| As a widely recognized agricultural pest,termites have a history of 250 million years.During the long evolutionary process,it gradually evolved from cockroaches and other cockroaches of the order Blattaria to termites of the order Isoptera.As a social insect,the colony of termites can be roughly divided into four social castes: queen,king,worker,and soldier.Workers are easily exposed to various pathogens during their foraging process.The complex immune system of termites ensures their healthy survival in an environment full of pathogens.In addition to their innate immune system,there are also co-immune mechanisms among individuals of termites and between termites and symbiotic microorganisms.Millions of symbiotic microorganisms inhabit the digestive tract of termites,most of which are colonized in the hindgut and mainly collaborate with the host to degrade lignocellulose.In recent years,studies have shown that the intestinal flora of lower termites can cooperate with the host to resist the infection of pathogens.However,there is still a lack of sufficient research on the synergistic effects of host and intestinal microorganisms in the immune process of higher termites.Therefore,we conducted this study to reveal the role of intestinal microorganisms in the innate immune response of higher termites.The research contents can be mainly divided into the following three parts:Firstly,in order to study the impact of antibiotics on intestinal microflora and individual termites,we administered four different types of antibiotics to termites,including kanamycin sulfate,ampicillin,streptomycin,and tetracycline.Based on the results of 16 s r DNA sequencing,we found that compared to the other three antibiotics,kanamycin has a wider range of action and a longer duration of action,mainly affecting the bacterial abundance of the phyla Firmicutes,Spirochetes,and Actinomycetes.After that,through q PCR,we proved at the experimental level that kanamycin has the most obvious inhibitory effect on total bacteria in the hindgut.In addition,we also studied the impact of kanamycin on the survival of termites.After 48 hours of treatment,the survival rate of termites did not significantly change,but there was a significant decrease after 72 hours.The results showed that the inhibitory effects of four antibiotics on intestinal microflora of termites varied,and kanamycin had the best inhibitory effect on intestinal microflora and did not affect the survival of termites at 48 hours.Secondly,in order to study the effect of pharmacological destruction of intestinal flora on termite resistance to Metarhizium robertsii,we measured the intestinal gene expression profile of termites treated with kanamycin.The results showed that there was almost no change in gene expression in the intestine of termites fed kanamycin.However,after infection with M.robertsii by termites whose intestinal flora has been pharmacologically destroyed,the gene expression in the body changes dramatically,and the differentially expressed genes are mainly concentrated in signal transduction pathways such as calcium ions.In order to further explore the mechanism of intestinal microflora in host immune response to M.robertsii.We conducted q RT-PCR identification of the differentially expressed genes of top20 in the transcriptome,and found that the expression of specific genes in the intestine of termites infected with M.robertsii,whose intestinal flora was pharmacologically destroyed,was significantly downregulated compared to that of normal individuals infected with M.robertsii.These genes include apolipoprotein apoa2,Hsp70,ATP synthase,and reverse transcriptionrelated genes.These genes may play an important role in the immune response of termites mediated by intestinal bacteria.In order to further verify the role of apolipoprotein apoa2 in the response of termites to the immune response of M.robertsii,we conducted RNA interference on the termite apoa2 and found that the mortality of termites that interfered with apoa2 significantly increased after infection with M.robertsii.The above results indicate that intestinal microflora plays a positive role in the response process of termites to M.robertsii,and the imbalance of intestinal microflora can cause functional disorders of termites in response to M.robertsii.Finally,in order to explore the possible role of intestinal microflora in the immune response of termites to M.robertsii,we combined metagenome sequencing and metatranscriptome sequencing to analyze the impact of M.robertsii infection on the structure and function of intestinal microflora in termites.After infection with M.robertsii,the proportion of intestinal microflora in termites is unbalanced,and the relative abundance of some nondominant bacteria such as Actinomycetes increases,while the transcription activity of bacteria other than Proteobacteria generally decreases.The functional annotation results at the bacterial level showed that the gene function of dominant bacteria such as Proteobacteria had almost no change after M.robertsii infection,while the gene function of Spirochetes had the most drastic changes and its transcription activity decreased by about 70% after M.robertsii infection.Among the differentially expressed genes between the two samples,we found that a bacterial derived gene involved in glutathione metabolism,glutamate-cysteine ligase(gcl),was upregulated by more than 10 times after infection with M.robertsii.The content of glutathione significantly decreased after pharmacological damage to the intestinal flora.Based on the above results,we speculate on a potential mechanism that intestinal bacteria may promote glutathione synthesis through upregulation of their own gcl and cooperate with the host to interact with M.robertsii.In summary,our research has revealed the potential role and mechanism of intestinal microflora in host immune response to fungal pathogens,providing an important reference for further research on the insect immune system. |
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