Towards the generation of gnotobiotic larvae as a tool to investigate the influence of the microbiome on the development of the amphibian immune system
Data files
Apr 03, 2023 version files 10.88 GB
Abstract
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.
Methods
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 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.
Development and Exposure Experiment with Batracochytrium dendrobatidis (Bd)
We collected skin swab samples to test for Bd 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 Bd 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 Bd load by using the cycle threshold (Ct) value to calculate genomic equivalences. We also adjusted the Bd plasmid copy numbers by accounting for dilution during the extraction process.
Usage notes
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.