Towards the generation of gnotobiotic larvae as a tool to investigate the influence of the microbiome on the development of the amphibian immune system

The immune equilibrium model suggests that exposure to microbes during early life primes immune responses for pathogen exposure later in life. While recent studies using a range of gnotobiotic (germ-free) model organisms offer support for this theory, we currently lack a tractable model system for investigating the influence of the microbiome on immune system development. Here, we used an amphibian species (Xenopus laevis) to investigate the importance of the microbiome in larval development and susceptibility to infectious disease later in life. We found that experimental reductions of the microbiome during embryonic and larval stages effectively reduced microbial richness, diversity, and altered community composition in tadpoles prior to metamorphosis. In addition, our antimicrobial treatments resulted in few negative effects on larval development, body condition, or survival to metamorphosis. However, contrary to our predictions, our antimicrobial treatments did not alter suscepti..., DNA extractions for 16s sequencing
On week five of tadpole development, we humanely euthanized N = 6 (Experiment 1) and N = 10 (Experiment 2) tadpoles per treatment group for 16S rRNA targeted amplicon microbiome sequencing analysis. We randomly selected tadpoles from each tank and weighed tadpoles to the nearest 0.1 g and measured snout-to-vent length (hereafter, SVL) to the nearest 0.1 mm to calculate body condition (mass/SVL). We euthanized the tadpoles using sterile MS-222 that we neutralized by adding sodium bicarbonate to pH 7.0. We then transferred the tadpoles to Powerbead Pro Tubes (Qiagen, Valencia, CA, USA). We homogenized the tadpoles using a tissue homogenizer (Mixer Mill MM 400; Retsch, Newtown, PA, USA) for 3 minutes, each at 25 hz. We then extracted DNA from the homogenized samples using the QIAmp PowerFecal Pro DNA Kit (Qiagen, Valencia, CA, USA). We included N = 4 water blanks for Experiment 1 and N = 5 water blanks for Experiment 2 for which we followed the same proto..., The raw sequencing data files are uploaded in a .fastq file format. All other data files are uploaded as Excel (.xlsx) files. Please see README files for more information.