Abstract: 【Aim】 Toll signaling pathway is an important component of insect innate immunity. Toll receptors play a key role in activating the immune response to pathogen infection. This study aims to explore the function of Toll receptor genes in resistance to microbial infection and maintaining the homeostasis of gut microbiota in Anopheles stephensi. 【Methods】 The Toll receptor genes in An. stephensi were identified by BLAST search using the published Toll sequences in Anopheles gambiae. The relative expression levels of Toll receptor genes in the fat body of healthy adults of An. stephensi and the adults infected with Beauveria bassiana and Erwinia carotovora subsp. carotovora (Ecc) for different time were detected by real-time quantitative PCR. After the double-stranded RNA of AsToll1A or AsToll5A was microinjected into the thorax of female adults of An. stephensi, the survival rates of the RNAi-treated mosquitoes after fungal infection and the total gut bacterial load and the expression levels of antimicrobial peptide genes in the RNAi-treated mosquitoes were detected. 【Results】 Eight Toll receptor genes, i.e., AsToll1A, AsToll5A, AsToll6, AsToll7, AsToll8, AsToll9, AsToll10 and AsToll11, were identified in An. stephensi. The real-time quantitative PCR analysis showed that AsToll5A had the highest expression level in the fat body of healthy female adults of the mosquito, followed by AsToll1A, and the expression levels of other Toll receptor genes in the fat body were extremely low. Compared to the control (injecting PBS), B. bassiana or Ecc infection significantly induced the transcription of AsToll1A and AsToll5A, whereas the expression levels of other Toll receptor genes during pathogen infection showed no significant change. Silencing AsToll1A or AsToll5A significantly reduced the resistance of An. stephensi adults to B. bassiana infection, and extremely significantly increased the total gut bacterial load in the adult midgut as compared to the dsGFP-treated control. Moreover, silencing AsToll1A remarkably inhibited the expression of antimicrobial peptide genes DEF1 and GAM1, while only the expression of GAM1 was down-regulated in the dsAsToll5A-injected mosquitoes. 【Conclusion】 The Toll receptors in An. stephensi are conserved in structure and function. AsToll1A and AsToll5A are highly induced in response to both fungal and Gram-negative bacterial infections, and also regulate the homeostasis of gut microbiota in An. stephensi.

Key words: Anopheles stephensi; Toll receptor, Beauveria bassiana, Gram-negative bacteria, RNA interference, gut bacteria