How To Culture Limnoterrestrial Heterotardigrades: A Case Study In The Echiniscus, Pseudechiniscus And Viridiscus Genera

Background: Tardigradologists have long tried to culture limnoterrestrial heterotardigrades without success. This is likely because they depend on specific conditions in their microhabitats. Most limnoterrestrial heterotardigrades are found associated with bryophytes and lichens (collectively cryptogams). In contrast to eutardigrades, many of which are easily cultured, heterotardigrades are found in cryptogams that prefer drier and sunnier habitats and that tend to grow on the vertical surfaces of boulders, cliffs, or tree trunks. By carefully observing heterotardigrades in the Echniscus and Viridiscus genera over many months, we determined that these organisms feed on chloroplasts and cytoplasm of both moss cells (typically moss protonema) and 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 most eutardigrade species. Taking these observations into account we varied food, water, and desiccation cycle conditions with the aim of developing a culturing protocol for heterotardigrades. Results: We have maintained laboratory cultures of several generations and counting of Echiniscus, Pseudechniscus and Viridiscus genera tardigrades using the following experimentally derived protocol: Both moss and algae from their natural habitats are required as food sources and a small layer of rain or spring water is added every morning and allowed to evaporate some overnight. Furthermore, the organisms are 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 provides a walking substrate for the tardigrades, and possibly a source of cations. Crucially, the cultures must be allowed to dry out completely every ten days for a period of at least three days. Moss in the culture dish significantly improves the chances of the tardigrades coming out of their desiccation-resistant states successfully. Conclusions: We conclude that periodic drying out, moss and algae are all required to successfully culture heterotardigrades. Furthermore, drying must occur slowly, and the moss protonema enables this by retaining moisture thereby slowing evaporation. We suspect our protocol will work for most limnoterrestrial heterotardigrades with minor tweaking of culturing conditions.