| The termite Odontotermes formosanus belongs to the Macrotermitinae subfamily of Termitidae under the order Blattodea and is the most species-rich genus of the subfamily Macrotermitinae,with a major distribution in Africa and Asia.As a social insect living in high-density colonies,it is exposed to a variety of pathogens throughout its life history and has therefore evolved a unique set of immune mechanisms.It has been shown that intestinal symbiotic flora can promote host innate immunity and cooperate with the host to build up the immune system to enhance survival in nature.The role of termite intestinal symbiotic flora in cellulose degradation has been confirmed,but its role in termite immunity and its mechanisms have not been reported.The termite hindgut has a variety of endosymbiotic bacteria and is a rich and complex ecological endosymbiotic environment.Revealing the changes in the gut flora after Metarhizium infestation of the host can provide insight into how the gut flora responds to the unfavorable environment of Metarhizium infestation during Metarhizium infestation and,more importantly,can significantly improve the effectiveness of our use of biocontrol for agricultural pest control by studying the important immune activities of insects,potentially providing more target genes for use in biocontrol.In this study,the fungus Metarhizium robberstii was used to infect termite Odontotermes formosanus.Based on the observation of changes in the intestinal microbes of termites by anatomical microscopy,light microscopy and electron microscopy,we analyzed the structural changes of intestinal flora before and after infection by 16 S rDNA sequencing and metagenome sequencing to identify the bacterial taxa that may play a key role in the resistance of higher termites to Metarhizium robberstii infection,and constructed the metabolic pathways of intestinal symbiotic flora before and after infection by metagenome sequencing to reveal the signaling pathways that undergo significant changes in order to resolve the specific role of intestinal symbiotic flora in the immunity of higher termites and their molecular mechanisms.The metabolic potential of uncultured bacteria was explored based on metagenomic data assembly of single microbial genomes,revealing the potential measures of unknown microbes against the invasion of Metarhizium robberstii.This study will provide a new understanding of the immune mechanism of termites on the one hand,which can understand the symbiotic mechanism of termite symbionts from another perspective and enrich the symbiosis theory,and on the other hand,it is expected to provide theoretical and practical guidance for biological control of termites.The main results are as follows:1.We observed the conditions in the gut of termites after Metarhizium robberstii invasion under dissecting microscope,light microscope and electron microscope,respectively.Firstly,the invasion of Metarhizium robberstii led to the death of the termites,and at the beginning of the infection Metarhizium robberstii entered the intestine and reached the hindgut,the area with the highest concentration of microorganisms.The invasion of Metarhizium robberstii leads to a disturbance of the intestinal microorganisms,which is reflected in an increase in the density of intestinal microorganisms.However,at the end of the infection,when the population was on the verge of death,the intestinal epithelial barrier was not destroyed,reflecting the protection of the intestinal tissues by the intestinal microorganisms from Metarhizium robberstii.2.Based on 16 S rDNA sequencing to analyze the structural changes of their intestinal flora before and after infection,a total of 1938 OTUs(Operational Taxonomic Units)were obtained.Among them,the number of OTUs that could be annotated to the database was 1865.The changes in the overall level of diversity and relative abundance of the intestinal colonies were mainly studied,as well as the trends of the phylum that changed significantly during the infection with Metarhizium robberstii.We found that a decrease in the abundance and diversity of the termite gut microbial community occurred at the beginning of the infection,followed by a slow recovery and eventual equilibrium.This reflects the measures taken by the gut flora at the colony level to cope with the unfavorable environment of Metarhizium robberstii infection.Most of the phyla did not change significantly,indicating that most of them were more resistant to Metarhizium robberstii,while three significantly changed phyla,Deferribacteres,Proteobacteria and Spirochaetes,were present,reflecting their susceptibility to Metarhizium robberstii infection.3.Metagenome sequencing using the Illumina HiSeq platform and gene prediction after quality control yielded 792,038 ORFs with a total length of 472.72 Mbp,including 214,957 intact genes.Annotation of the pathways in the KEGG database revealed a significant down-regulation of overall function within the mid-infection period,including energy metabolism subsystems(carbon sequestration pathway,methane metabolism,nitrogen metabolism,oxidative phosphorylation and sulfur metabolism)and cell motility(bacterial chemotaxis,bacterial motor protein and flagellar assembly,subsystems ABC transporter,phosphotransferase system(PTS)and transporter(functional class: membrane transporter)).However,protein export and ribosome-related pathways are activated at the beginning of infection,suggesting a means of resistance to Metarhizium robberstii in this regard.Meanwhile,we annotated to 3 immune pathways: NOD-like receptor signaling pathway,antigen processing and presentation and Th17 cell differentiation.The analysis showed that these immune pathways were mainly involved by the phya Firmicutes,Bacteroidetes,Proteobacteria,and Spirochaetes in the intestinal symbiotic bacteria.The expression of Hsp90 gene was found in all three pathways,implying that Hsp90 is a conserved gene involved in innate immunity in termite intestinal symbionts.In addition,we performed the assembly of single microbe genomes based on metagenomic data,and isolated a total of 218 microbial genome drafts(MAGs),of which 36 were of high quality,to explore the evolutionary patterns of termite gut microbes by phylogenetic analysis.Annotation of each MAG with its closest species according to the species-level genome bins(SGB)database yielded two known strains Trabulsiella odontotermitis,Acinetobacter oleivorans,while the vast majority of microbial genomes belonged to unknown species(u SGB).Subsequent metabolic functional annotation of high-quality microbial genomes unearthed potential functions of uncultured strains. |
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