Tips and tricks how to culture water bears: simple protocols for culturing eutardigrades (Tardigrada) under laboratory conditions

Abstract

Tardigrades are microinvertebrates inhabiting almost all aquatic and terrestrial ecosystems throughout the world. They are known for their ability to enter into cryptobiosis and to survive extreme environmental conditions (e.g. lack of water, very high and low temperatures, high doses of radiation, vacuum space). Thanks to these abilities, tardigrades are excellent model organisms for various types of studies, e.g. ecological, ethological, physiological, astrobiological, biotechnological or medical, or even in integrative taxonomy. For most of these studies well-established tardigrade cultures are essential. Here we present a review of methods/protocols used in tardigrade culturing in the past. Based on this data and on our several years of experience in tardigrade culturing, we tried different methods and developed new ones that seem to be optimal. Here, we propose our own simple protocols for culturing herbivorous, omnivorous and carnivorous eutardigrade species in environmental chambers as well as in room conditions. We also describe methods for culturing rotifers, nematodes and algae, used as food sources for tardigrades. Moreover, many years of tardigrade culturing allowed us to describe the problems that may occur during culturing, explain their causes and propose solutions. We believe that these simple protocols will be very useful for many scientists planning tardigrade applications in their studies.

The tardigrade Hypsibius exemplaris has the active mitochondrial alternative oxidase that could be studied at animal organismal level

“…Accordingly, fast reproduction and simple culture protocol [e.g. 37 , 38 ] as well as not sophisticated procedure of monitoring mitochondria functioning in intact animals including the whole-body respiration and mitochondria coupling, could enable analysis of the affected animals’ physiology and facilitate fast screening of pharmacologically active compounds. Moreover, H .…”

“…Z151 strain of the parthenogenetic species Hypsibius exemplaris was purchased from Sciento (Manchester, United Kingdom) and cultured according to the published protocol [ 37 ]. To maintain the culture, specimens were kept in POL EKO KK 115 TOP + climate chamber (photoperiod 12h light/12h dark, 20 o C, relative humidity of 50%) on Petri-dishes (55 mm in diameter) with bottom scratched by sandpaper to allow tardigrade locomotion.…”

Verification of Hypsibius exemplaris Gąsiorek et al., 2018 (Eutardigrada; Hypsibiidae) application in anhydrobiosis research

“…Chlorella vulgaris Beijerinck 1890 [51] (SAG211-11b strain) was served as a food once per week after the dish cleaning. Animals were transferred to a new culture dishes every few months (for details see [52]). The algae strain was kindly provided by Marcin Dziuba (Department of Hydrobiology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland) and was obtained from the culture collection of algae (Sammlung von Algenkulturen (SAG)) at the University of Göttingen, Germany.…”

How to culture limnoterrestrial heterotardigrades

“…The ability to rear tardigrades under laboratory conditions enables replicable experiments to study various aspects of their fascinating biology (Suzuki, 2003). There is, however, no unified protocol for culturing all tardigrade taxa, and available protocols proposed to date concern eutardigrades only (Greven, 2018;Roszkowska et al, 2021). The published methods differ depending on the specific environmental requirements of different species (Arakawa et al, 2016;Hohberg, 2006;Morek et al, 2020).…”

“…These methods differ primarily in the type of food they use, walking substrate, photoperiod, and ambient temperature (Altiero & Rebecchi, 2001;Roszkowska et al, 2021). Aside from having a suitable, oxygenated water source, the most important requirement for successfully culturing tardigrades appears to be their food source (Bryndová et al, 2020;Sayre, 1969).…”