How to culture limnoterrestrial heterotardigrades

Abstract

Zoologists have long tried to culture the limnoterrestrial heterotardigrades associated with bryophytes and lichens without success. By carefully observing heterotardigrades in the genera Echiniscus, Pseudechiniscus, and Viridiscus over many months, we determined that these organisms feed on chloroplasts and cytoplasm from the cells of moss (typically moss protonema), and on the single-celled green algae associated with the moss (typically Chlorella vulgaris). We also determined that the cryptogams they associate with, and hence the heterotardigrades themselves, spend more time in a dried state than do most eutardigrades, which are more easily cultured. Taking these observations into account, we varied food, water, and desiccation cycle conditions with the aim of developing a viable culturing protocol for heterotardigrades. We used this experimentally derived protocol to maintain laboratory cultures of the tardigrades Echiniscus sp., Pseudechiniscus aff. P. ramazzotti and Viridiscus viridianus, which regularly produced new eggs and hatchlings. Both moss and algae from their natural habitats were used as food sources, and a small layer of rain or spring water was added every morning and allowed to partially evaporate overnight. Furthermore, the organisms were more likely to lay eggs on a dense mat of moss protonema, grown by inserting tips of moss branches into a solidified KCM–agar medium. The medium also provided a walking substrate for the tardigrades, and possibly a source of cations. Crucially, the cultures were allowed to dry out completely every 10 days for a period of at least 3 days. Moss in the culture dish significantly improved the chances of the tardigrades coming out of their desiccation-resistant states successfully. The ability to culture heterotardigrades makes these organisms available for modern genomics and other studies with implications for understanding desiccation resistance mechanisms beyond those studied in a few model eutardigrade species.