The Optimization Of Germ-free Zebrafish Model And The Comparison Of Two High-throughput Sequencing Platforms In Intestinal Microbial Function Study

The digestive tract of vertebrates is a home to numerous symbiotic microorganisms,which are also known as a very important"organ"of the host.The microbiota plays important roles in various physiological processes,including modulation of immune system,enhancement of intestinal barrier,absorption or digestion of nutrients,inhibition of pathogens and host behavior and neurodevelopment.Germ-free animal technology and microbial sequence technology are vital platforms to understand the exact functions of commensals.However,these two techniques still face some challenges in practice.The first question is how to determine the inoculation conditions of germ-free animal models.The second question is how to confirm that sequencing data can reflect the function of microorganisms.In view of the above two questions,this paper explores the following two aspects:1.Germ-free or gnotobiotic animals act as simple and powerful tools for elucidating the causality relationships between hosts and their microbial residents Colonizing germ-free?GF?zebrafish with specific bacterial species provides the possibility of understanding the influence on host biological processes including gene expression,development,immunity and behavioral responses.However,the colonization conditions of microorganisms in different studies are varied,including colonization time,colonization concentration and exposure time.The responses of GF zebrafish to various colonization conditions remain unclear.To address this issue,we explored the responses of GF zebrafish by using two bacterial species at varying concentrations,colonization time points and exposure duration.Therefore,we mono-associated GF zebrafish with Escherichia coli DH5?or Bacillus subtilis WB800N at concentrations ranging from 10² to 10⁷ CFU/mL either at3 day post fertilization?dpf?or 5 dpf for 24 or 48 hours.We evaluated the responses of GF zebrafish by analyzing the survival rate,colonization efficiency,nutrients metabolism,intestinal cell proliferation,innate immunity,stress and behavior responses by comparing it to conventionally raised zebrafish?CONR?and GF zebrafish.?i?The results indicated that the final bacteria concentrations ranging from 10⁴ to 10⁶ CFU/mL are relatively appropriate inoculation concentrations.?ii?The E.coli DH5?mainly induced the expression of genes related to nutrients metabolism,cell proliferation and immunity,while B.subtilis WB800N mainly upregulated the expression of genes related to immunity and stress responses.This study provides useful information on bacterial colonization of GF zebrafish and the interaction between the host and microbiota.2.With the rapid development of amplicon sequencing technology,high-throughput sequencing based on 16S rRNA gene has become a powerful tool for exploring the bacterial composition in an ecosystem.At present,there are two kinds of high-throughput sequencing based on bacterial 16S rRNA gene.One is total microbial sequencing technology based on bacterial 16S rRNA gene and another is active microbial sequencing technology based on bacterial 16S rRNA.Total microbial sequencing technology can reflect the existing microbial members rather than selecting the active members.Active microbial sequencing technology can capture subtle changes in microbial reacting to the environment but microbial RNA lacks stability and gene copy numbers may vary.In order to investigate the advantages and disadvantages of 16S rRNA gene scequence technology and 16S rRNA scequence technology,zebrafish model with excess fat accumulation were performed.Wide-type zebrafish?WT?fed with control diet?CON?,high-carbohydrate diet?HC?or high-fat diet?HF?and two kinds of gene knockout zebrafish(cpt1b^-/-and ppar?b^-/-)fed with a control diet were involved in the experiment.After four weeks of treatment,intestinal microbiome of zebrafish was analyzed using 16S rRNA gene sequence?DNA-based?and 16S rRNA sequence?RNA-based?.?i?The results indicated that diet treatments especially HF diet donated more to lipid accumulation of zebrafish and microbial composition than host genetics.?ii?Compared with total microbial sequencing,the active microbiota reflect more consistent alterations with the phenotype of zebrafish,which was also verified by microbiota transplantation to germ-free zebrafish.