Analysis Of Intestinal Microbial Diversity Of Five Species Of Grasshoppers And Isolation And Identification Of Cellulose Degrading Bacteria

Grasshoppers,as the general name of the class Insecta,Orthoptera and Acridoidea,are the important pests in agriculture and animal husbandry.Swarms of grasshoppers can cause great harm to agriculture and animal husbandry because of wide distribution,large feeding capacity,wide feeding habits,strong breeding ability and fast migration.A large number of microorganisms inhabit in the intestinal tract of grasshoppers,and these microorganisms interact with the host and influence each other to form a coevolutionary relationship.In this process,a variety of population structures and biological functions have been formed between microorganisms.The study of grasshoppers' digestion of cellulose and function of intestinal microbes provides a new idea for the green utilization of crop straw.This research mainly carried out the work from the following three aspects:1.In this study,a small fragment library was constructed through the Illumina Hi Seq sequencing platform,and paired-end sequencing was used to measure the 16 S r RNA gene sequence of the bacteria in the intestines of five grasshoppers to analyze the microbial diversity.There are 14 samples of five species of grasshoppers.The results show that a total of seven phyla,12 classes,21 orders,43 families and 57 genera are annotated in their intestines.At the level of phylum,Proteobacteria accounted for the highest proportion,Firmicutes was the second.At the genera level,Klebsiella accounted for the highest proportion in the intestinal microbes of the five species grasshoppers.Alpha diversity analysis showed that the intestinal microbial species richness of five species grasshoppers from high to low was Acrida cinerea,Sphingonotus mongolicus,Trilophidia annulate,Atractomorpha sinensis and Calliptamus abbreviates.The species diversity of the intestinal microbes of the five species grasshoppers from high to low Sphingonotus mongolicus,Trilophidia annulate,Acrida cinerea,Calliptamus abbreviates and Atractomorpha sinensis.In Beta diversity analysis,PCo A analysis and UPGMA cluster analysis found that the samples of the same species did not all gather together.This is due to two reasons: individual differences and different collection time.Lef Se difference analysis of samples between groups showed only four biomakers were screened in the intestines of two species of grasshopper.Among them,the biomakers in the intestine of Atractomorpha sinensis are Rickettsiales,Anaolasmataccae and Wolbachia,and the biomaker in the intestine of Sphingonotus mongolicus is Pantoea.The results of KEGG function prediction showed that the functional genes involved in carbohydrate metabolism accounted for the largest proportion in the intestinal flora.2.The cellulose digestibility and hemicellulose digestibility of five species of grasshoppers were determined by anthrone colorimetry and tercichrophenol colorimetry,respectively.The results shows that the digestibility of cellulose is higher than that of hemicellulose.The cellulose digestibility from high to low was in the order of Sphingonotus mongolicus(67.91%),Calliptamus abbreviates(60.23%),Acrida cinerea(56.97%),Atractomorpha sinensis(54.86%),and Trilophidia annulate(49.87%).The digestibility of hemicellulose from high to low is Sphingonotus mongolicus(47.51%),Acrida cinerea(39.28%),Calliptamus abbreviates(32.18%),Trilophidia annulate(19.25%)and Atractomorpha sinensis(17.77%).3.By using traditional culture method,physiology biochemistry experiment and 16 S r RNA gene sequencing,11 strains of cellulose degrading bacteria were identified from the intestines of Trilophidia annulate,Calliptamus abbreviates,Atractomorpha sinensis and Sphingonotus mongolicus.Among them,four strains of Bacillus were isolated from the intestine of Trilophidia annulate;five strains of bacteria were isolated from the intestine of Atractomorpha sinensis,one of which was identified as Bacillus,and the remaining four strains were identified as Aeromonas;one strain was isolated from the intestinal tract of Calliptamus abbreviates,which was identified as Klebsiella;one strain was isolated from the intestine of Sphingonotus mongolicus and it was identified as Klebsiella.Through the determination of cellulase activity and filter paper enzyme activity,it was found that the cellulase activity of Bacillus was higher than that of the other two genera,and the filter paper enzyme activity had little difference.