Abstract: 【Aim】 Heat shock proteins (Hsps) are a kind of anti-stress proteins that are rapidly produced when organisms are under adverse environmental conditions, which can help them survive in adverse environments. This study aims to explore the mechanism of Hsp90AB in the growth and development of Anopheles sinensis under insecticide stress and in the deltamethrin resistance, laying a foundation for further study of the mechanism of Hsp90AB and the prevention and control of An. sinensis. 【Methods】 Based on the transcriptome and genome data of An. sinensis, the full-length cDNA sequence of AsHsp90AB was cloned by using RT-PCR and analyzed by bioinformatics. The expression profiles of AsHsp90AB in both deltamethrin-susceptible strain (WX-LS) and deltamethrin-resistant strain (WX-LR) at different developmental stages (egg, 1st-4th instar larvae, female pupa, male pupa, female adult and male adult), and in different female adult tissues (salivary gland, midgut, Malpighian tubules, fat body, integument and ovary) of An. sinensis were detected by RT-qPCR. The 4th instar larva and 3-day-old female adult of WX-LS of An. sinensis were stressed with the median lethal concentration (LC₅₀)(0.0016 mg/L) of deltamethrin using the larval immersion method (baseline susceptibility assay) and the WHO contact bioassay method (0.05% deltamethrin-coated bottles), respectively, and the expression level of AsHsp90AB was detected by RT-qPCR. After the AsHsp90AB silence by RNAi with microinjection method in WX-LR female pupa, the expression level of AsHsp90AB was detected by RT-qPCR. The median knockdown time (KT₅₀), knockdown rate and survival rate of female adult treated with deltamethrin by 0.05% deltamethrin-coated bottles via the WHO contact bioassay method at 48 after RNAi were observed and calculated. 【Results】 The full-length cDNA sequence 2 678 bp of AsHsp90AB (GenBank accession no.: PP405608) in An. sinensis was cloned, with an open reading frame of 2 154 bp in length encoding 718 amino acids. The predicted theoretical molecular weight and isoelectric point of AsHsp90AB were 81.65 kD and 4.94, respectively. AsHsp90AB has the characteristic motif of the Hsp90 family and contains the MEEVD motif as a cytoplasmic type. Phylogenetic analysis showed that AsHsp90AB of An. sinensis clustered together with the Hsp90s from Anopheles and then with the Hsp90s from Culex, indicating that they were closely related. AsHsp90AB was expressed during the whole developmental stage in both WX-LS and WX-LR, and was highly expressed in adult males and the ovaries of adult females of WX-LS. AsHsp90AB exhibited high expression in the 4th instar larva and the highest expression level in female adult antennae of WX-LR. The expression levels of AsHsp90AB in the 4th instar larva and 3-day-old female adult of WX-LS of An. sinensis after exposure to deltamethrin stress were upregulated by 1.37-fold-2.61-fold and 1.91-fold-2.58-fold, respectively, while that in the knocked down female adult was downregulated by 72.51%. The expression of AsHsp90AB in female pupa of WX-LR of An. sinensis was inhibited by RNAi and its expression level significantly decreased at 24, 48 and 72 h after RNAi by 44.00%, 61.09% and 55.95%, respectively, as compared with that in the dsEGFP control group. The female adult of WX-LR of An. sinensis injected with dsAsHsp90AB began to be knocked down at 10 min after 0.05% deltamethrin-coated bottle treatment, while that in the dsEGFP control group began to be knocked down at 30 min after 0.05% deltamethrin-coated bottle treatment. The knockout rate gradually increased during 1 h of 0.05% deltamethrin-coated bottle treatment, and the knockout rates of the two groups were significantly different. The KT₅₀ value of female adult in the dsEGFP control group was (50.000±1.667) min and that in the dsAsHsp90AB treatment group was (30.000±0.600) min. The survival rate of female adult of WX-LR of An. sinensis after 0.05% deltamethrin-coated bottle treatment and transferred to the recovery bucket for 24 h after recovery was significantly increased by 24.5% as compared with that in the dsEGFP control group. 【Conclusion】 AsHsp90AB was highly expressed in WX-LR of An. sinensis, and the mortality of An. sinensis under the application of deltamethrin was increased when AsHsp90AB was silenced. This study provides a valuable reference for the potential function of AsHsp90AB in the growth and development as well as the deltamethrin resistance of An. sinensis.

Key words:  Anopheles sinensis, heat shock protein gene, deltamethrin, insecticide stress, insecticide resistance, RNAi