Analysis of the bacterial diversity in intestines of Hepialus gonggaensis larae

Abstract:

[Objective] We investigated the intestinal microbial diversity in the larval gut of Hepialus gonggaensis, an economically important insect. [Methods] We used morphological, physiological, chemotaxonomic characteristics and 16S rRNA analysis method, and the molecular method of PCR-DGGE (denaturing gradient gel electrophoresis) analysis based on the sequence of 16S rRNA V3 region gene. [Results] By the traditional isolation method, 8 genera of bacteria were identified from 11 isolated bacterial populations. The dominant bacteria in intestine belonged to enterobacter. By 16S rRNA V3 region gene DGGE method, eleven distinct bands were obtained from 16S rDNA amplificons. The bands were purified, sequenced. The sequences aligned with GenBank database and showed that they were belonged to 8 different genera of bacteria. Phylogenetic analysis showed that the sequences of bacteria belonged to the Proteobacteria and Firmicutes. The most dominant bacteria group was Carnobacterium in the gut and Bacillus followed by it. The different patterns were observed in different instars larvae guts from DGGE profiles, which might be related to their physiological development stages. [Conclusion] 8 genera were obtained from intestine of H. gonggaensis by traditional culturing method and 16S rDNA analysis method respectively, but the two groups were not exactly same, and the dominant group was different also. This suggested that a combination of molecular and traditional culturing methods can be used to analyze and monitor the diversity of intestinal microflora effectively, and that will give us more information of microorganism diversity.

Key words:Hepialus gonggaensis;16S rRNA; normal isolation culture; denaturing gradient gel electrophoresis (DGGE); specie diversity