Identification of putative muscarinic acetylcholine receptor genes in Bactrocera dorsalis and functional analysis of Bdor-mAChR-B.

Muscarinic acetylcholine receptors (mAChRs) play important roles in the insect nervous system. These receptors are G protein-coupled receptors, which are potential targets for insecticide development. While the investigation of pharmacological properties of insect mAChRs is growing, the physiological roles of the receptor subtype remain largely indeterminate. Here, we identified three mAChR genes in an important agricultural pest Bactrocera dorsalis. Phylogenetic analysis defined these genes as mAChR-A, -B, and -C. Transcripts of the three mAChRs are most prevalent in 1-d-old larvae and are more abundant in the brain than other body parts in adults. Functional assay of Bdor-mAChR-B transiently expressed in Chinese hamster ovary cells showed that it was activated by acetylcholine (EC, 205.11 nM) and the mAChR agonist oxotremorine M (EC, 2.39 μM) in a dose-dependent manner. Using the CRISPR/Cas9 technique, we successfully obtained a Bdor-mAChR-B knockout strain based on wild-type (WT) strain. When compared with WT, the hatching and eclosion rate of Bdor-mAChR-B mutants are significantly lower. Moreover, the crawl speed of Bdor-mAChR-B knockout larvae was lower than that of WT, while climbing performance was enhanced in the mutant adults. Adults with loss of function of Bdor-mAChR-B showed declined copulation rates and egg numbers (by mated females). Our results indicate that Bdor-mAChR-B plays a key role in the development, locomotion, and mating behavior of B. dorsalis.