Dataset Information

Best practices for germ-free derivation and gnotobiotic zebrafish husbandry.

ABSTRACT: All animals are ecosystems with resident microbial communities, referred to as microbiota, which play profound roles in host development, physiology, and evolution. Enabled by new DNA sequencing technologies, there is a burgeoning interest in animal-microbiota interactions, but dissecting the specific impacts of microbes on their hosts is experimentally challenging. Gnotobiology, the study of biological systems in which all members are known, enables precise experimental analysis of the necessity and sufficiency of microbes in animal biology by deriving animals germ-free (GF) and inoculating them with defined microbial lineages. Mammalian host models have long dominated gnotobiology, but we have recently adapted gnotobiotic approaches to the zebrafish (Danio rerio), an important aquatic model. Zebrafish offer several experimental attributes that enable rapid, large-scale gnotobiotic experimentation with high replication rates and exquisite optical resolution. Here we describe detailed protocols for three procedures that form the foundation of zebrafish gnotobiology: derivation of GF embryos, microbial association of GF animals, and long-term, GF husbandry. Our aim is to provide sufficient guidance in zebrafish gnotobiotic methodology to expand and enrich this exciting field of research.

SUBMITTER: Melancon E

PROVIDER: S-EPMC5568843 | biostudies-other | 2017

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiota to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat mass than germ-free (GF) or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure, and respiratory quotient using a novel isolator-housed metabolic cage system, which allows long-term measurements without contamination risk. This demonstrated that microbiota-released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and GF mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well integrated into appetite control. We also observed no significant difference in energy expenditure after normalization by lean mass between the different microbiota groups, suggesting that cumulative small differences in energy balance, or altered energy storage, must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: In GF mice, the respiratory exchange ratio (RER) was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly, the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied GF mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a GF-like drop in liver glycogen storage during the light phase and both liver and plasma metabolomes of OligoMM12 mice clustered closely with GF mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle and which are absent in the OligoMM12 consortium.

Project description:Background: Germ-free or axenic organisms are valuable tools for studying immunity, digestion, and development in different hosts. Although most of these studies have been conducted on mice, recently, germ-free invertebrate models (e.g. Drosophila and Apis) are used due to their easy husbandry, low cost for production, maintenance and the high number of individuals per generation they produce. However, a limitation of using these insects is the simple bacterial community present in their guts. The gut of the American cockroach Periplaneta americana displays a complex gut bacterial community composed of hundreds of species. Using P. americana, we developed a germ-free omnivorous invertebrate model to investigate how gut bacteria stimulate and shape normal gut development and metabolism. To determine if the insect host is directly affected by the presence of specific members of their bacterial community, gnotobiotic cockroaches were generated by inoculating a set of various P. americana gut-endemic Gram-negative (Bacteroidetes; n=11) and Gram-positive (Firmicutes; n=2) bacterial strains into germ-free insects. Additionally, we were able to recover the ‘normal’ bacterial-induced gut phenotype by co-housing germ-free cockroaches with wildtype P. americana to produce gut-bacteria conventionalized insects. Changes in gene expression profiles from two distinct regions (midgut and hindgut) of P. americana guts were quantified by RNA-Seq analysis of the germfree, gnotobiotic and conventionalized insects. Basic transcriptomics description: High-resolution transcriptome profiling of germ-free, gnotobiotic, and conventionalized treated P. americana midgut and hindguts. Ca. 43 million reads were obtained for each treatment. A de-novo assembly of all sequence reads was performed by Trinity assembler. Transcriptome assembly yielded 369,082 gene models and 554,155 isoforms. After running Trinotate pipeline, 65,047 (12 %) these transcripts matched an annotated product in at least one of the reference databases used (Uniprot, pfam, KEGG, COG). Additionally, 1,008 putative bacterial genes were annotated in the P. americana genome and ultimately excluded from these analyses. After bacteria decontamination, 553,147 assembled isoforms were used for transcript quantification and differential expression analysis using the DESeq2 pipeline. DESeq2 analysis detected 6,730 and 3,958 differentially expressed transcripts among the germ-free, gnotobiotic and conventionalized treatments in P. americana hindgut and midgut, respectively.

Dataset Information

Best practices for germ-free derivation and gnotobiotic zebrafish husbandry.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets