Creation of a Technology Platform for Fighting Mosquito Bites

Objective: Octopamine receptor (OctR) based deterrents are a potentially better way of preventing mosquito bites, because the mechanism of action is not toxicity which encourages resistance. The OctR belongs to a class of primitive heterotrimeric G protein-coupled receptors (GCPR) found only in invertebrates and plays an important role in biting-related behaviors. This study focuses on developing a testing system that will accelerate the discovery of ecofriendly deterrents free of the perils of current repellent technologies. Materials and Methods: The Anopheles gambiae mosquito which is the vector for malaria was recently reported to have two splice variants of an OctR gene named AgAOctR and AgBOctR that have N-terminal halves in common. The full length AgBOctR sequence was obtained by removing the C-terminal half of the AgAOctR with restriction enzymes and ligating its remaining N-terminal region with the unique half of the synthesized AgBOctR sequence. The AgBOctR gene was then functionally expressed in mammalian cells via calcium phosphate-mediated transfection. Radioligand binding was then used to detect the functionally expressed proteins in transfected cells and stable clones were selected. Fluorescent detection of changes in intracellular calcium was used as a measure of activation of the Gq-PLC-IP3-Ca2+-mediated signaling pathway. Results: Differential restriction digest followed by size determination via agarose gel electrophoresis of the newly created AgBOctR sequence was used to verify its length and composition. When expressed in mammalian cells the AgBOctR gene was found to code for a protein that specifically binds an octopamine receptor radioligand, while no specific radioligand binding was detected in untransfected cells. Further these AgBOctR-expressing cells were activated by octopamine with a higher potency than tyramine indicating that the gene truly encodes for an octopamine receptor rather than a closely related tyramine receptor. That OctR signaling could be readily detected via monitoring of the Gq-PLC-IP3-Ca2+ signaling pathway suggests that AgBOctR is specifically an alpha-like OctR, because beta-like OctRs utilize a different signaling pathway. Conclusions: A technology system has been created that will allow us to correlate the mosquito OctR activity of compounds with their ability to prevent mosquito biting. This will accelerate the discovery of innovative mosquito deterrents that prevent infectious disease transmission.