Diversity Analysis And Function Study Of Intestinal Microbiota In Three Springtail Species

Springtails are a group of lower micro-animals with primary wingless and entognathous features,and belong to Arthropoda,Hexapoda.Springtails have a great number of variety and are distributed widely.Their main foods are dead branches and fallen leaves,humus,bacteria and fungi from the soil.The complex intestinal microflora of springtails can help the host decompose and utilize ingested organics.In order to investigate the gut microbial diversity and related functions of springtails,this study uses Sinella(Coecobrya)oligoseta,Proisotoma minuta and Tomocerus missus as experimental animals.Microflora in the adult intestinal contents of 3 springtail species were analyzed and compared by 16 S r DNA amplicon sequencing,and function prediction of the gut microbial genes was conducted using Tax4 Fun method.Simultaneously,the culturable intestinal bacteria of 3 springtail species were isolated by traditional methods,and some bacteria capable of degrading soluble starch,carboxymethyl cellulose sodium and pectin were screened from the culturable intestinal bacteria,as well as the initial evaluation of enzyme production capacity and molecular identification of polysaccharide-degrading bacteria.Additionally,Aeromonas,a type of common pathogen in animal intestine,was also screened from the culturable intestinal bacteria,and their analysis of multilocus sequence typing(MLST)and tests of virulence genes and drug susceptibility were conducted,respectively.The results of this study were as follows:Firstly,among the 3 springtail species,T.missus showed the highest adult gut microbial diversity,while S.(C.)oligoseta showed the lowest adult gut microbial diversity.At the phylum level,Proteobacteria,Firmicutes and Bacteroidetes were the most predominant microbiota,followed by Actinobacteria in the adult intestinal microbiota of 3 springtail species.At the genus level,the relative abundance of Pseudomonas in the gut of S.(C.)oligoseta(16.21%)was significantly higher than those in the guts of P.minuta(0.87%)and T.missus(1.37%).The relative abundance of Vibrio in the gut of P.minuta(25.81%)was significantly higher than those in the guts of S.(C.)oligoseta(3.35%)and T.missus(0.004%).Secondly,based on KEGG pathway annotations,we predicted that the highest relative abundance function genes in adult gut microbiota of 3 springtail species may be involved in the metabolisms of carbohydrates and amino acids,and the function genes involved in infectious diseases and drug resistance were significantly higher than other genes related to human diseases.Thirdly,a total of 47 strains of polysaccharide-degrading bacteria screened out from the 106 strains of culturable intestinal bacteria,include 37 strains of amylase producing bacteria mainly belonging to Microbacterium and Bacillus;16 strains of cellulase producing bacteria mainly belonging to Cellulosimicrobium and Bacillus;3 strains of pectinase producing bacteria belonging to Bacillus and Paenibacillus.Tm-L4 strain is able to produce amylase,cellulase and pectinase,and it has 99.79% sequence similarity with Paenibacillus amylolyticus by 16 S r DNA sequence alignment.Fourthly,3 strains of Aeromonas caviae were screened out from the culturable intestinal bacteria of S.(C.)oligoseta and P.minuta.The MLST analysis showed their housekeeping genes belong to new sequence type(ST),i.e.ST653,and the corresponding allele types are gyr B456,gro L438,glt A465,met G462,pps A503 and rec A499.The PCR amplification of their virulence genes showed the positive genes respectively were lip,ela,alt,hly A and fla.The test of drug susceptibility showed this 3 strains were resistant to ampicillin,ampicillin-sulbactam,tetracycline and nalidixic acid,while were sensitive to the 12 antibiotics including azithromycin,chloramphenicol,and amikacin.The above results suggested that,in the adult intestines of 3 springtail species,S.(C.)oligoseta,P.minuta and T.missus,the core microbiota are the same at the phylum level,but obviously different at the genus level.The influencing factors should include their genetic traits and the abundance and diversity of microbes in the habitat of each species.The most bacteria in the intestines microflora of 3 springtail species were involved in energy metabolism and nutrition function,which is helpful for them to play the role of decomposers in soil ecosystem.The screened bacteria capable of degrading starch,cellulose and pectin,with a feature of enzyme production relatively higher,are promising to be developed into probiotics,feed additives or enzyme engineering bacteria,and exert certain values in the industries of food,aquaculture and enzyme.Some pathogenic or drug-resistant microorganisms may also be existed in the intestines of 3 springtail species.The new type strain of Aeromonas caviae was successfully obtained and the tests of virulence genes and drug susceptibility indicated their potential pathogenicity and antibiotic resistance or sensitivity,which provides theoretical basis for the precaution and antibiotic usage of related diseases caused by Aeromonas caviae in springtails.