DOI: 10.5061/dryad.t76hdr849
title: Towards the generation of gnotobiotic larvae as a tool to investigate the influence of the microbiome on the development of the amphibian immune system

description: 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 susceptibility to the lethal fungal
 pathogen Batrachochytrium dendrobatidis (Bd) in the
 adult life stage. While our treatments to reduce the microbiome
 during early development did not play a critical role in determining
 susceptibility to disease caused
 by Bd in X. laevis, they nevertheless indicate
 that developing a gnotobiotic amphibian model system may be
 highly useful for future immunological investigations.
description: <strong>DNA extractions for 16s
 sequencing</strong> 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
 protocol for extraction, substituting molecular-grade water instead of a
 tadpole sample. We then shipped the samples on ice to the Idaho State
 University Molecular Research Core Facility for 16S rRNA
 sequencing.  We received fastq files from Idaho State
 University that were demultiplexed and had primers/adapters removed. These
 fastq files have been uploaded.
 <strong>Development and Exposure Experiment with
 <em>Batracochytrium dendrobatidis
 </em>(<em>Bd</em>)</strong> We
 collected skin swab samples to test for <em>Bd</em> presence
 and infection intensity using standardized swabbing techniques. During the
 infection experiment, we continued to collect mass, SVL (as described
 above), and skin swab samples for diagnostic testing every two weeks until
 the termination of the experiment. We extracted DNA from our swabs using
 the DNeasy Blood and Tissue DNA Extraction Kit (Qiagen, Valencia,
 California, USA). We then quantified <em>Bd</em> DNA on the
 swabs using real-time quantitative polymerase chain reaction (qPCR). We
 used an internal positive control and a dilution set of plasmid standard
 (Pisces Molecular, Boulder, CO, USA) to quantify Bd load. We
 determined <em>Bd</em> load by using the cycle threshold (Ct)
 value to calculate genomic equivalences. We also adjusted
 the <em>Bd</em> plasmid copy numbers by accounting for
 dilution during the extraction process.  
description: 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.