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BACKGROUND: Historically the main source of laboratory Xenopus laevis was the environment. The increase in genetically altered animals and evolving governmental constraints around using wild-caught animals for research has led to the establishment of resource centres that supply animals and reagents worldwide, such as the European Xenopus Resource Centre. In the last decade, centres were encouraged to keep animals in a "low microbial load" or "clean" state, where embryos are surface sterilized before entering the housing system; instead of the conventional, "standard" conditions where frogs and embryos are kept without prior surface treatment. Despite Xenopus laevis having been kept in captivity for almost a century, surprisingly little is known about the frogs as a holobiont and how changing the microbiome may affect resistance to disease. This study examines how the different treatment conditions, "clean" and "standard" husbandry in recirculating housing, affects the skin microbiome of tadpoles and female adults. This is particularly important when considering the potential for poor welfare caused by a change in husbandry method as animals move from resource centres to smaller research colonies.
RESULTS: We found strong evidence for developmental control of the surface microbiome on Xenopus laevis; adults had extremely similar microbial communities independent of their housing, while both tadpole and environmental microbiome communities were less resilient and showed greater diversity.
CONCLUSIONS: Our findings suggest that the adult Xenopus laevis microbiome is controlled and selected by the host. This indicates that the surface microbiome of adult Xenopus laevis is stable and defined independently of the environment in which it is housed, suggesting that the use of clean husbandry conditions poses little risk to the skin microbiome when transferring adult frogs to research laboratories. This will have important implications for frog health applicable to Xenopus laevis research centres throughout the world.
212942/Z/18/Z Wellcome Trust , BB/R014841/1 Biotechnology and Biological Sciences Research Council (GB), E3 Research England, NC/P001009/1 National Centre for the Replacement, Refinement and Reduction of Animals in Research
Fig. 1. Experimental design. Schematic outlining the housing conditions, developmental stages, and experimental set up used in this experiment
Fig. 2. Alpha diversity across the data set. Alpha diversity (represented by the Shannon Index) across the data set, representing the diversity of species within each sample group. For each sample, the alpha diversity was calculated based on 10 random subsamples of 5000 OTUs. Significant differences between groups are based on an independent two-sample t-test (** = p ≤ 0.01; * = p ≤ 0.05; ns = not significant). Full alpha diversity values for each individual sample, including observed diversity and Chao1 Index, can be found in Supplementary Table 1
Fig 3. Principal coordinates analysis (PCoA) of all samples in the data set. Axis 1 and Axis 2 represent the coordinates of the greatest sources of orthogonal variation within the data and represent 27.9 and 9.4% of variation in the entire data set respectively. Whilst results from biological replicates and distinct tanks are generally consistent, Axis 1 represents the difference in diversity between the adult frogs as compared to both tadpoles and environmental water samples. Axis 2 shows close clustering between adults housed in standard and clean conditions, and tadpoles and their environmental water samples for clean conditions only. However, a significant change in diversity is seen for tadpoles from standard conditions, despite water samples from standard tanks showing similar profiles to those from clean tanks.
Fig 4. Barplot showing the distribution of bacteria at the phylum level of taxonomy. In each case, the top 15 phyla are highlighted, with all remaining phyla shown in grey.
Fig. 5. Comparison between genera identified on adult frogs, tadpoles and in water controls. Venn diagrams showing the overlap between unique genera identified in samples from standard conditions compared to clean conditions for a) adult frogs, b) tadpoles and c) water samples.
Fig. 6. Heatmap showing the top 50 species identified across the data set. OTU counts from biological replicates amongst the different replicate tanks were combined to give a single count, which was normalised so that all samples had the same total. Cells are coloured based on the percentage of the total OTU count (on a log10 scale), with more abundant OTUs shown in red
Fig. 7. Relative abundance (%) of core microbiome members shared between clean and standard housing conditions for adult, ii) tadpole, and iii) environmental water samples. OTUs were combined at the Genus level and were identified as core microbiome members based on an average relative abundance greater than 1% in both Clean and Standard conditions. Comparative relative abundance (on a log10 scale) is plotted for clean and standard conditions to highlight similarities and differences between the housing conditions
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