neurotransmitter whose core function is to carry signals from in cholinergic synapses, including at neuromuscular junctions. In these synapses, once the signal has been successfully passed, AChE breaks down the neurotransmitter, effectively halting the signaling and allowing the by-products to be recycled into new neurotransmitters. Given its vital role, many pesticides inhibit AChE, preventing it from breaking down acetylcholine. The resultant increased half-life of the neurotransmitter prolongs its post-synaptic effects and can cause hyperactivity, uncoordinated movements, tremors, convulsions and/or paralysis of the insect (Colović et al., 2013; Fukuto, 1990; Lang et al., 2012). In line with our molecular dataset, Corbel et al. (2009) found in Aedes that acetylcholine is not efficiently hydrolyzed by AChE in the presence of DEET, indicating that the repellent can operate as an AChE inhibitor. Thus, if DEET impedes AChE production, as our experiments suggest, it could act as a mild pesticide to further deter ticks from coming into contact with the repellent or from remaining in its vicinity. In addition to P450 s and AChEs, several other gene groups were also suppressed by DEET. Noteworthy was an immediate downregulation of transcripts related to linoleic acid metabolism. In humans, linoleic acid synthesis is converted to mono-hydroxyl products by certain P450 s (Ruparel et al., 2013); thus, the activity of transcripts in this pathway appear coupled to that of P450 s. At later time points, transcription factors, immunoglobulin-like transcripts and epidermal growth factors were all repressed. These groups have diverse functions and therefore further studies are needed to shed light into their role (if any) in DEET repellence. On the other hand, both metallopeptidase and phosphatidylinositol 3/4-kinase were induced by DEET exposure. Phosphatidylinositol 3/4-kinase is implicated in the regulation of a broad range of cellular activities (Funaki et al., 2000). Metallopeptidases are associated with neural repair/regeneration after damage in humans and the degradation of the extracellular matrix (Chuang et al., 2010; Estrada-Pena and Mans, 2014; Kiryu-Seo et al., 2000). It is possible that these transcripts are involved in activation of mechanisms aimed to repair damage caused by DEET exposure, including the potential detrimental effects of neural hyperactivity due to AChEs inhibition (see above).