Abstract: 【Aim】 This study aims to elucidate the molecular characters of chitin synthase (CHS) gene in Apis mellifera and uncover the function of CHS in immune response of A. mellifera worker larvae to Ascosphaera apis stress. 【Methods】 The molecular characters,
conserved motifs and structural domains of the A. mellifera CHS protein were predicted and analyzed using relevant bioinformatics software. Phylogenetic analysis of amino acid sequences of CHS proteins in A. mellifera and other insects was performed with MEGA X software. The dsRNAs of CHS and egfp were synthesized via in vitro transcription method, and fed to the A. apis-stressed 3-day-old larvae of A. mellifera workers to perform RNAi. RT-qPCR was used to determine the expression levels of CHS and immune-related genes abaecin, apidaecin, birc5, defensin-1 and PGRP-S2 in the 5-day-old larval gut of A. mellifera workers in response to A. apis stress. 【Results】 The CHS protein of A. mellifera contains 1 572 amino acids belonging to 20 types of amino acids, among which the numbers of amino acids with positive and negative charge are 177 and 169, respectively. CHS has the molecular weight of 178.77 kD, isoelectric point of 6.65 and cellulose synthase CESA3 catalytic structural domain. Three motifs (Motif 1, Motif 2 and Motif 3) and two structural domains (Chitin_synth_2 and Glyco_trans_2_3) were identified in CHSs in 13 insect species such as A. mellifera and A. cerana. Phylogenetic tree showed that the CHSs in A. mellifera and A. cerana of Hymenoptera showed the highest amino acid sequence identities, and clustered into one branch with the bootstrap value of 100. The expression level of CHS in the 5-day-old larval gut of A. mellifera in the dsCHS-fed group was significantly down-regulated as compared to that in the dsegfp-fed control group, with the interference efficiency of 29.40%. In the dsCHS-fed group, the expression levels of abaecin, birc5 and defensin-1 in the 5-day-old larval gut of A. mellifera were extremely significantly up-regulated, that of PGRP-S2 was significantly up-regulated, while that of apidaecin was extremely significantly down-regulated, as compared with those in the dsegfp-fed control groups. 【Conclusion】 A. mellifera CHS may be an intracellular hydrophilic transmembrane protein. The amino acid sequences of CHSs in A. mellifera and other insects are highly conservative, and those in A. mellifera and A. cerana are the most highly conservative. CHS affects A. mellifera worker larval immune response to A. apis stress.

Key words: Apis mellifera, Ascosphaera apis, RNA interference, chitin synthase, immune response