| Holotrichia parallela larvae are the severe agricultural pests, they feed on plant roots and organic matter of high cellulose content. Its hindgut typically contains a wide diversity of microorganisms, which may play an important role in food digestion of the host. Here, the microbial abundance and diversity of different developmental stages of the H. parallela were investigated, and we used a culture-independent method to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the larval hindgut. Furthermore, we isolated a large number of cellulolyitc and xylanolytic bacteria from the hindgut of H. parallela larvae, which were expected to be applied to the biodegradation of cellulose and hemicellulose.1. In our study, the microbial abundance and diversity of different life stages of the scarab larvae, H. parallela, were investigated using bacteria culture-independent method based on the sequence of bacteria16S rRNA V1-V3region gene. After comparing microbial community richness and composition among these different samples, the results were as follows:(1) Among the different life stages of the H. parallela larvae, the hindgut microbial diversities of the second instar, third instar larvae were significantly higher than the microbial diversity of the first instar larval hindgut.(2) Adult, ovary, egg, first-instar, second-instar and third-instar larvae libraries included the similar phyla composition, where Bacteroidetes were the most important groups, followed by Firmicutes and Proteobacteria. However, the soil library was dominated by Proteobacteria, Bacteroidetes and Candidate division TM7.(3) Many bacteria residented in hindgut of H. parallela larvae were from parent-to-offspring transmission, OTU220, OTU461from Clostridia, OTU105, OTU856, OTU1130from Betaproteobacteria,OTU114, OTU774, OTU1157, OTU931from Bacteroidetes were common in LC, OL, FL, SL, and TL samples, respectively, they are strong candidates for being members of H. parallela larvae core microbiome.2. The morphology and ultrastructure of the hindgut fermentation chamber of H. parallela, were examined using scanning electron microscopy, and the weight of third instar larvae was determined. The results showed that a special lobe-like structure was formed in the wild-caught and lab-reared third-instar larvae. The lobe-like structure of wild-caught population constituted by bacteria and the bacteria-covered cuticular intima, however, we could not observed the obviously cuticular intima structure in the lab-reared population. Furthermore, we found that there is a significant difference between the lab-reared populations (p<0.05). The weight of the third instar larvae was the highest under the unsterile soil and eggs treatment group, followed by sterile soil and unsterile eggs, unsterile soil and sterile eggs treatment groups. If the soil and the surface of eggs were sterilized, the larval weight became the lowest. Our results showed that the eggshell surface and environmental microbial populations significantly affect the growth and development of H. parallela larvae.3. We used the homologous cloning strategy with degenerate primers to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of H. parallela larvae. PCR with degenerate primers using metagenomic DNA from H. parallela larvae hindgut content yielded amplicons for beta-galactosidase (GH2), beta-1,4-endoglucanase (GH8), endo-xylanase (GH10,11), alpha-galactosidase (GH36) enzymes. All of these genes were grouped into101OTUs. Among them,24,27,19,14and17OTUs were identified in the GH2,8,10,11, and36families, respectively. Of these reads,93.94%,10%,52.48%,28.95%, and19.32%of GH2, GH8, GH10, GH11, and GH36sequences were<80%identical with those of known GH enzymes, respectively. Moreover,10.61%,3.33%, and18.42%of GH2, GH8, and GH11sequences had<60%identities with the available sequences in the NCBI database. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela, and many of them originate from unknown micro-organisms. Based on phylogenetic analysis, fragments from cellulase family (GH8) were most associated with the phylum of Proteo bacteria, and sequences from hemicellulase families (GH2, GH10, GH11and GH36) were related to enzymes from Bacteroidetes and Firmicutes.4. From the hindgut contents of H. parallela,93cellulolytic bacterial strains were isolated after enrichment in CMC medium. Among these isolates, a novel bacterium, designated HP207, with high endoglucanase productivity was selected for further study. This bacterium was identified as Pseudomonas sp. based on the results of the16S rRNA gene analysis, morphological characteristics and biochemical properties. The composition of the optimal fermentation medium was as follows (in g/L):CMC-Na (15), wheat bran (40), yeast extract (15), NaCl (10), KH2PO4(0.94) and K2HPO4(1.9). The optimal initial medium pH, temperature, medium volume, inoculum size and incubation period were7.0,30℃,100mL/250mL (v/v),3%(v/v) and24h, respectively. The maximum endoglucanase yield of1.432U/mL (expressed as enzyme activity) was obtained at the shake flask level. Moreover, this enzyme was also highly thermostable; approximately55%of the original activity was maintained after pretreatment at70℃for1h.5. From the hindgut contents of H. parallela,103xylanolytic bacterial strains were isolated. Among these isolates, a highly xylanolytic bacterium, designated HP455, was selected for further study. This bacterium was identified as Sphingobacterium sp. based on the results of the16S rRNA gene analysis. The endo-xylanase (xyn455) gene of the glycoside hydro lase (GH) family10and β-xylosidase (xyl455) gene of the GH43family were cloned and expressed in vitro from this highly xylanolytic bacterium. Both the Xyn455and Xy1455enzymes could degrade the arabinan, they are bifunctional enzymes with both endo-xylanase/β-xylosidase and a-L-arabinofuranosidase activities. And we also found that the in vitro degradation of xylan by Xyn455and Xy1455showed a strong synergistic hydrolysis action between these two enzymes. Furthermore, Xy1455enzyme also exhibits high xylose tolerance and a broad pH stability. These results suggest that the recombinant Xyn455and Xy1455enzymes are potential candidates to be used in feed additives, paper and other industries. |
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