关键词: 中华按蚊, 化学感受蛋白, 拟除虫菊酯, 丙烯菊酯, CRISPR/Cas9； 空间驱避

Abstract:  【Aim】 To clone the chemosensory protein 3 gene AsCSP3 of Anopheles sinensis, and analyze its function in allethrin repellency. 【Methods】 The full-length cDNA sequence of AsCSP3 was cloned using RT-PCR. The expression levels of AsCSP3 at different developmental stages of pyrethroid-resistant and -susceptible A. sinensis, including 4th instar larvae, early female pupae, late female pupae, newly emerged female adults, and female adults on the 1st, 3rd, 6th, and 9th day after emergence, in different tissues such as the antennae, head (with antennae removed), thorax (with legs removed), abdomen, and legs of the 3-day-old female adults of pyrethroid-resistant and -susceptible A. sinensis, and in the 3-day-old female adults of pyrethroid-resistant A. sinensis at different time points after fumigation with 0.05% permethrin were detected using qRT-PCR. The binding affinities of the recombinant AsCSP3 protein to allethrin, deltamethrin, and permethrin were assessed using a fluorescence competitive binding assay. Moreover, an AsCSP3 mutant was generated using the CRISPR/Cas9-mediated knock-in method, and the repellent effects of allethrin and deltamethrin on the AsCSP3 mutants were compared with those on the wild-type A. sinensis. 【Results】 The cloned full-length cDNA sequence of AsCSP3 of A. sinensis encodes 183 amino acids, which contains conserved cysteines of chemosensory proteins. AsCSP3 was highly expressed in the late female pupae and newly emerged female aduls. AsCSP3 was primarily expressed in the antennae and legs of the 3-day-old female adult. In the 3-day-old female adult of pyrethroid-resistant strain of A. sinensis, the expression level of AsCSP3 in the legs was significantly lower than that in the pyrethriod-susceptible strain of A. sinensis， while its expression level in the antennae and head (with antennae removed) was notably higher than that in the pyrethroid-susceptible strain of A. sinensis. After induction with allethrin, the expression level of AsCSP3 significantly decreased from 2 h and remained significantly lower than that of the untreated control group up to 24 h. The recombinant AsCSP3 protein could bind to both deltamethrin and allethrin, but could not bind to permethrin. Mutation of the AsCSP3 neither affected the growth, development, fecundity, or blood-feeding behavior of A. sinensis, nor did it affect the contact repellency of deltamethrin, however, weakened the spatial repellency of allethrin, resulting in 1.77-fold higher number of take-off and 1.97-fold longer flying time within 3 min in wild-type A. sinensis than those in AsCSP3 mutants, respectively. 【Conclusion】 There is a potential association between AsCSP3 and the spatial repellency of allethrin.

Key words: Anopheles sinensis, chemosensory protein, pyrethroids, allethrin, CRISPR/Cas9, spatial repellency