Pinpointing the microbiota of tardigrades: What is really there?
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Abstract
Microbiota are considered significant in the biology of tardigrades, yet their diversity and distribution remain largely unexplored. This is partly due to the methodological challenges associated with studying the microbiota of small organisms that inhabit microbe‐rich environments. In our study, we characterized the microbiota of 31 species of cultured tardigrades using 16S rRNA amplicon sequencing. We employed various sample preparation strategies and multiple types of controls and estimated the number of microbes in samples using synthetic DNA spike‐ins. We also reanalysed data from previous tardigrade microbiome studies. Our findings suggest that the microbial communities of cultured tardigrades are predominantly composed of bacterial genotypes originating from food, medium, or reagents. Despite numerous experiments, we found it challenging to identify strains that were enriched in certain tardigrades, which would have indicated likely symbiotic associations. Putative tardigrade‐associated microbes rarely constituted more than 20% of the datasets, although some matched symbionts identified in other studies. We also uncovered serious contamination issues in previous tardigrade microbiome studies, casting doubt on some of their conclusions. We concluded that tardigrades are not universally dependent on specialized microbes. Our work underscores the need for rigorous safeguards in studies of the microbiota of microscopic organisms and serves as a cautionary tale for studies involving samples with low microbiome abundance.
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dies
This was super helpful, thank you. And it's interesting to see that most of the suspected contaminants have also been seen as contaminants elsewhere! Thank you for running these analyses.
Any thoughts on what approaches your group will take in the future to avoid this? One thing we ended up finding post-study is that looking for transcripts, i.e. actual expression of genes, ended up being a decent proxy for whether a bacterium was present. Because it is suggestive of a microbe actually being alive and growing. It's not a perfect correlation, but it was surprisingly tight.
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M. radiatus
Any ideas as to why Burkholderiaceae and M. radiatus were so sensitive to these experimental procedure differences?
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We show that the vast majority of bacterial signal in tardigrade microbial community profiles, whether sequenced by us or other authors, originates from sources other than the tardigrades themselves
Thank you so much for doing this careful study. It's so important to get this right, and I know it can be challenging to wade through conflicting observations. Your overview of this in the intro is also very helpful. We also struggled with similar issues for the tick microbiome, and it took quite a bit of work to experimentally consider the "negative result" (see here: https://www.biorxiv.org/content/10.1101/198267v1). Major kudos to you for your persistence on this question!
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