Effects Of Caffeine On The Intestinal Flora Of Ectropis Obliqua And Isolation Of Related Microorganisms

Ectropis obliqua is one of the main pests that harm tea plants,seriously restricting the economic value of tea.Caffeine is the most abundant alkaloid in tea leaves and is toxic to insects.However,E.obliqua not only absorb and digest the nutrients of tea,but also gradually form the special ability of metabolizing caffeine during the process of long-term co-evolution with tea plants.Some studies have revealed that a number of insects possess specific gut microorganisms,some of which significantly contribute to detoxification of phytotoxins and pesticides,but it is not clear whether the intestinal symbiotic bacteria of E.obliqua are involved in the detoxification of caffeine.Therefore,this study intends to use next generation sequencing technology and the traditional culture method to preliminarily explore the relationship between the intestinal flora and caffeine-detoxification of E.obliqua.The main results are as follows:1.Using next generation sequencing technology,we analyzed the intestinal flora structure of E.obliqua larve fed with different foods,at the phylum level,Firmicutes and Proteobacteria were the dominant phylum in CK group(not containing caffeine artificial diet)and CF group(containing 0.5% caffeine artificial diet).Under caffeine stress,the relative abundance of Firmicutes in CF group decreased to 59.39% compared with that in CK group(88.04%)(P<0.005),while the relative abundance of Proteobacteria increased from 6.35% to 20.11%(P<0.05).At the genus level,Enterococcus(83.40%),Pseudomonas(1.10%)and Weissella(0.71%)were the dominant bacteria in CK group,while Enterococcus(49.90%),Weissella(1.85%)and Sphingomonas(1.41%)were the dominant bacteria in CF group.Enterococcus was the most dominant bacteria in both groups,but under caffeine stress,its relative abundance decreased significantly from 83.39% to 49.90%(P<0.05).In addition,the relative abundance of pseudomonas decreased significantly,while sphingomonas increased significantly and became the dominant bacteria.High concentration caffeine had little effect on the structure of intestinal flora composition of E.obliqua larve,but it could change the relative abundance of intestinal flora.According on the OTU sequences obtained after homogenization,a total of 3,285 OTUs were detected in CF group,while only 2,085 OTUs were detected in CK group;The heat map clustering at genus level showed that the differences in intestinal bacterial structure between the two groups were significant.These results indicated that the high concentration caffeine caused the intestinal flora diversity of E.obliqua larvae increased significantly.The Shannon index,Simpson index,Chao1 index and ACE index of E.obliqua larval intestinal flora in CF group were higher than those in CK group,and there were significant differences between the two groups(P<0.005;P<0.05;P<0.05;P<0.005),which indicated that high concentration caffeine can significantly increased diversity and richness in the intestinal microbial of E.obliqua larvae.2.11 strains of bacteria and 16 strains of fungi were isolated from the gut of E.obliqua larvae on the media containing caffeine as the sole source of carbon and nitrogen.After 16 S r DNA and ITS r DNA sequence analysis,it have reliminarily identified that 11 strains of bacteria were Acinetobacter bouvetii,Acinetobacter haemolyticus,Janibacter melonis,Kocuria sp.,Deinococcus sp.,Bacillus sp.,Stenotrophomonas maltophillia,Streptomyces microflavus,Staphylococcus sciuiri;16 strains of fungi were Penicillium sp.,Penicillium commune,Penicillium paxilli,Penicillium mallochii,Penicillium chrysogenum,Penicillium citrinum,Penicillium copticola,Mucor sp.,Pyrenochaeta unguis-hominis,Purpureocillicum lilacinum,Talaromyces funiculosus,Aspergillus versicolor,Cladosporium anthropophilum,Plectosphaerella cucumerina.Among them,the similarity of strain CF-FG and MH155323(Mucor sp.)was only 82.88%,which was a potential new species and further research was needed.The ability of bacteria to degrade caffeine was found to be weak,while some fungi could metabolize caffeine to a certain extent.The caffeine-degradation rates of strain CF-F9 Penicillium sp.(36.31%),strain CF-FF1 Penicillium mallochii(35.37%)and strain CF-FF Penicilium paxilli(33.56%)were relatively high.In summary,this study showed that high concentration caffeine significantly affected the diversity and abundance of intestinal flora of E.obliqua larvae.Intestinal symbiotic microorganisms of E.obliqua larvae might participate in the metabolism of caffeine,and could cooperate with the host to improve the ability of detoxification caffeine.Thus,the intestinal flora could improve their host's defense ability.