Diversity Analysis Of Three Species Grasshoppers Gut Microbiome And Isolation Of Cellulose Degrading Bacteria

Grasshoppers belong to a large category of agricultural pests,but there were few research on the composition of grasshopper intestinal microflora,community diversity and functional bacteria.The main components in grasshoppers food are cellulose and hemicellulose.Due to grasshoppers large appetite and high utilization rate,the intestinal contents of grasshoppers have the potential to be developed into bioreactor,at the same time,it can improve straw utilization efficiency and turn waste into treasure.In this study,Aiolopus tamulus,Oedaleus asiaticus and Shirakiacris shirakii were taken as the research objects,using the method of constructing a 16 S rRNA gene library and Illumina Miseq sequencing technology.The V3-V4 region of the midhindgut of the adult was introduced into high-throughput sequencing for the first time,and the intestinal microbial diversity among different species was studied.At the same time,the digestibility of cellulose and hemicellulose of three species grasshoppers was determined,and cellulose degrading bacteria in midgut and hindgut were isolated and identified.The main research results were as follows:1.The main taxonomic elements anoted by the genne sequencing results of intestinal microorganisms of 3 speceises grasshoppers belongs to 7 phyla,12 classes,20 orders,42 families and 54 genera.2.The relative abundance of Proteobacteria and Firmicutes was more than 1% in the intestinal microbial communities of the three grasshoppers,which were dominant bacteria.Actinobacteria,Cyanobacteria and Fusobacteria exist in the intestinal tracts of the three species grasshoppers in trace amounts.However,Bacteroidetes was not detected in the intestinal tract of Shirakiacris shirakii,Tenericutes was only detected in the intestinal tract of Oedaleus asiaticus,and was not detected in the intestinal tracts of the other two species grasshoppers.3.At the level of genus,there were three dominant genera of Aiolopus tamulus,including Klebsiella,Enterococus,Enterobacter,and 6 dominant genera of Oedaleus asiaticus.Specifically,it included Klebsiella,Enterococcus,Pantoea,Wolbachia,Enterobacter,Lactococus,and three dominant genera of Shirakiacrisshirakii,namely Klebsiella,Lactococus,and Staphylococcus.4.Diversity analysis showed that the species richness in the intestinal tract of Shirakiacris shirakii was the highest,while the diversity of the microbial community in the intestinal tract of Aiolopus tamulus was the highest.Different species of grasshoppers have different intestinal microflara structures,among them,there was little difference of intestinal microflora structure between Shirakiacrisshirakii and Aiolopus tamulus,and at the same time,time differences will also lead to changes in the intestinal microflora structure of the same species of grasshoppers.5.The contents of cellulose hemicellulose in feces and wheat seedlings of the three species grasshoppers were determined by adopting moss black phenol colorimetry and anthrone colorimetry.The digestibility of cellulose/hemicellulose among Aiolopus tamulus,Shirakiacris shirakii and Oedaleus asiaticus were 43.95/17.21%,44.12/47.62% and38.01/24.99%,respectively.There was no significant difference in cellulose digestibility among the three species grasshoppers(P>0.05),the hemicellulose digestibility of Shirakiacris shirakii was significantly higher than the other two species grasshoppers(P<0.05).In addition,by analyzing the correlation between digestibility and microorganisms,we found Klebsiella,Enterococcus and Lactococcus were all significantly related to cellulose digestion.6.With sodium carboxymethyl cellulose as the sole carbon source,three strains capable of producing transparent circle were screened from the intestinal tract of Aiolopus tamulus,and they were separated and purified.The cellulase activity was measured by DNS method and re-screened.Combined with morphological observation,physiological and biochemical identification and molecular biology,the strains H-1,H-2 and H-7 were preliminarily identified as Bacillus cereus.7.Two strains capable of producing transparent circles were screened from the intestinal tract of Shirakiacris shirakii.Cellulase activity was measured by DNS method and re-screened.The two strains were identified according to morphology,physiological and biochemical characteristics and molecular biology.The strains S-3 and S-5 were preliminarilyidentified as Bacillus cereus.