Pinpointing the microbiota of tardigrades: what is really there?

Abstract

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.

Implementing high-throughput insect barcoding in microbiome studies: impact of non-destructive DNA extraction on microbiome reconstruction

“…For these reasons, it is important to estimate the absolute abundance of the microbiome rather than just report the relative abundance of microbial clades. Unfortunately, with some notable exceptions (Hammer et al, 2017; Sanders et al, 2017; Ravenscraft et al, 2019; Surmacz et al, 2024), it is rarely done in experimental studies. We argue that the available tools for abundance estimation, including quantitative PCRs and spike-ins (Props et al, 2017; Tourlousse et al, 2017; Jian et al, 2020; Harrison et al, 2021), should become a part of standard microbiome analyses.…”