Abstract: 【Objective】 The objectives of this study are to examine microbial communities from the digestive tract of Bombus lantschouensis reared in the laboratory, and to analyze the specific bacteria spatio-temporally in different developmental stages of B. lantschouensis, which is the important foundation and basis for further studying the function of symbiotic bacteria in bumblebee. 【Method】The fragment of the bacterial 16S rDNA gene was PCR-amplified with the universal eubacterial primers 774F and 1391R and a 16S rDNA clone library of gut bacteria was constructed. Each single clone was picked and sequenced. The sequences were checked for chimeras, sequences of chimeric origin were removed from further analysis. Sequences obtained were analyzed by BLASTn and matched with valid reference sequences in the NCBI (National Center for Biotechnology Information) to determine the bacterial species types. The 16S rDNA gene primers of the specific bacteria Gilliamella apicola and Snodgrassella alvi were designed based on sequencing of bacterial taxonomic groups obtained from the clone library, then constructed recombinant plasmid using the specific bacteria primers and diluted the plasmid to 10 folds series for 5 concentration gradients after concentration determination. Total genomic DNA was extracted from egg, larva, pupa, and 0, 5th, 10th, 15th, 20th days worker bees. The bumblebee β-actin gene was selected as the reference gene, the quantitative real-time PCR (qPCR) was used to explore the quantity variation of symbionts during the different developmental stages of B. lantschouensis.【Result】A total of 213 clones were picked and sequenced, after Chimeras checking, 202 sequences were obtained and then divided into 16 OTUs. The sequence similarity with GenBank sequences was between 93%-99%. The phylogenetic analysis found that the sequences fell into 6 major lineages within the domain bacteria. Among them, 45% and 30% were respectively G. apicola and S. alvi, 10% was Bifidobacterium, 5% was Fructobacillus fructose, 2% was Lactobacillus, 2% was Flavobacterium aciduliphilum, and 6% was mixture of uncommon bacteria types. The qPCR result showed that G. apicola and S. alvi were detected in all development stages, the two bacterial species were similar in the quantity variation patterns with decreasing after increasing, finally reached the steady state. The quantity of G. apicola and S. alvi in egg, larva and pupa was very low, in 5th day significantly higher than those in other development stages, and then gradually reduced, stabilized after 15th day, the bacteria quantity in 20th day showed no difference with 15th day.【Conclusion】Four common symbiotic bacteria including G. apicola, S. alvi, F. fructose and Bifidobacterium were identified in the digestive tract of B. lantschouensis reared in laboratory. G. apicola and S. alvi are dominant bacterium among them. G. apicola and S. alvi both have horizontal and vertical transmission. The quantity of G. apicola and S. alvi in egg, larva and pupa was very low, and increased rapidly after worker bees emergence, stabilized after 15 days. The temporal variation of two dominant bacteria in B. lantschouensis may be related to the function of symbiotic bacteria in bumblebee.

Key words: bumblebee , symbiotic bacteria , gut microbiota , qPCR