关键词: 大分舌蜂, 肠道细菌, 细菌多样性, 幼虫, 滞育预蛹, 发育, 高通量测序

Abstract: 【Aim】 Based on the important role of insect gut microorganisms in host health, growth and development, this study aims to preliminarily explore the diversity and differences of gut bacterial communities in different instar larvae and diapause prepupae of Colleles gigas. 【Methods】 The V3-V4 gene fragment of 16S rRNA was amplified by PCR using the bacterial DNA extracted from the gut contents of the 1st-5th instar larvae and diapause prepupae of C. gigas collected from the field, and was sequenced by IlluminaMiseq second-generation high-throughput sequencing technology. Based on the obtained sequence data, the composition, abundance and diversity of gut bacteria of larvae and diapause prepupae of C. gigas were analyzed by bioinformatics methods. 【Results】 Bacteria of 15 phyla, 23 classes, 43 orders, 80 families and 128 genera were detected in the gut bacterial communities of C. gigas larvae, of which the main phylum, order, family and genera were Proteobacteria (accounting for 93.74%), Rickettsiales (accounting for 68.68%), Anaplasmataceae (accounting for 68.64%), and Wolbachia (accounting for 68.64%), respectively. Beta diversity analysis showed that the gut bacterial community changed with the development of larvae, and could be divided into three groups: early instar group (1st-3rd instar larva), late instar group (4th-5th instar larva) and diapause prepupa group. Alpha diversity analysis showed that there was a significant difference in the gut bacterial diversity between the diapause prepupa group and the late instar group and early instar group, while the gut bacterial diversity between the late instar group and the early instar group showed no significant difference. The linear discriminant analysis results showed that there was significantly dominant class in both diapause prepupa group and early instar group, being Alphaproteobacteria and Gammaproteobacteria, respectively, but no dominant class existed in the late instar group. At the order level, Enterobacteriales and Pseudomonadales were dominant in the early instar group, and Rickettsiales was dominant in the diapause prepupa group. At the family level, Enterobacteriaceae and Moraxellaceae were dominant in the early instar group, and Anaplasmataceae was dominant in the diapause prepupa group. At the genus level, Enterobacter and Acinetobacter were dominant in the early instar group, and Wolbachia was dominant in the diapause prepupa group. The results of functional gene annotation also showed the characteristics of the three groups. 【Conclusion】 There are significant differences in the community structure of gut bacteria in different instar larvae and diapause prepupae of C. gigas. The bacterial diversity decreases gradually from the early instar group to the late instar group and then to the diapause prepupa group, and this may be related to the feeding characteristics and adaptation of gut microorganisms to the gut environment. This study lays a foundation for the study of gut microorganisms of soil-nesting wild bees and also provides a new angle and direction for the protection of this kind of wild bees.

Key words: Colleles gigas, gut bacteria, bacterial diversity, larva, diapause prepupa, development, high-throughput sequencing