Abstract:

【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.

Key words: Clostera restitura, intestinal bacteria, cultivable bacteria, 16S rDNA gene sequence, community structure