A Study On The Diversity Of Animal Gut Microbial Symbionts And The Host-microbe Interactions Based On Eupolyphage Sinensis Walker And Euprymna Scolopes

This thesis reports a systematic study on the diversity of symbiotic microbe in animals and the host-microbe interactions.In this study,the diversity of gut microbes of traditional Chinese medical insect Eupolyphaga sinensis Walker was investigated;and two closely related bactericidal permeability-increasing proteins(BPIs)of the squid Euprymna scolopeswere characterized,and their functions in the squid-vibrio system were identified.We explored the microbial community structure and diversity of gut microbes of Eupolyphaga sinensis Walker,applying culture-dependent method and High-throughput sequencing techniques.In the culture-dependent study,224 strains were isolated from the female adult gut of Eupolyphaga sinensis Walker.The results of fingerprinting analysis including ARDRA?RFLP and Eric-PCR on the 16S rDNA of isolates suggest that the gut microbe has abundant genetic diversity.Seventy one representative strains were chosen for 16S rDNA sequencing.With the sequencing results and according to the phylogenetic analysis,the 71 strains were identified to species and categorized into 10 genera,which are Bacillus,Staphylococcus,Loctobacillus,Streptomyces,Pseudomonas,Providencia,Citrobacter,Psedocitrobacter,Enterobacter and Klebsiella,respectively.Within them,Bacillus was the dominant group,occupying a percentage of 33%,followed by Pseudomonas and Citrobacter,which occupy 20%and 18%respectively?In the High-throughput sequencing study,the variable V4 regions of 16S rRNA gene were analyzed to explore the community structure and diversity of both bacteria and archaea in the egg,larva gut and adult gut of Eupolyphaga sincnsis Walker.Results reveal that the richness of bacteria in the gut was notably higher than that in the egg,and also higher in the larva gut than that in the adult gut.No significant difference between the larva and adult gut occurred for archaea.The diversity of gut bacteria was abundant(Shannon index>9.15,Simpson index>0.98)and much higher than that of egg bacteria(Shannon index = 4.84,Simpson index = 0.73).The bacteria in the investigated samples were categorized into 12 phylums,24 classes,39 orders,60 families,and 71 genera.The dominant groups include Bacteroidetes,Firmicutes and Proteobacteria,all occupying a ratio higher than 22%.The archaea was categorized into 3 phylums,7 classes,8 orders,11 families,and 13 genera.The most dominant group was Methanobrevibacter,accounting for a proportion up to 22%.The sequencing results show that the diversity of gut symbiotic microbes of Eupolyphaga sinensis Walker was high.Within the gut microbes these genera such asPhascolarctobacterium,Clostridium and Bacteroides were found in the gut of Eupolyphaga sinensis Walker,Which was identified as“Phylo-functional core of gut microbiota" in human.Most of the identified bacteria existed in both egg and gut of Eupolyphaga sinensis Walker,but a few of them were only found in the gut.PCR products were not observed using the universal archaeal primer for microbe DNA amplification in eggs,but sequencing results of the PCR products amplified by universal bacterial primer show that small amount of Archaea(2.2%)occurred in the egg.These results suggest that the community of gut symbiotic microbes may be jointly controlled by both vertical parental transmission and horizontal environmental transmission,respectively,influenced by many internal and external factors,and evolved together with the host.Gut microbes show high similarity in general while differences do occur among individuals.These results imply that the gut symbiotic microbes change dynamically in response to change in growth stage and characteristics of individuals.We studied the host-microbe interactions with two BPI gene sequences,with high structural similarity,of the squid from RNA sequencing data.We characterized the two host proteins(EsBPI2 and EsBPI4)and identified their functions.These proteins have molecular characteristics typical of other animal BPIs,are closely related,and nest phylogenetically among other invertebrate BPIs.Affinity purified,EsBPIs have potent antimicrobial activities against the squid's Gram-negative symbiont,Vibrio fischeri,which coionizes the light-organ crypt epithelia.Activity of both proteins was abrogated by heat treatment and coincubation with specific antibodies.Pretreatment under acidic conditions similar to those presented during symbiosis initiation rendered the Vibrio fischeri more resistant to the proteins' antimicrobial activity.Confocal immunocytochemistry of juvenile animals localized both EsBPIs not only to the symbiotic organ,but also to other epithelial surfaces that interact directly with environmental seawater.The proteins were distinguished by their intracellular distributions.Further,whereas EsBPI4 was restricted to epithelia,EsBPI2 also occurred both in the blood and in a transient juvenileorgan that mediates hatching from the egg;the EsBPI2 labeling of this organ was lost as it underwent the typical post-hatch degradation.The data provide evidence that these BPIs play different defensive roles early in the life of Euprymna scolopes,modulating interactions with the symbiont and protecting host tissues from colonization by environmental bacteria.This study describes new functions for BPIs,including a biochemical mechanism by which marine animals can resist uncontrolled fouling by members of the bacterioplankton,and a role in the modulation of a beneficial symbiotic association.