思茅松毛虫肠道微生物群落组成和结构动态变化研究

Insect gut microbial communities researches have been making a lot of progress, but the rules of the gut microbial communities dynamics with insect system development process and the composition and structure of gut microbial communities in insect foregut, midgut and hindgut have not been done in-depth research yet. There are very few systematic and comprehensive studies on the relationship among diet, gut environment and microbial communities, on the mechanism about core bacterial microbiota how to establish the gut microbial communities, and on the robustness of gut microbial communities. Dendrolimu kikuchii is a representative defoliator of coniferous trees. In our early study, the Dendrolimu kikuchii gut aerobic or facultative aerobic bacteria were isolated and cultured, but the dynamic variation rule of gut microbial communities still failed to be revealed. In this project, by using Dendrolimu kikuchii as the research material, and by following the multi-disciplinary theories such as entomology, microbiology, ecology, metagenomics and bioinformatics, and by applying the technologies and methods such as anaerobic culture, 16S rDNA amplicons library building, the next generation sequencing, polyphasic taxonomy, database mining, analysis of species and community and insect artificial breeding, the dynamic changes of the foregut, midgut and hindgut microbial community composition and structure in different instars and different feedings will be studied, the core microbiota impact on community establishment and community robustness will also be deliberated. The results will provide the scientific basis for the succession of gut microbial community under the combined influences of insect phylogeny, insect gut environment and diet, and provide the scientific reference for the new pest management strategy.

昆虫肠道微生物研究成果颇丰。但是，昆虫前中后肠肠道微生物群落组成和结构、昆虫系统发育过程中肠道微生物群落动态变化规律未做深入研究；食物、肠道和肠道微生物的关系、核心类群在建群中作用机制以及肠道微生物群落的鲁棒性等研究不系统和全面。我们前期对思茅松毛虫可培养（兼性）好氧微生物进行了研究，但是，没能揭示其肠道微生物群落组成、结构和动态变化规律。本课题以思茅松毛虫为对象，交叉昆虫学、微生物学、生态学、宏基因组学和生物信息学的理论，利用微生物培养、16S rDNA扩增子库、高通量测序、多相分类、数据库挖掘、群落分析和昆虫人工饲养等技术和手段，研究思茅松毛虫不同龄期、不同食性和前中后肠肠道微生物群落组成、结构和稳定性的动态变化，揭示昆虫肠道环境在昆虫系统发育和食物作用下，肠道微生物群落的演替规律，揭示肠道微生物群落建群机制和鲁棒性，这将丰富昆虫肠道微生物群落学的研究，也为害虫管理提供科学依据。

通过对思茅松毛虫肠道细菌和真菌的分离培养和鉴定，细菌16S rRNA扩增子、16S rRNA基因和真菌ITS序列测序研究发现：思茅松毛虫肠道细菌类群丰富，但真菌类群较少，前中后肠微生物群落组成和结构类似，克雷伯氏菌属和肠球菌属是思茅松毛虫肠道微生物核心类群，存在于所有龄期的前中后肠；思茅松毛虫肠道微生物的群落实在3龄滞育后建立起来的，表现出1-3龄肠道微生物群落组成类型少，结构简单但相似，4-7龄肠道微生物群落组成类型丰富，结构复杂但相似；通过对思茅松转录组和人工饲喂研究发现：思茅松的进化分布和产树脂基因表达与思茅松毛虫食性相关，食物类型与思茅松毛虫肠道微生物群落组成和结构相关，在所有龄期和肠道部位都分离到枯草芽孢杆菌（Bacillus subtilis），该菌是否与思茅松树脂代谢相关需要进一步研究；通过思茅松毛虫饲喂抗生素后对思茅松毛虫肠道细菌16s rRNA扩增子库和思茅松毛虫生长发育的分析发现：思茅松毛虫肠道细菌群落有极强的鲁棒性，对今后“除共生”策略防治思茅松毛虫较为不利。