Functional characterization of Ixodes neuropeptide receptors

Neuropeptidergic systems control feeding behaviors in animals, including arthropods. Given the wide variety of pathogens transmitted by ticks during hematophagy, there is an urgency to understand the neural mechanisms responsible for tick feeding behavior. We characterized three Ixodes signaling systems that are involved in feed-ing regulation in other arthropods: neuropeptide CCHamide (CCHa), short neuropeptide F (sNPF) and sulfakinin. RNAs encoding the preproneuropeptides and their re-ceptors were characterized and cDNAs for the receptors were expressed in HEK293T cells by transient transfection. Activation of the receptors by synthetic peptides was monitored by a calcium release (FLIPR) fluorescence assay. There was a single receptor (CCHaR) activated by CCHa (NH2-SCKMYGHSCLGGH-amide) containing a disulfide bond with an EC50=12 pM, while a scrambled cyclic peptide was inactive at 1 μM. Of the two Ixodes NPY-like receptors, NPYLR1A was activated by sNPF (NH2-GGRSPSLRLRF-amide) with an EC50=1.9 nM. NPYLR1B did not respond to 10 μM sNPF. A single sulfakinin receptor was activated by a sulfated sulfakinin (NH2-SDDY(SO3H)GHMRF-amide) with an EC50=220 pM but not by 1 μM non-sulfated sulfakinin. The Ixodes GPCRs were able to couple to endogenous HEK293T G-protein(s). Surprisingly, human but not Ixodes GNAQ restored CCHaR responsiveness in HEK293T cells with GNAQ/GNA12 disruptions. Quantitative RT-PCR analysis indicated that all three receptors were expressed in the synganglion. CCHaR/CCHa were found at high levels in the midgut from unfed ticks, and CCHaR expression in the mid-gut was confirmed by RNAScope in situ hybridization. These results establish ligand-receptor identities for three central neuropeptide systems in Ixodes and set the stage for structure-function and physiological investigations.