The role of kinin neuropeptides in tick midgut: A fluorescently labeled kinin maps the cognate receptor to muscles and triggers peristalsis

As obligatory blood-feeding arthropods, ticks transmit multiple pathogens causative of diseases to a wide range of hosts. Understanding the digestion process and its regulation in ticks is crucial as both influence pathogen transmission and tick reproductive success. Previously, for the cattle fever tick Rhipicephalus microplus we demonstrated the activity of tick kinins on their cognate recombinant G protein-coupled receptor. Here, we determined the physiological role of the kinin receptor in the tick midgut using a fluorophore-labeled kinin. A tetramethylrodamine-labeled endogenous kinin 8 peptide sequence, (TMR-RK8), identical in Rhipicephalus sanguineus and R. microplus, activated the R. microplus kinin receptor expressed in CHO-K1 cells in a dose-dependent manner. In addition, it labeled the recombinant receptor in these cells as shown by confocal microscopy, while a labeled peptide with the scrambled-sequence of kinin 8 (TMR-Scrambled) did not. Females of R. sanguineus were then dissected to isolate intact midguts for functional experiments. Confocal analysis after applying TMR-RK8 to midguts ex-vivo unequivocally revealed the kinin receptor in circular and longitudinal muscles. This labeling was not observed in midguts treated with the TMR-Scrambled control. Critically, TMR-RK8 retained activity when applied to the midgut, inducing significant peristalsis. The TMR-Scrambled peptide did not elicit peristalsis in the midgut. These findings underscore the importance of the kinin receptor in midgut physiology as the myotropic function of kinins in eliciting tick midgut peristalsis was established. In addition, as GPCRs are poorly immunogenic, our innovation in applying a fluorescent ligand for receptor localization instead of antibodies further advances tick endocrinology.