Methodological optimization for obtaining insect odorant receptor interactomes

Abstract

Insect behavior affects ecosystems and human welfare, as seen in the recent cases of declining honeybee colonies and the Zika outbreak. As olfaction is the major sensory input regulating insect behavior, it is important to understand insect olfaction. Intriguingly, insects use a unique family of odorant receptors (ORs) distinct from the well-studied vertebrate or nematode counterparts, making it difficult to comprehend the fundamentals. Especially for protein？protein interactions, available information is extremely scarce mostly due to methodological hurdles. To address this issue, a platform for discovering novel insect OR-interacting proteins is devised in the model insect, Drosophila melanogaster. The platform is essentiallycomposed of improved tissue manipulation methods incorporated in immunoprecipitation-mass spectrometry (IP-MS) technique. Using Orco on behalf of the insect OR family, detailed protocols including adult olfactory tissue enrichment, sample preparation for immunoblotting, IP for effective IP-MS, etcetera have been optimized. Subsequent IP-MS followed by data analyses and electrophysiological recordings has validated the Orco interactome. In conclusion, the platform has generated a meaningful Orco interactome and therefore is anticipated to successfully aid future studies on insect ORs.