Comparative Analysis Of The Diversity Of Gut Bacteria Of Two Gynaephora Species

Gynaephora spp.(Lepidoptera: Erebidae: Lymantriinae)are one group of major pests of alpine meadow on the Tibet Plateau in China.They not only feed on high quality forage as a tough competitor of livestock,but also change the vegetation community structure of alpine meadow,which means it has become a restrictive factor and barrier for the sustainable utility and healthy development of alpine meadow.Their outbreak in many areas and continuous damage of grassland caterpillars show good adaptation to extreme ecological environments such as low oxygen,low temperature and strong ultraviolet radiation in the alpine meadow.In this thesis,the composition,diversity and function of gut bacteriain Gynaephora menyuanensis and Gynaephora alpherakii inhabiting different altitudes in alpine meadow were compared and analyzed based on the isolation and culture methods and high-throughput sequencing technology,so as to dissect the ecological mechanisms of Gynaephora's adaptation to high-altitude environments from a perspective of gut bacteria.The main results are as follows:1.Gut bacteria of fifth-instar larvae of two Gynaephora species were isolated,cultivated,and purified based on six kinds of culture media.Species identification of gut bacteria was conducted by using morphological characteristics of bacterial colony and 16 S sequencing techonology.The results showed that five culture media can be used to isolate Gynaephora gut bacteria.A total of five species of gut bacteriawere isolated from G.menyuanensis,of which three species(M1,M2,M3)belonged to Staphylococcus of Firmicutes,and two species belonged to Lelliottia(M4)and Leckerella(M5)of Proteobacteria.A total of 6 gut bacteria were isolated from G.alpherakii,three of which belonged to Staphylococcus(N1 and N5)and Enterococcus(N4)of Firmicutes,and the remaining three belonged to Serratia(N2 and N6)and Pseudomonas(N3)of Proteobacteria.Serratia is known to have a positive role in assisting insects to degrade cellulose and on lignin,suggesting that this bacterium may also assist G.alpherakii to degrade plant fibers.2.A total of 57 OTUs were obtained from 16 S amplicon sequencing of gut bacteria from G.menyuanensis,which were annotated as: 4 phyla,of which the dominant phylum was Proteobacteria;7 classes,of which the dominant classes were Alphaproteobacteria and Gammaproteobacteria;12 orders,of which the dominant orders were Rickettsiales and Pseudomonadales;15 families,of which the dominant families were Rickettsiaceae and Pseudomonadaceae;13 genera,of which the dominant bacteria were Wolbachia and Pseudomonas;and 7 species,of which the dominant species were Enterococcus tyrococcus,Staphylococcus sciuri,and Methylotenera mobilis.The sequencing results of the gut metagenome of G.menyuanensis were annotated as: 5 kingdoms,of which Bacteria(71.8%)and Eumycetes(12.1%)showed the highest relative abundance;27 phyla,of which Proteobacteria,Firmicutes,and Bacteroidetes were the dominant phyla;51 classes,of which Alphaproteobacteria,Gammaproteobacteria and Bacillus were the dominant classes;107 orders,of which Rickettsiales,Enterococcales,and Bacillus were the dominant orders;178 families,of which Anaplasmataceae and Enterobacteriaceae were the dominant families;and 236 genera,of which Wolbachia and Escherichia were the dominant genera.3.A total of 91 OTUs were obtained by 16 S amplicon sequencing of G.alpherakii,which were annotated as: 5 phyla,of which Firmicutes showed the highest relative abundance;7 classes,of which Bacilli and Gammaproteobacteria were the dominant classes;10 orders,of which Lactobacillus and Bacillus were the dominant orders;11 families,of which Carnobacteriaceae and Enterococcaceae were the dominant families;9 genera,of which Carnobacteriales and Enterococcaceae were the dominant genera;8 species,of which Enterococcus casseliflavus and Bacillus muralis were the dominant species.The sequencing results of the gut metagenome of G.alpherakii were annotated as: 5 kingdoms,of which Bacterial(57.3%),Eumycetes(17.7%)and Virus(11.6%)showed the highest abundance;27 phyla,of which Proteobacteria,Bacteroidetes,and Firmicutes were the dominant phyla;56 classes,of which Gammaproteobacteria,Bacillales,and Cellulosimicrobiales were the dominant classes;119 orders,of which Enterobacteriales,Bacillales,and Cellulophagales were the dominant orders;200 families,of which Enterobacteriaceae,Arthroceae,and Cyclobacteriaceae were the dominant families;and 262 genera,of which Escherichia,Agrobacteriales,and Cecembia were the dominant genera.4.Many annotated genes obtained from metagenomic sequencing of gut bacteria were related with metabolism,carbohydrate transport,amino acid synthesis and so on,indicating that the gut bacteria may be involved in physiological processes such as carbohydrate metabolism,material synthesis,material degradation and absorption of the host,and thus play an important role in ecological adaptation of Gynaephora to extreme environments of alpine meadow.16 S amplicon sequencing showed that Wolbachia and Pseudomonas were the most abundant species in gut bacteria of G.menyuanensis,while Carnivora and Enterococcus in gut bacteria of G.alpherakii.This difference in the most abundant gut bacteria may be associated with adaptation of the two Gynaephoras species to different altitudes.The ? diversity of gut bacteria in G.menyuanensis was not significantly higher than that of G.alpherakii(P>0.05),but nine genera including Acinetobacter,Achromobacter,Blastomonas,Hydrogenophaga,Methanobacterium,Wolbachia,Enterococcus,Pseudomonas,and Carnivorawere significantly different between the two Gynaephora species(P<0.01)when the relative abundance of gut bacteria were not considered.Therefore,it was proposed that these nine generas may be associated with the adaptation of the two Gynaephora species to different altitude environments.