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BACKGROUND: Anopheles sinensis is the most widely distributed mosquito species and is the main transmitter of Plasmodium vivax malaria in China. Most previous research has focused on the mechanistic understanding of biological processes in An. sinensis and novel ways of interrupting malaria transmission. However, the development of functional genomics and genetics-based vector control strategies against An. sinensis remain limited because of insufficient site-specific genome editing tools.RESULTS: We report the first successful application of the CRISPR/Cas9 mediated knock-in for highly efficient, site-specific mutagenesis in An. sinensis. The EGFP marker gene driven by the 3 × P3 promoter was precisely integrated into the odorant receptor co-receptor (Orco) by direct injections of Cas9 protein, double-stranded DNA donor, and Orco-gRNA. We achieved a mutation rate of 3.77%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed by polymerase chain reaction (PCR) amplification and sequencing. The Orco mutation severely impaired mosquito sensitivity to some odors and their ability to locate and discriminate a human host.CONCLUSION: Orco was confirmed as a key mediator of multiple olfactory-driven behaviors in the An. sinensis life cycle, highlighting the importance of Orco as a key molecular target for malaria control. The results also demonstrated that CRISPR/Cas9 was a simple and highly efficient genome editing technique for An. sinensis and could be used to develop genetic control tools for this vector.

Background The olfactory system plays a crucial role in regulating insect behaviors. The detection of odorants is mainly mediated by various odorant receptors (ORs) that are expressed in the dendrites of olfactory neurons of chemosensilla. Anophelessinensis is a major malaria vector in Eastern Asia and its genome has recently been successfully sequenced and annotated. In this study, we present genome-wide identification and expression profiling of OR genes in different chemosensory tissues of An.sinensis. Methods The OR genes were identified using the available genome sequences of An.sinensis. A series of bioinformatics analyses were conducted to investigate the structure, genome distribution, selective pressure and phylogenetic relationships of OR genes, the conserved domains and specific functional sites in the OR amino acid sequences. The expression levels of OR genes were analyzed from transcriptomic data from An.sinensis antennae, proboscis and maxillary palps of both sexes. Results A total of 59 putative OR genes have been identified and characterized in An.sinensis. This number is significantly less than that in An.gambiae. Whether this difference is caused by the contraction or expansion of OR genes after divergence of the two species remains unknown. The RNA-seq analysis showed that AsORs have obvious tissue- and sex-specific expression patterns. Most AsORs are highly expressed in the antennae and the expression pattern and number of AsORs expressed in antennae are similar in males and females. However, the relative levels of AsOR transcripts are much higher in female antennae than in male antennae, which indicates that the odor sensitivity is likely to be increased in female mosquitoes. Based on the expression patterns and previous studies, we have speculated on the functions of some OR genes but this needs to be validated by further behavioral, molecular and electrophysiological studies. Further studies are necessary to compare the olfactory-driven behaviors and identify receptors that respond strongly to components of human odors that may act in the process of human recognition. Conclusions This is the first genome-wide analysis of the entire repertoire of OR genes in An.sinensis. Characterized features and profiled expression patterns of ORs suggest their involvement in the odorous reception of this species. Our findings provide a basis for further research on the functions of OR genes and additional genetic and behavioral targets for more sustainable management of An.sinensis in the future. Graphical Abstract

Female adult anautogenous mosquitoes rely heavily on diverse chemical stimuli to locate and discriminate hosts for feeding through their olfactory system. Odorant binding proteins are believed to be the first proteins to interact with semiochemicals in the olfactory sensory system, but their functions in host seeking have not been extensively explored. Here, we used transposon-based transgenesis and CRISPR/Cas9-based genome editing to localize and functionally characterize the odorant binding protein AsOBP1 in the malaria mosquito Anopheles sinensis and uncovered its roles in host-seeking behavior of female mosquitoes. AsOBP1 is specifically expressed in the cuticle and sensilla trichodea of antennae of adult mosquitoes. Although AsOBP1−/− mutant females alighted on and probed blood hosts, the AsOBP1 mutation severely impaired host-seeking behavior. The in vitro competition assay identified nine of 83 human odorants, among which 1-tetradecanol and heptanal enhanced the attraction of mice and human hands to An. sinensis mosquitoes. The olfactory sensitivity of AsOBP1 mutants to these two compounds was reduced. Our study reveals that AsOBP1 is required for the host seeking of An. sinensis through detecting human odor, such as 1-tetradecanol and heptanal, and provides a potential target for malaria control.