Miller AJ et al. (2023), Towards the generation of gnotobiotic larvae as...

XB-ART-59812

Philos Trans R Soc Lond B Biol Sci 2023 Jul 31;3781882:20220125. doi: 10.1098/rstb.2022.0125.

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

Miller AJ, Gass J, Jo MC, Bishop L, Petereit J, Woodhams DC, Voyles J.

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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. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

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	Figure 1. . Microbial richness of groups of Xenopus laevis larvae treated with antimicrobial cocktail or sham control solutions and reared in sterile and non-sterile conditions. (a,d) The number of unique operational taxonomic units (OTUs, a common measure for richness) in tadpoles following five weeks of development in sterile and non-sterile conditions. (b,e) Shannon diversity index values, which measure richness and evenness of OTUs in a community, for groups of X. laevis tadpoles following five weeks of development in sterile and non-sterile conditions. (c,f). Inverse Simpson diversity values, which are an index of dominance of OTUs, for groups of X. laevis tadpoles following five weeks of development in sterile and non-sterile conditions. Box and whisker plots show median values with upper and lower quartiles and maximum and minimum values.
	Figure 2. . Non-metric multidimensional scaling (NMDS) ordination of groups of tadpoles treated with antimicrobial cocktail or sham control solutions and reared in sterile and non-sterile conditions. (a) Groups of tadpoles after antimicrobial (AMX; purple points and ellipse) or sham control treatments (no AMX; grey points and ellipse) and reared with sterile food (darker shade of purple and grey points) or non-sterile food (lighter shades of purple and grey points). (b) NMDS ordination of treatment groups of tadpoles after a single treatment of antimicrobial 1 (AMX1; purple points and ellipse) or sham control treatments (no AMX; grey points and ellipse) or antimicrobial 2 (AMX2; blue points and ellipses) that were administered once (light blue points), twice (medium blue points) or three times (dark blue points).
	Figure 3. . Rarefied total OTU abundances of microbes from multiple phyla in Xenopus laevis larvae treated with antimicrobial cocktail or sham control solutions and reared in sterile and non-sterile conditions. (a) Group mean and (b) individual absolute abundances in seven microbial phyla.
	Figure 4. . Development, body condition and survival in groups of Xenopus laevis treated with antimicrobial cocktail or sham control solutions and reared in sterile and non-sterile conditions. (a,d) Percentage of froglets (total number of frogs/initial number of embryos; not including tadpoles used for 16 s sequencing) of Xenopus laevis that successfully completed metamorphosis (% with 95% Clopper–Pearson confidence intervals). (b,e) Body condition of tadpoles taken five weeks after embryo arrival (calculated as mass/snout-to-vent length). (c,f) Survivorship of X. laevis tadpoles during development. Box and whisker plots show median values with upper and lower quartiles and maximum and minimum values.
	Figure 5. . Body condition after metamorphosis, change in body condition over time in an inoculation experiment, and intensity of infection (pathogen load) after exposure to Batrachochytrium dendrobatidis (Bd). (a) Body condition of frogs taken after rearing in sterile and non-sterile conditions and three weeks after metamorphosis (calculated as mass/snout-to-vent length). (b) Body condition for all groups over time in weeks following Bd exposure (dots indicate mean intensity of infection as determined with quantitative PCR. Error bars indicate standard error of the mean). (c) Intensity of infection (pathogen load) after exposure for all groups throughout the Bd infection. Pathogen load was calculated as log(genomic equivalents (GE) + 1). Error bars indicate standard error of the mean.

References [+] :

Al Nabhani, Imprinting of the immune system by the microbiota early in life. 2020, Pubmed