Abstract: Insect odorant receptors (ORs) play critical roles in the peripheral olfactory system and are involved in such vital life events in insects as feeding, mating and oviposition. With the development of sequencing technologies and bioinformation tools, insect OR genes have been widely identified in recent years. In this article, we comprehensively reviewed the research methods of insect OR genes, and their principles, advantages and disadvantages. Furthermore, we summerized the numbers and proportions of research methods published for identification and functional studies of ORs since 2015. Besides, we listed the research advances of ORs in serious insect pests. A total of 2 543 and 5 111 OR genes had been respectively identified through genome and transcriptome sequencing analyses during 2015 to 2019. Gene expression and protein localization analysis are used to analyze the expression specificities of OR genes in different tissues and developmental stages. The in situ hybridization and immunohistochemistry are used for analyzing cellular and tissue localization, while the semi-quantitative reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) are often used to study the spatiotemporal expression profiles of OR genes. Otherwise, cryo-electron microscopy (Cryo-EM) is employed to elucidate the OR micro-crystal structure and to demonstrate the interactions between critical amino acid residues and ligands. Multiple approaches are developed for functional characterization of ORs. In vitro heterologous expression systems are commonly used to study the function of insect ORs, representing 75.76% of the research methods published from 2015 to 2019. The most abundantly used in vitro systems are through heterologous expression in Xenopus oocytes with two-electrode voltage clamp system (43.94%), followed by transgenic Drosophila with single sensillum recording (SSR) technique and cell line expression systems with calcium imaging. In vivo research methods include RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease Cas9 (CRISPR/Cas9) gene edting technique. The latter has made great breakthroughs in OR deorphanization, and has a great prospective application for further studies. Finally, we suggest the future research directions for insect ORs including, (1) studying the molecular mechanism of olfaction in serious insect pests and natural enemies; (2) elucidating the molecular mechanism of synergism to insect sex pheromones and host plant volatiles; (3) analyzing the micro-crystal structure of ORs and explore the specific recognition mechanisms of ORs and odorants; and (4) developing behavioral regulation products in insects through RNAi technique.

Key words: Odorant receptor, gene identification, expression and localization, two-electrode voltage clamp, single sensillum recording, RNA interference, gene editing technology