| Gut microbiota has diverse ecological and evolutionary effects on their hosts. However, the ways in which it responds to environmental heterogeneity remain poorly understood. In this study, we surveyed intestinal microbiota of Holotrichia parallela larvae that from different geographic regions, and examined how environmental factors account for the variation of gut community composition between natural population. Furthermoer, by applying aerobic cultivation conditions we isolated and identified cellulose-degrading bacteria from the gut of H. parallela and cloned genes fragments encoding cellulose, to evaluate the nutritional contributions of gut microbiota to scarabs.1. Bacterial16S rRNA gene clone libraries were constructed and clones were subsequently screened by Denaturing Gradient Gel Electrophoresis (DGGE) and sequenced. The result showed that the bacterial16S rRNA gene sequences grouped into205OTUs, and identified as Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria and Fusobacteria sequences.After comparing microbial community richness and composition among these host populations, several feature of the gut microbiota can be found:(1). One prominent feature of the gut microbiota of H. parallela larvae was the high prevalence of Firmicutes and Proteobacteria sequences. Sequences affiliated with Firmicutes (mainly Clostridia) were detectable in all of the natural populations, and they were the most prominent group in the hindgut microbiota of the ten populations. Sequences related to Beta-, Gamma-and Deltaproteobacteria were also present in all natural populations, although at relatively low proportions. However, bacteria belonging to other phyla (Bacteroidetes, Actinobacteria, and Fusobacteria) were unevenly distributed across the host populations. Bacteria belonging some phyla were only detected in several population but absent from other natural populations.(2). Six OTUs were shared between the ten natural populations, and the remaining OTUs were unevenly spread across the host populations, with many appearing in only1host population among the ten sampled.(3). Species richness also varied across the host locations. The bacterial community from the TJ population (38.8) possessed the least species richness, while the FJ (86.5) population had the highest species richness. The∫-LIBSHUFF analysis also showed that most of the ten clone libraries were significantly different from each other.(4) Spearman nonparametric correlation test and redundancy discriminate analysis(RDA) showed that the bacterial diversity (Chaol estimator) and community structure variation that we observed can be explained by soil pH, organic carbon and total nitrogen, and the climate factors (e.g., mean annual temperature, January low temperature, mean annual precipitation) of the locations where the populations were sampled.2. Applying aerobic cultivation conditions,207strains of aerobic and facultatively anaerobic cellulolytic bacteria ifrom the gut of H. parallela larvae. These bacterial isolates were assigned to21genotypes by amplified ribosomal DNA restriction analysis (ARDRA). A partial16S rDNA sequence analysis and standard biochemical and physiological tests were used for the assignment of the21representative isolates. Our results show that the cellulolytic bacterial community is dominated by the Proteobacteria (70.05%), followed by the Actinobacteria (24.15%), the Firmicutes (4.35%), and the Bacteroidetes (1.45%). Pseudomonas, Ochrobactrum, Rhizobium, Cellulosimicrobium, and Microbacterium were the predominant groups, but members of Bacillus, Dyadobacter, Siphonobacter, Paracoccus, Kaistia, Devosia, Labrys, Ensifer, Variovorax, Shinella, Citrobacter, and Stenotrophomonas were also found. Furthermore, some cellulolytic bacteria, such as Siphonobacter aquaeclarae, Cellulosimicrobium funkei, Paracoccus sulfuroxidans, Citrobacter freundii, and Pseudomonas nitroreducens are reported to be cellulolytic for the first time in this study.3. Using the homologous cloning strategy with degenerate primers and chromosome walking techniques, two genes fragments encoding cellulase were cloned from one bacterial strain Rhizobium radiobacter isolated from the gut of H. parallela larvae. One gene is glycosyl hydrolase family (GH)3family, and temporarily called celⅢ. Sequence analysis showed that the celⅢ encoded products shared sixty percent similarity to the beta-glucosidase from Grosmannia clavigera kw1407. The other gene is glycosyl hydrolase family (GH)8family, and temporarily called celⅧ. Sequence analysis showed that the celⅢencoded products shared sixty percent similarity to the endoglucanase from Agrobacterium sp. ATCC31749. The presented results suggests scarab larvae have potential to assist the bio-fuel industry by providing new sources of (hemi)cellulolytic bacteria and bacterial (hemi)cellulolytic enzymes.
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