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.


Introduction
There is at present no standard method for tardigrade culturing, and such a standard, unified approach for all tardigrade taxa will never be proposed. This is due to the fact that different tardigrade species have different environmental requirements. In the past many different culture protocols were proposed by different authors. The first information on tardigrade culturing (probably of Pseudobiotus species), based on keeping them in an aquarium, was published by Von Wenck (1914) and Marcus (1929). Nowadays, different authors use different culture methods not only for different species, but also for the same species (for a review see Altiero et al. 2018). The main differences concern the walking substrate, type of food, ambient temperature and photoperiod (for more details see Table I and the Discussion section).
Such a diversity of applied protocols, for e.g. culturing of the same species, could be potentially problematic for comparing the results and conclusions of different experiments focused on different aspects of tardigrade biology. Therefore, here we propose rearing protocols for stock cultures of five eutardigrade species (terrestrial and freshwater) with different food preferences, i.e. herbivorous, omnivorous and carnivorous. The protocols were developed and, based on our observations, constantly applied and continually improved by us over the past several Table I. Summary of the different freshwater and terrestrial eutardigrade species (in taxonomic order) and culture conditions used by different authors (species names according to Degma et al. 2009Degma et al. -2020; genus abbreviations according to Perry et al. 2019; some species names were updated according to modern literature, i.e. Hys. dujardini/Hys. exemplaris, Mil. tardigradum/ Mil. inceptum, Pam. cf. richtersi/Pam. fairbanksi and Mil. cf. alpigenum/Mil. alpigenum   years. We describe useful tips and tricks for eutardigrade culturing and discuss possible problems that may arise during such laboratory culturing, as well as propose possible solutions. We think that the presented protocols can be useful for different types of studies, e.g. ecological, ethological, physiological, astrobiological, biotechnological or medical, or even in integrative taxonomy.

Origin of the cultured species
The commercial culture of parthenogenetic Hys. Tardigrades were extracted from the samples and studied following the standard methods as described in Dastych (1980). Tardigrade taxonomy follows Bertolani et al. (2014) and later updates for Isohypsibiidae (Gąsiorek et al. 2019). Genus abbreviations follow Perry et al. (2019).

Equipment for keeping, cleaning and feeding eutardigrade cultures
Two climatic chambers were used: a POL EKO KK 115 TOP+ (POL-EKO Aparatura) climatic chamber with controlled temperature and relative humidity (RH) (climatic chamber 1) (Figure 1), and a POL ST1 BASIC (POL-EKO Aparatura) climatic chamber with controlled temperature and photoperiod (climatic chamber 2). The tardigrade cultures were also maintained under room conditions (see section 2.3). All species were reared on plastic vented Petri dishes with one of three combinations of diameters (Ø) and heights: 35 mm and 11 mm, 55 mm and 13 mm or 90 mm and 13 mm, respectively. The bottom of each Petri dish was scratched with sand paper (grid size from P-100 to P-240) to allow tardigrade locomotion. Each Petri dish was filled with a culture medium containing spring water (Żywiec Zdrój: bicarbonates: 121.06 mg/L, fluorides: 0.07 mg/L, Mg 2+ : 5.37 mg/ L, Ca 2+ : 36.39 mg/L, Na + : 7.79 mg/L) mixed with double distilled water (ddH 2 O) in a 1:3 ratio (hereafter referred to simply as culture medium). Sterile plastic disposable (Pasteur) pipettes (3 mL) were applied to exchange culture medium, and automatic pipettes (20-200 µL) were used to add food and extract tardigrades and/or their eggs.

Details on culture conditions for each eutardigrade species
As detailed in Table II, in chamber 1 (see section  2.2) the cultures were performed in complete darkness, at 17°C and at 40% RH (defined as culture regime CR 1), whereas in chamber 2 they were performed at 20°C with 12 h/12 h (L/D) photoperiod, at an RH of ca. 50% (defined as CR 2). It is also possible to grow this culture at room temperature if there is no access to a climatic chamber. We reared tardigrades at room temperature, which ranged from 21 to 24°C, with RH estimated at 40-60% and light conditions naturally regulated (by seasonal changes) in a day/night cycle (defined as CR 3). However, we do not recommend this third method because these conditions are very variable. Table II summarises all culture conditions for each tardigrade species that we tested.
Macrobiotus polypiformis was cultured in Ø 55 mm Petri dishes under CR 2 and 3 with similar rates of success. Initially the species was cultured under CR 3 during autumn, winter and spring, whereas during summer (due to high temperatures in the laboratory) specimens were transferred to the chamber 2 (under CR 2 conditions), and since that time we have continued to culture them successfully using that regime. Algae, rotifers and nematodes were used as food, as we noticed that juveniles fed on algae and that adults had a mixed diet.

Algae
The green alga C. vulgaris strain SAG211-11b was obtained from the culture collection of algae (Sammlung von Algenkulturen (SAG)) at the University of Göttingen, Germany. Algae were cultivated under sterile conditions in Wright's Cryptophytes (WC) medium (Guillard & Lorenzen 1972) (Figure 2a-b). Each turbidostat comprises a 2 L bottle filled 2/3 full with autoclaved liquid WC medium, two inflow channels and a culture excess outflow. One of the inflows was used to supply fresh WC medium by a peristaltic pump at rate of 800 mL/day; filtered (with 0.2 µm filter) air was pumped, by an air pump at the bottom of the chemostat, through the other inflow to mix the culture and Tips and tricks how to culture water bears supply it with CO 2 . When the medium was pumped into the turbidostat, the excess culture was pushed out (due to the overpressure induced by air pumped into the bottle), and collected in another flask. The turbidostats were held in a walk-in phytotron (Conviron), with a constant temperature of 20°C, 160 µmol photons per m 2 /s light intensity and a 16 h/8 h (L/D) photoperiod.
To concentrate the algae in the outflow flask, they were left to settle for 2 days, and the excess medium was decanted afterwards. The settled algae were then transferred without a sterile regime to glass bottles (not completely twisted off) and left for 2-3 weeks so that the algae began to aggregate on the bottom of the bottle. This stock of aggregated algae was further used for feeding the tardigrades.

Rotifers
The rotifer L. inermis strain 1.A2.15 originates from a wastewater treatment plant (WWTP) located in southern Poland and was obtained from a sludge sample derived from the WWTP aeration chamber. The sample was divided into 1 mL portions that were poured into a 24-well tissue culture plate and kept in a SANYO MLR-350 Versatile Environmental Test Chamber (Sanyo Electric Co Ltd) at 15°C for 3 weeks. Then, single rotifers were individually transferred with a micropipette into 24-well tissue culture plates filled with spring water (Żywiec Zdrój), and 10 µL of 2% NOVO (nutrition powder, patent Pajdak-Stós et al. 2017) suspension was added as food source. The plate was transferred to a walk-in climatic chamber with a constant temperature of 20°C and kept in darkness. The strains were controlled every few days and the best proliferating strains were then selected for further culturing.
The selected strain 1.A2.15 was transferred into Ø 55 mm plastic Petri dishes filled with spring water and fed weekly with a pinch of NOVO powder. Since then the cultures have been constantly incubated in the same walk-in climatic chamber in darkness at 20°C. When the density of cultures reached ca. 5000 individuals/mL, the cultures were transferred to Ø 90 mm Petri dishes. Once a week the cultures were checked, half the culture volume from each dish was removed and made up with the fresh spring water, and they were fed with NOVO powder. Strain 1.A2.15 is now constantly cultured in the laboratory of the Aquatic Ecosystems Group, Institute of Environmental Sciences, Jagiellonian University in Kraków.

Nematodes
The wild-type Bristol N2 strain of C. elegans was obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota (Duluth, Minnesota, USA). Standard methods are used for the maintenance and manipulation of the strain under sterile conditions (Brenner 1974). Briefly, animals were grown monoxenically on solid nematode growth medium (NGM; NaCl 50 mM, peptone 0.25%, CaCl 2 1 mM, cholesterol 5 μg/mL, KH 2 PO 4 25 mM, MgSO 4 1 mM and agar 1.7%) using Escherichia coli Migula, 1895 (strain OP50) as the food source. The E. coli strain was grown in liquid Luria broth (LB) medium. An overnight LB culture of E. coli was used to seed the lawn on NGM prepared on Ø 100 mm plates. The plates were incubated overnight at 37°C, allowed to cool before animals were transferred and then incubated at room temperature in the dark. Specimens of C. elegans reproduce quickly and in large numbers. A sterilised spatula was used to move a chunk of agar with animals onto freshly seeded NGM plates, and after 4-5 days animals were harvested by washing them off the plate with the tardigrade culture medium and used as food for tardigrades.

Tardigrade feeding and culture cleaning
All the tardigrade cultures were cleaned, and tardigrades were fed, once per week. This schedule seems to be optimal because at shorter intervals it was observed that not all food was consumed and the medium was still fresh and relatively clean. Tardigrades were fed (with algae and/or rotifers and nematodes) according to the quantities presented in Table III, after cleaning of cultures. As the rotifers tend to concentrate on the bottom of vials, before feeding tardigrades the rotifer cultures were thoroughly mixed. Culture medium was poured onto plates with nematodes (ca. 20-30 mL, depending on the number of tardigrade cultures that needed to be fed) and medium with suspended nematodes was transferred to a 50 mL falcon tube prior to feeding.
Some differences concerning food intake were observed among taxa. For example, Pam. experimentalis specimens consumed food much faster than did specimens of other species, even when cultured at the same density as two Pam. fairbanksi populations. Moreover, some examples of cannibalism were sometimes observed (most often, larger specimens fed on juveniles). A different situation was observed in the case of Mac. polypiformis, which almost never consumed all food offered during the week. In the cultures of herbivorous species (especially in darkness), it was observed that after a week some algae were faded (yellow-brown in colour) and the fading process seemed to occur faster when the number of specimens in culture was higher. This indicates that the tardigrades were at least partly responsible for this process, i.e. by sucking/killing algae cells.
Usually, after a week, a thin iridescent filmprobably the accumulation of metabolic wastes and bacteria -was observed on the surface of the water in almost all dishes (Figure 3a-b). This thin film was always firstly removed during the cleaning procedure. It was removed delicately using the Pasteur pipette, along with the upper part of the culture medium, with special attention paid to not removing the animals or their eggs. Later, fresh culture medium was added and the cultures were then strongly mixed using the pipette. After the animals and their eggs fell to the bottom, floating food remains, old exuviae and dead animals were removed. During this procedure a portion of empty eggs (completely transparent) from the cultures of Macrobiotus and Paramacrobiotus species were also removed. At the same time, if necessary, eggs of Paramacrobiotus species were collected to establish new cultures.
The procedure for establishing new cultures from the eggs begins with transferring eggs to Ø 55 mm Petri dishes with a smooth bottom (not scratched), with a small amount of rotifers as food for hatching juveniles. In these Petri dishes eggs continued to hatch, and once per week juveniles were transferred to new culture Petri dishes (that were scratched on the bottom). Hatching carried out in smooth dishes makes it easier to catch juveniles and transfer them to new cultures. Table IV presents a list of the most frequent problems we observed in our cultures, as well as their possible solutions.

200-300
Milnesium inceptum Nematodes, rotifers 200 µL of nematodes (ca. 3500 specimens in 1 mL) and 2 mL of rotifers (ca. 7500 specimens in 1 mL) 300-400 *These are values that we find optimal per dish, i.e. animals do not show any signs of population overcrowding and remain active. Smaller and larger numbers of animals per dish are also possible, but in that case dishes should be checked more frequently to detect problems (such as anoxia, lack of food, cessation of reproduction, etc.) in time.

Discussion
The available culture protocols concerning Eutardigrada species differ in detail regarding medium, walking substrate, food, culture containers, environmental conditions (e.g. ambient temperature or photoperiod) and cleaning/feeding schedule. The majority of cultured species are freshwater or terrestrial taxa from the superfamilies Hypsibioidea Pilato 1969, Isohypsibioidea Sands et al. 2008or Macrobiotoidea Thulin 1928. However, one of the most commonly reared taxa is Milnesium Doyère, 1840, belonging to the family Milnesiidae Ramazzotti 1962. As shown in Table I, the number of successfully cultured taxa is high, but it is even higher taking into account all the other species to which protocols used for one species have been later adapted. For example, the protocol proposed by Stec et al. (2015) for Mac. paulinae Stec, Smolak, Kaczmarek & Michalczyk, 2015 (Pilato, Binda and Catanzaro, 1991) and Pam. lachowskae Stec, Roszkowska, Kaczmarek & Michalczyk, 2018, 2018a, 2018b, 2018c, 2018dNowak & Stec 2018;. Also, the protocol suggested for Hys. exemplaris was used later for Mac. shonaicus Stec, Arakawa andMichalczyk, 2018 (Stec et al. 2018e). This clearly suggests that for species with similar food and environmental requirements, culturing methods can be considered more or less universal.
Carnivorous species are fed mainly with small rotifers or/and nematodes of a few species, whereas herbivorous tardigrades are fed mainly with different species of unicellular freshwater algae (for more details see Table I). Our observations indicate that some species (like Mac. polypiformis) may have a mixed diet, which was also observed for Mac. shonaicus Stec, Arakawa and Michalczyk, 2018, for example (Sugiura et al. 2020a). Although a few different species of algae, nematodes and rotifers have been proposed as food for different Eutardigrada species, the most often used were algae: Chlorococcum sp. or Chlorella vulgaris Beijerinck 1890; nematodes: Caenorhabditis elegans (Maupas, 1900) and rotifers: Lecane inermis (Bryce, 1892). Other types of food proposed for some tardigrade species were bacteria, diatoms, cyanobacteria, other tardigrades or even fungi (Nelson et al. 2010;Guidetti et al. 2012;Roszkowska et al. 2016;Morek et al. 2020;Bryndová et al. 2020). Moreover, the same type of food (the same prey species or algae) has been proposed for different tardigrade taxa, which shows that tardigrades are not monophagous and some are even omnivorous, which was also previously suggested by several authors (e.g. Schill et al. 2011;Kosztyła et al. 2016;Bryndová et al. 2020;Kihm et al. 2020). However, it should be mentioned that lifespan and reproductive success can be strongly affected by food type (Bryndová et al. 2020).
The majority of cultures are maintained on plastic Petri dishes with a thin layer of agar on the bottom of the dish. Some authors used other plastic containers, or glass, such as watch glasses, bottles or Erlenmeyer

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M. Roszkowska et al. flasks. Because tardigrades are unable to crawl on smooth glass or plastic surfaces, different researchers use agar or algae as a walking substrate, or scratch the bottom of dishes with sand paper (Table I).
The agar solution (which ranges from 1.2% to 3%), container type and size of the Petri dishes probably are not very important factors in the success or failure of tardigrade cultures. According to our observations, the agar layer is not a perfect solution because it dissolves in water (used as culture medium) after some time; it also peels off from the dishes and falls apart into smaller fragments, which makes cleaning and observing tardigrades very difficult. The other popular walking substrate, i.e. algae, used also as food, is employed only for herbivorous species. Again, according to our observations, it is not a good solution, especially if the algae are planktonic and not overgrowing the bottom, because when a tardigrade accidentally falls down to the bottom of the dish it most likely will die because of its inability to return to the algae.
As mentioned, some authors use scratched Petri dishes, the same method as used in the present study. This is a very useful method because it is free from the disadvantages of both approaches described above, i.e. it never encounters the problems caused by fragmented agar and it avoids the death of tardigrades due to their inability to return to algae.
The most often used media are different types of spring water or a mixture of spring water and ddH 2 O; however, some authors used tap water or distilled water only. In other cases the type of water used was not specified.

Problem
Reason Solution Tardigrades are not moving, they lie still and are crescent shaped (Figure 4a) -Too many tardigrades on one plate -Divide the animals into two dishes; exchange culture medium for fresh -There was a shortage of oxygen -Exchange culture medium for fresh -Wrong diet -Animals brought into light condition sometimes do not move for a while -Try a different type of food -Wait a few minutes Tardigrades' bodies are straight and transparent; animals are not moving (Figure 4b) -Critical shortage of oxygen (anoxibiosis) -Immediately exchange culture medium (it may take up to several days for the animals to regenerate, and some of them may not survive; if necessary change the medium once a day) -Animals are dead The largest animals are alive but they are not walking on the dish, only lying and moving ( Figure 4c) -Animals ate too much -No action required Tardigrades are becoming more transparent, not milky-white as they should be ( Figure 4d) -Animals are not eating -Change the food (try different type of food) or exchange the medium; sometimes it is good to change the dish to a new one or try to add a photoperiod if the animals are constantly in the dark Tardigrades eat smaller individuals -More food is needed -Give more food per dish, or transfer adult specimens to a separate dish Tardigrades do not reproduce -The species may be dioecious -Complete darkness is not optimal for the species -Temperature is not optimal -More specimens should be placed in one dish -Add a photoperiod or place the culture in light conditions -Try increasing/decreasing the temperature by a few degrees (similar to the natural environmental conditions of the species) Some tardigrades are in the shape of a tun (Figure 4e) -Inappropriate conditions in the chamber -Try increasing/decreasing the temperature, or add a photoperiod if the animals are constantly in the dark -Try a different type of food -Wrong diet -Change the dish to a new one -Dish too "old" with some fungi appearing Some tardigrades form larger groups on the dish (Figure 4f) -Animals are likely mating -No action required The culture conditions varied in different studies, with ambient temperature being the main controlled factor. Temperatures used in different culture protocols varied from very low (4-7°C) to quite high (25°C); the most frequently applied temperatures were in the range of 14°C to 20°C. The lowest temperatures were used in cultures of the Antarctic species Acutuncus antarcticus (Richters, 1904) or the high mountain species Grevenius monoicus (Bertolani, 1982), in line with the natural conditions under which these species live. In contrast, the highest temperatures were used in cultures of terrestrial Milnesium species and Ramazzottius varieornatus Bertolani & Kinchin, 1993 and of freshwater Thulinius ruffoi (Bertolani, 1982), Thu. stephaniae (Pilato, 1974), Hys. exemplaris and Gre. myrops (du Bois-Reymond Marcus, 1944 But, of course, the impact of temperature is strongly species-dependent, which has been confirmed by other authors (e.g. Sømme & Meier 1995;Ramlov & Westh 2001;Li & Wang 2005a, 2005bHengherr et al. 2009;Zawierucha et al. 2018;Neves et al. 2020). Nevertheless, as shown in Table I, some tardigrade species have been cultured with success at different temperatures. The same tardigrade species have also been developed properly at different temperature and/or food regimes, although some morphological structures were larger at lower temperatures and smaller at higher temperature (Kosztyła et al. 2016). According to our observations, controlling the ambient temperature is essential, and at higher temperatures (above 25°C) tardigrades cease activity (probably due to anoxia) whereas at lower temperatures (below 16°C) they reproduce much slowly. These aspects certainly require more detailed studies in the future.
In turn, according to our observations, photoperiod and relative humidity seem not to be crucial for tardigrade cultures. The same can be inferred based on the literature (see Table I and papers cited herein) because humidity values are almost never reported, and the photoperiod is also not reported or different regimes are proposed. This is understandable especially in the case of humidity, because

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M. Roszkowska et al. all of the cultures are maintained permanently in a liquid medium. The photoperiod seems not to be very important because many tardigrades are blind. However, a positive or negative phototaxy was observed for some species, e.g. by Marcus (1929), Baumann (1961) and Beasley (2001), and also by our team in the case of Mac. polypiformis. It is also very important, and should be considered before establishing tardigrade cultures, that some tardigrades can be cultured as isogenic strains because they are parthenogenetic while others need to be cultured in pairs, in small groups or in dense populations because they are dioecious.
In our study we used simple methods of mass culturing of herbivorous, omnivorous and carnivorous tardigrades, allowing us to avoid the problems, discussed above, with walking substrate (agar or algae). We also discussed in detail the amount of food which is necessary and a quite simple schedule of cleaning and feeding. Moreover, we listed possible problems that may occur when culturing some taxa (e.g. dioecious species, anoxia, cannibalism, etc.) and proposed easy solutions. The culturing of terrestrial Heterotardigrada is still a challenge, as a good protocol has not been established yet; this is because tardigrades are usually cultured in a liquid medium, but most terrestrial heterotardigrades prefer very dry and sunny conditions. Nevertheless, we hope that our simple methods will help beginners to establish tardigrade cultures. This simple approach for culturing many different taxa of tardigrades can accelerate more advanced ecological, ethological, physiological, astrobiological, biotechnological, taxonomical and molecular studies on tardigrades.