Regulatory factors controlling tick salivary glands (SGs) are direct upstream neural signaling pathways arising from the tick's central nervous system. Here we investigated the cholinergic signaling pathway in the SG of two hard tick species. We reconstructed the organization of the cholinergic gene locus, and then used in situ hybridization to localize mRNA encoding choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) in specific neural cells in the Ixodes synganglion. Immunohistochemical staining revealed that cholinergic axonal projections exclusively reached type I acini in the SG of both Ixodes species. In type I acini, the rich network of cholinergic axons terminate within the basolateral infoldings of the lamellate cells. We also characterized two types (A and B) of muscarinic acetylcholine receptors (mAChRs), which were expressed in Ixodes SG. We pharmacologically assessed mAChR-A to monitor intracellular calcium mobilization upon receptor activation. In vivo injection of vesamicol-a VAChT blocker-at the cholinergic synapse, suppressed forced water uptake by desiccated ticks, while injection of atropine, an mAChR-A antagonist, did not show any effect on water volume uptake. This study has uncovered a novel neurotransmitter signaling pathway in Ixodes SG, and suggests its role in water uptake by type I acini in desiccated ticks.

Biology Centre Institute of Parasitology Czech Academy of Sciences České Budejovice Czech Republic

Department of Entomology Kansas State University 123 Waters Hall Manhattan KS USA

Faculté de Pharmacie Université de Limoges Limoges France

Faculty of Science University of South Bohemia České Budejovice Czech Republic

UMR BIPAR INRAE Ecole Nationale Vétérinaire d'Alfort ANSES Université Paris Est Maisons Alfort France

UMR SPE 6134 CNRS Université de Corte Pascal Paoli Corse France

UMR Virologie INRAE Ecole Nationale Vétérinaire d'Alfort ANSES Université Paris Est Maisons Alfort France

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Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick-Pathogen Interactions