The DNA Composite Barcode Technology Was Used To Study The Intestinal Microbial Diversity Of Aphids

As is known to all that there are plenty of microbes in the intestinal of insect.So far,the studies regarding of the intestinal microbial communities,functions,influencing factors and the symbiotic relationship with the host are all based traditional taxonomy method.The traditional taxonomy method through observing the morphology characte-ristics and physiological index of microbial to identify species,it's heavenly relying on experienced taxonomists.But studies have shown that more than 99%of the microorganisms can not be cultured and isolated in laboratory.This flaw may be resulted in a majority of microbial resources have not yet known.DNA metabarcoding is a new method,which combined DNA barcoding and high-throughput sequencing technique,can be efficient and accurate identified species and assessed microbial diversity.To some extent,this method can make up for the deficiency of the traditional culture and molecular identification method,and can be greatly used to study the insect intestinal microbial.Grasshopper is a one of the main agricultural pests,their intestines contains a large number of microorganisms.At present,a lot of research on the grasshopper intestinal microbial morphology and classification,but about the microbial function,application and the relationship with the grasshopper is less.Therefore,we need to study the microbial community structure and diversity,and the relationship between grasshopper and symbiotic microbial.In this study,we aimed to obtain the community constructure composition and founction of intestines symbiotic microbial in 8 grasshopper species,including 8 individuals from 8 species collected in the Qinling Mountain(namely,Trilophidia annalata,Atractomorpha sinensis,Acrida cinerea,Chorthippus chinensis,Pedopodisma tsinlingensis,Fruhstorferiola omei,Euchorthippus unicolor and Xenocatantops brachycerus),and 3 individuals from 3 species collected in the campus of Shaanxi Normal University(namely,Trilophidi annulata,Atractomorpha sinensis and Acrida cinerea).We employed alpha diversity,beta diversity,and cluster analysis to evaluate differences of the grasshoppers intestinal microbial diversity.The main results are as follows:(1)16S rRNA gene were sequenced on an Illumina MiSeq platform,after a series of processing got 611,485 high quality sequences,and 3,421 OTUs.However,internal transcribed spacer(ITS)were sequenced on 454 FLX+ platform,got 130,600 high quality sequences,and 2,786 OTUs.(2)The analysis of the area system is shown that 23 phyla,58 classes,108 orders,and 375 genera were identified by the 16S rRNA sequences,and 5 phyla,16 classes,29 orders,and 40 genus were identified by the ITS sequences.This is probably because the database of bacterial is more completeness than the database of fungus.(3)The results of the analysis of the community structure were shown that the Actinobacteria,Proteobacteria,Tenericutes and Cyanophyta were the most common taxas,with lower occurrences of Bacteridetes,Frimicutes and Gemmatimonadetes by the 16S rRNA.The Tremellales and Auriculariales were the most common orders by the ITS.At same time,it suggested that the two orders belong to invaid fungui of grasshoppers.(4)Alpha diversity analysis revealed that the microbial community richness and diversity of the Oedipodidae were relatively high,and the microbial community richness and diversity of the Catantopidae is relatively low.The result may be related to their different diets.(5)Beta diversity analysis revealed that the grasshopper intestinal microbial community structure of the Catantopidae showed no significant different,in contrast,the gut microbial community structure of Acrididae in the different environments had a relatively low similarity.(6)Cluster analysis showed that the intestinal microflora of Oedipodidae and Catantopidae have similar community structure,but the other family have obvious community variations.The Rhodococcus and Brevundimonas of the bacteria identified by 16Sr RNA gene were the dominant bacteria,Bacillus and Wolbachia were the sub-dominant bacteria in the intestinal of grasshoppers at the genus level.Whereas,the Bullera?Ochrolechia and Exophiala were the dominant fungal identified by ITS,Penicilliumo and Fusarium were the inferior genus of the grasshoppers' intestinal fungal.It also showed that the microbial populations of the grasshoppers' intestinal were highly diverse in terms of number of species and their community structure composition,although the difference of the microbial communities varied with the grasshoppers host species and the surrounding environment.