Pinpointing the microbiota of tardigrades: what is really there?

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Microbiota have been proposed as an important aspect of tardigrade biology, but little is known about their diversity and distribution. Here, we attempted to characterize the microbiota of 44 cultured species of tardigrades using 16S rRNA amplicon sequencing, using different specimen pooling strategies, various DNA extraction kits, and multiple types of controls. We also estimated the number of microbes in samples using synthetic DNA spike-ins. Additionally, we reanalyzed data from previous studies.

Our results suggest that the microbial community profiles of cultured tardigrades are dominated by bacterial OTUs and genotypes originating from food, medium, or laboratory reagents. We found microbial strains consistently enriched in certain tardigrades (relative to the culture media and controls), which indicates likely symbiotic associations, but the reads representing putative true tardigrade-associated microbes rarely exceeded 20% of the datasets. Some of the identified tardigrade-associated microbes matched symbionts identified by other studies. However, we also identified serious contamination issues with previous studies of tardigrade microbiome, making some of their conclusions questionable. We conclude that tardigrades are not universally dependent on specialized microbes and highlight the necessary safeguards in future studies of the microbiota of microscopic organisms.

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  1. 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.

  2. 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: Major kudos to you for your persistence on this question!