Study On Diversity Of Gut Flora Of Apriona Germari (Hope) (Coleoptera: Cerambycidae) Larvae, By Molecular Methods

Apriona germari (Hope) is an important pest of many kinds of trees, and the destructive pests distribute almost all over the world. Researches have showed that intestinal bacteria have very close relationship to the well-being of the insect host in many aspects, e.g. nutrient and metabolic activities, and especially immune functions. A substantial part of intestinal bacteria of this beetle are still to be thoroughly described for the reason that less than 1% of the microbes in natural environments could be successfully isolated by culture-dependent techniques under laboratory conditions. Fortunately, in the last decades, several 16S rDNA-based molecular methods have been developed for bacteria identification.The gut flora of A. germari larvae was investigated by three 16S rDNA-based molecular approaches to provide novel insights into the uncultured microbes in guts and help attempt to control the pest. These methods were denaturing gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (RFLP) analysis of traditional 16S rDNA clone library and directly randomly sequencing of normalization 16S rDNA clone library. DGGE analysis inferred 22 different bacteria from 15 genera by sequencing principal bands and an uncultured Proteobacterium and a Klebsiella appeared in every sample. Band E and L identified as Bacillus sp. and an uncultured Klebsiella sp. were the strongest bands. DGGE band patterns showed that gut flora in summer was more complex than in winter generally. A traditional 16S rDNA clone library and a normalization bacteria 16S rDNA clone library were established. Restriction fragment length polymorphism (RFLP) analysis of traditional clone library clustered tested 175 clones into 48 different groups, and the available sequences representing each RFLP-type were closely related to 48 different bacteria in 22 genera. The predominant bacteria were species in genera Klebsiella, Lactococcus and Entorococcus, which accounted for 24.6%, 19.4% and 17.1% of the screened clones. RFLP had higher solution than DGGE. Directly randomly sequencing of 82 clones from normalization library resulted in 79 different bacteria from 32 genera 17 families of and 7 phyla or sub-phyla. Among them, eight genera of Acetobacter, Arcobacter, Brevundimonas, Hydrogenophaga, Kluyvera, Pantoea, Grimontella and Nesterenkonia were not covered neither by direct DGGE nor RFLP techniques. Additionally, the two clone libraries provided much more bacterial diversity information than DGGE analysis. Phylogenetic analysis of all the obtained sequences was performed using the CLUSTAL W, and PHYLIP (version 3.67) using neighbor-joining algorithms. From the tree, it showed that some of the gut flora in this study had less relationship with other sequences in evolutionary development history. In conclusion, the results of all three molecular methods revealed an unexpected largely diversity of microbes associated with the gut and in the intestinal contents of A. germari larvae. More than 140 bacteria species from 9 phyla or sub-phyla, 22 families and 44 genera were detected via molecular techniques in present study.It proved that direct DNA extraction and amplification of the 16S rDNA has the advantage of huge database entries for bacterial assemblies over the culture-dependent technique and all three molecular methods of DGGE, RFLP and normalization techniques could complement each other to provide comprehensive bacterial information and the normalization technique was efficient tool to explore gut bacterial community as DGGE and RFLP.