Inferring Microbial Interactions in the Gut of the Hong Kong Whipping Frog () and a Validation Using Probiotics.

The concerted activity of intestinal microbes is crucial to the health and development of their host organisms. Investigation of microbial interactions in the gut should deepen our understanding of how these micro-ecosystems function. Due to advances in Next Generation Sequencing (NGS) technologies, various bioinformatic strategies have been proposed to investigate these microbial interactions. However, due to the complexity of the intestinal microbial community and difficulties in monitoring their interactions, at present there is a gap between the theory and biological application. In order to construct and validate microbial relationships, we first induce a community shift from simple to complex by manipulating artificial hibernation (AH) in the treefrog . To monitor community growth and microbial interactions, we further performed a time-course screen using a 16S rRNA amplicon approach and a Lotka-Volterra model. Lotka-Volterra models, also known as , predict the dynamics of microbial communities and how communities are structured and sustained. An interaction network of gut microbiota at the genus level in the treefrog was constructed using Metagenomic Microbial Interaction Simulator (MetaMIS) package. The interaction network obtained had 1,568 commensal, 1,737 amensal, 3,777 mutual, and 3,232 competitive relationships, e.g., has a commensal relationship with . To validate the interacting relationships, the gut microbe composition was analyzed after probiotic trials using single strain (, and , respectively) and a combination of , and , because of the cooperative relationship among their respective genera identified in the interaction network. After a 2 week trial, we found via 16S rRNA amplicon analysis that the combination of cooperative microbes yielded significantly higher probiotic concentrations than single strains, and the immune response (interleukin-10 expression) also significantly changed in a manner consistent with improved probiotic effects. By taking advantage of microbial community shift from simple to complex, we thus constructed a reliable microbial interaction network, and validated it using probiotic strains as a test system.