An optimised molecular-based method for ecological study of tardigrades in soils

To improve approaches for studying soil tardigrades, we compared three tardigrade extraction methods, including centrifugal flotation, Baermann funnel, and Whitehead tray, and found that the Whitehead tray method outperformed the two others. We optimised the Whitehead tray extraction condition under various soil pre-treatment processes, sample sizes, light exposure, and extraction time. We found that extending light exposure of non-sieved soils facilitates the extraction of tardigrades. We sequenced the DNA extracted from isolated tardigrade individuals and environmental soil samples, and found that tardigrade DNA extracts yielded a 173-fold increase in the proportion of tardigrade sequences and a 12-fold improvement in the estimation of tardigrade diversity. • The Whitehead tray method outperformed centrifugal flotation and the Baermann funnel in soil tardigrade extraction. • Prolonged light exposure of non-sieved soils enhanced tardigrade extraction. • Tardigrade extractions improved tardigrade detection in samples where soil environmental DNA failed. • Extracting DNA from tardigrade individuals increased sequence recovery and diversity detection.