Biodiversity And Metabolic Functions Of Gut Microbiota In Silkworm Bombyx Mori

Microorganisms play a crucial role during the development of animals.They impact the growth,the resistance,the fecundity and the longevity of animals.A large numbers of microbes have been detected in tissues of mammal,bird,fish and even invertebrates like insects.With the rapid development of ’omic’ technologies,the microbe-host interactions have been extensively studied.Considering that lepidopteran insects harboring numbers of microorganisms in their alimentary tracts,the communication between gut microbes and their host could be very sophisticated and not well studied yet.Manipulating the growth of the insect host by their gut microbes gives great ecological and economic advantages in agriculture.To make the potential use of gut microbes in pest control and commercial applications,a systematic investigations of lepidopteran microbiome,including the distribution,community dynamics,variation among host species,and metabolic preferences are needed.In this study,we use the model insect of Lepidoptera,silkworm(Bombyx mori),mulberry feeding lepidopteran insects(Acronicta major,Euproctis similis,and Glyphodes pyloalis),and other lepidopteran species that already have the metagenomic sequencing data to explore the bacterial dynamics,metabolic potentials,vitamin provisions,and detoxification functions of lepidopteran microbiota.We collected gut samples from the silkworm Bombyx mori in different life stages to investigate their fungal and bacterial communities.As a result,571,980 fungi reads were obtained from the metagenomic data(DNA sequencing),while 1,448,300 reads(DNA sequencing)and 872,724 reads(RNA sequencing)were obtained from gut bacteria samples.The number of bacteria and fungi in the silkworm alimentary tract was increasing with the development of larvae,and decreased after the pupation.We found that community structure of gut bacteria in different larval stages were divided into two distinct groups,1st and 2nd larval instars(Early instars)and 3rd-5th larval instars(Late instars).Moreover,the bacterial RNA sequencing data revealed that some bacteria are transit in the gut because they showed high abundance in DNA sequencing data and low level in RNA sequencing data,such as the genus Auriemonas.Other high abundance species in DNA sequencing data revealed similar trends in RNA data,which indicate that these bacterial species might interact with their host insects.Shotgun metagenomic sequencing of gut bacteria in silkworm strain P50 and Qiufeng × Baiyu revealed a large array of functional genes in gut microbiota.Metabolism of carbohydrates,amino acids,vitamins and harmful chemicals such as plant defense chemicals and xenobiotics comprise functional genes of silkworm gut microbiota.An investigation of high abundance bacteria showed that these dominate species prefer to utilize carbohydrates than lipids in the gut.Except for Enterococcus,most of dominate species contain biosynthesis genes of group B vitamins.They could be a potential vitamin supplier to their host.Furthermore,to assess the biodiversity of lepidopteran insects living in the same habitat and feeding on same plants with silkworm larvae,the large dagger moth A.major,the yellow tail moth E.similis,and the mulberry pyralid G.pyloalis were collected and used for gut community analysis.The result showed that gut microbiota of lepidopteran insects are highly variable among species,even feeding on the same diet.Moreover,functional prediction of these gut microbiota revealed that the larval biomass is correlated with the function of gut microbiota.We further downloaded the sequencing data of other 5 lepidopteran species and analyzed together with the mulberry-feeding caterpillars.Clustering analysis revealed that the function of gut microbiota was divided into two distinct clusters:the large-sized group(>300 mg)and the small-sized group.The impact of different antibiotics on silkworm gut bacteria were tested and polymyxin B selectively inhibited the growth of gram negative bacteria,whereas penicillin G acts conversely.Accordingly,these two antibiotics were used to change the ratio of G+/G-bacteria in silkworm gut.We observed that silkworm with higher abundance of G-bacteria was more resistant to insecticide chlorpyrifos.The result of 16S rRNA amplicon sequencing showed that the abundance of Stenotrophomonas maltophilia was decreased significantly in polymyxin B-treated group.Afterwards,we inoculated this species into germ-free silkworm larvae.The survival rate,detoxifying enzyme activity and relative gene expression of larvae under chlorpyrifos treatment revealed that S.maltophilia could enhance host resistance to chlorpyrifos.Result of GC and metabolomic analysis demonstrated that S.maltophilia did not degrade chlorpyrifos in silkworm gut.In contrast,it provides essential amino acids to host larvae,which indicate that S.maltophilia might work indirectly to manipulate the host insecticide resistance.In this study,amplicon-based metagenomic approaches were performed to explore the distribution,the community and the dynamic of silkworm gut microbes.This is the first time attempting to systematically reveal features of silkworm gut microbiome.These results demonstrated that bacterial community of early instar larvae is distinct from that in the late instar.Shotgun metagenomic analysis showed that carbohydrate metabolism is the major pathway in silkworm gut bacteria,whereas vitamin biosynthesis and biodegradation of harmful chemicals are also identified.Furthermore,by comparing the gene functions between silkworm and other lepidopteran insects,correlations between larvae biomass and gut bacteria functions were revealed.According to the shotgun sequencing data,a Stenotrophomonas maltophilia-mediated resistance against chlorpyrifos in silkworm and its possible mechanisms were discussed.These results establish a model for exploration of host-microbe interactions between lepidopteran insects and their host;present an experimental evidence that of lepidopteran host can benefit from their gut bacteria;and provide the theoretical basis for using beneficial bacteria in sericulture industry.