【目的】了解仁扇舟蛾(Clostera restitura)肠道共生细菌的主要类群及其群落结构特征,明确仁扇舟蛾幼虫前、中和后肠可培养细菌的种类及群落结构组成,为进一步研究特异功能细菌的生理生化特征和功能提供基础。【方法】选取仁扇舟蛾5龄幼虫进行肠道解剖,分离前、中和后肠不同肠段,研磨稀释10^-1~10^-6,分别涂布于NA和LB固体培养基上,每个处理重复3次,28 ℃培养72 h。后对前、中和后肠可培养细菌进行分离纯化培养,提取细菌基因组DNA,利用 16S rDNA 基因序列分析和PCR扩增技术,获得细菌基因组序列,后与NCBI GenBank数据库进行Blast同源性比对,参考序列相似度98%以上近缘序列的物种信息,结合菌落形态特征和生理生化特性鉴定细菌种类,利用MEGA 7.0软件,采用邻接法(Neighbor-Joining)构建系统进化树。【结果】从仁扇舟蛾5龄幼虫的前、中和后肠中共分离获得22株可培养细菌,分属于变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和放线菌门(Actinobacteria),共13个属,22个种。其中:假单胞菌属(Pseudomonas)4株,罗尔斯通菌属(Ralstonia)3株,芽孢杆菌属(Bacillus)3株,嗜麦芽寡养单胞菌属(Stenotrophomonas)2株,气球菌属(Aerococcus)2株,其余微小杆菌属(Exiguobacterium)、微球菌属(Micrococcus)、短小杆菌属(Curtobacterium)、伯克氏菌属(Burkholderia)、类芽孢杆菌属(Paenibacillus)、节杆菌属(Arthrobacter)、短状杆菌属(Brachybacterium)和两面神菌属(Janibacter)各1株。细菌的种类存在差异,前、中和后肠不同肠段的细菌种类分别为5、10和7种。3个肠段共有的细菌种类是皮氏罗尔斯菌(Ralstonia pickettii)和斯氏假单胞菌(Pseudomonas stutzeri)。【结论】仁扇舟蛾5龄幼虫肠道的可培养细菌种类较为丰富,其中以变形菌门和厚壁菌门为优势菌群。不同肠段中可培养细菌的多样性和群落结构存在差异,其中以中肠可培养细菌种类较为丰富。此次从仁扇舟蛾肠道中分离到与单宁降解相关的细菌,分析表明仁扇舟蛾肠道菌群与宿主的取食习性有关,肠道细菌可能在克服植物次生代谢物质的化学防御上发挥作用。

【Objective】This study aimed to analyze the diversity and community structure of culturable bacteria in the foregut, midgut and hindgut of 5th instar larvae of Clostera restitura. It also lays a foundation for studying the physiological and biochemical characteristics and functions of specific functional bacteria.【Method】Sterile dissections of the intestinal tract of the 5th instar larvae,C. restitura, were performed to separate the foregut, midgut and hindgut, and homogenized by hand. The gut homogenates were poured on nutrient agar (NA) and lysogeny broth (LB) media in triplicate after serial dilution (10^-1 to 10^-6) and incubated at 28 ℃ for 72 h. Single colonies were picked and repeatedly streaked on LB medium to obtain pure cultures. The bacterial solutions of each colony were processed for DNA extraction, and bacterial gene sequences were obtained using 16S rDNA gene sequence analysis and PCR amplification technology. The sequence match of each 16S rRNA gene sequence was assigned using NCBI BLAST and refers to the species information of adjacent sequences with sequence similarity above 98%. At the same time, morphological observation and physiological and biochemical characteristics of bacterial colonies were also employed for identification of bacterial species. Finally, the phylogenetic tree was constructed by neighbor-joining (NJ) with MEGA 7.0.【Result】Twenty-two cultivable bacteria were isolated from the intestinal tract of the 5th instar larvae of C. restitura, which were grouped into three phyla: Proteobacteria, Firmicutes and Actinobacteria, belonging to 13 genera and 22 species. Among them, there were four strains of Pseudomonas, three strains of Ralstonia, three strains of Bacillus, two strains of Stenotrophomonas, and two strains of Aerococcus; the remaining strains were Exiguobacterium,Micrococcus,Curtobacterium,Burkholderia,Paenibacillus,Arthrobacter,Brachybacterium and Janibacter. There were differences in the bacterial species in the different intestinal segments. The species of bacteria in the foregut, midgut and hindgut were 5, 10 and 7, respectively. The common bacteria were Ralstonia pickettii and Pseudomonas stutzeri in different intestinal segments.【Conclusion】The results indicate that the species of cultivable bacteria in the intestinal tract of the 5th instar larvae of C. restitura are relatively rich, mainly colonized by bacteria in the phyla Proteobacteria and Firmicutes, and there are differences in the diversity and community structure of cultivable bacteria among different intestinal segments. Among them, the midgut bacterial species were relatively rich. In this study, the bacteria related to tannin degradation were isolated from the C. restitura gut, indicating that the gut bacterial community of C. restitura is related to the diet of the host, and the intestinal bacteria may play a role in overcoming the chemical defense of plant secondary metabolites.