Collection and Lab Maintenance
Overview
Tardigrades are a phylum of animals with more than 1000 described species. Similar to many other phyla in the animal kingdom, tardigrades are diverse and are adapted to many different lifestyles and habitats. Accordingly, they have been found nearly everywhere on earth, in a wide variety of habitats, including oceans, ponds, moss, glaciers, and even volcanoes. Studies have shown that the most suitable sites to collect aquatic tardigrades are from sediments, floating vegetation, and running lotic waters. Whereas limnic/freshwater tardigrades can be collected via moss, lichens, and algal encrustations (Nelson et al., 2015). Overall, they need to inhabit moist environments to ensure gaseous exchange and prevent desiccation (Lemloh et al., 2011). Therefore, an environment with at least a water film is necessary when raising them. In addition to the water film, tardigrades need to be able to utilize locomotion where ever they are raised. Daniel Stec et al’s study in 2015 showed one way to aid locomotion by scratching the Petri dishes, which tardigrades were reared in, with fine sandpaper (Stec et al., 2015). This is important because locomotion is necessary for animals to find food. Moreover, tardigrades can also survive extreme conditions, such as very low temperatures, even if they are not adapted to it (Flinn Scientific, 2016). They have many mechanisms, including cryptobiosis, that allow them to do so. Given this diversity and ability to survive extreme conditions, it is likely that one universal method of rearing and culturing is not possible with tardigrades.
One species of tardigrades that has been best studied is Hypsibius exemplaris (sometimes called H. dujardini). It has been cultured for decades and has been recently used in research as a model organism. This is due to the fact that the species is relatively easy to culture. It is vegetarian and can survive on a single species of algae. Gabriel et al’s study (2007) raised Hypsibius exemplaris in 250 ml Erlenmeyer flasks that contained 150 ml of Chalkley’s Medium (5 ml of each of the following stock solutions per liter in dH20: NaCl, 2 g/100 ml dH20; KCl, 0.08 g/100 ml dH20; CaCl2, 0.12 g/100 ml dH20), enhanced with 2% soil extract. The tardigrades were fed 3-5 ml of concentrated cells from a Chlorococcum sp. culture. Each flask was sealed with clingfilm or parafilm to prevent contamination. The flasks were placed in a cool shaded place at 10-20℃. Sub-cultures were created every 4-6 weeks as necessary. The study also highlights a key point in the maintenance of tardigrades, embryo collection. Without embryos, strains of tardigrades cannot be maintained. To collect the embryos, small cultures of tardigrades were kept in Petri dishes with commercially bottled spring water at room temperature in a shaded area. The embryos were then removed from the parents by slicing the parental exuvia with 25 gauge hypodermic needles. The embryos were then fixed with absolute methanol and paraformaldehyde and then sonicated. After allowing the embryos to recover on ice, they were mounted on slides containing 0.2% gelatin, 0.02% chrome alum, 0.1% polylysine, and 1 mM azide (Gabriel et al., 2007).
Challenges
It is crucial to note that H. exemplaris is only one example of the vast diversity of tardigrades. Many other species will have different requirements depending on their natural habitat and diet. For example, carnivorous species require animals such as nematodes and rotifers in their diet. Rearing these tardigrades is even more complicated because conditions need to be optimized for each of the other animal species included in the diet. This method is also non-axenic and could cause contamination in future studies such as DNA analysis. Another challenge for raising diverse tardigrades is the diversity of life cycles across different tardigrade species. The lab culture described above (H. exemplaris) has a relatively short life cycle. Most other tardigrade species have a longer life cycle, e.g. with some egg incubation periods measured in months rather than in days/weeks. Life cycle estimates are further complicated and can be inaccurate because tardigrades can enter a state that approximates suspended animation when they undergo cryptobiosis. Also, the natural habitat of many tardigrades is characterized by alternating cycles of moisture and dryness and extreme fluctuations in temperature. Whether or not tardigrades require or have some fitness advantage from these alterations is unknown, but is important in questions of rearing and culturing. Therefore, because of the diversity of different dietary preferences, natural habitats, and life histories across tardigrade species, it is likely impossible to come up with a single method of rearing and culturing. The number of culturing conditions needed may approach the number of different types of tardigrades.
Fig. 1. Tardigrade Maintenance. Graphical representation of method extracted from Suzuki’s Life History of Milnesium tardigradum Doyère (Tardigrada) under a Rearing Environment (2003).
Reason for hope
Even though each tardigrade species may need to have its own rearing protocol, there have been some reports of success in raising different types of ‘wild’ tardigrades, which may provide some insights about different conditions to test in culturing newly-captured tardigrades. For example, one study determined that tardigrades can be raised in Petri dishes coated with agar (Suzuki, 2003). Agar was used because it is difficult for tardigrades to move across plastic surfaces without their mouthparts getting stuck to the bottom of the dishes. This method, however, works better if water is added to freshly prepared agar plates because this prevents gaps in agar plates, hence preventing trapped tardigrades. The method they used is summarized as follows: 1) An agar plate was prepared in a 3cm dish with water. 2) Tardigrades were added to the dish. 3) Rotifers in KCM solution were used as a food source 4) The water was changed each time food was added to the plate and the Tardigrades were checked daily (Fig. 1) (Suzuki, 2003).
Another study used small dishes that were placed in a wet chamber which was made up of a large Petri dish with a wet filter paper film cover. The cover was used to prevent the desiccation of the tardigrade cultures (Altiero and Rebecchi, 2001). Some drops of water, 0.6 ml of bacto-agar (substrate for walking), moss leaves, and bacteriophagous nematodes (food) were added in dishes containing carnivorous terrestrial species. Only 0.7 ml of spring water and drops of water containing the alga, Scenedesmus acutus, were added to the dishes that contained herbivorous limnic species. The alga was used as both a substrate and a food source. Both cultures of tardigrades were checked three times a week. This study also tested what temperature was preferred by different tardigrade species/strains. Two strains of Macrobiotus richtersi (diploid and triploid), the species Macrobiotus joannae, and Diphascon cf. scoticum were raised at 14℃ or 20℃, whereas Isohypsibius monoicus was reared at 4℃ or 14℃. The results indicated that the diploid strain of M. richtersi seems to prefer 20°C and the triploid strain seems to prefer 14℃. The other species did not show any significant preference for any particular temperature (Altiero and Rebecchi, 2001).
Overall, none of these methods are easy to implement at home so the most home-friendly version of the methods was chosen for our study.
Personal Experiences
To potentially obtain and rear tardigrades at home, moss, lichens, and algae were our habitats of choice. Samples of moss, lichens, and algae were collected from various individuals across the world (Fig. 2). The samples were collected from diverse substrates, such as trees, logs, water, ground, and walls (Fig. 3). The samples were kept in dry storage and then later rehydrated before screening. Rehydration is necessary to reverse anhydrobiosis, which is a drought-induced dormant state that tardigrades can enter (Nelson et al., 2015). After collecting possible tardigrade habitats, the next logical step would be to look for them.
Tardigrade culture or maintenance wells were prepared 24 hours before isolation. A milliliter of water and a drop of algae were added to two wells (in a 24-well plate) for each moss/lichen sample to be screened. The moss/lichen samples were rehydrated by placing them in a yogurt cup and adding commercially bottled spring water or rainwater. After 24 hours, the liquid from each moss/lichen sample was extracted and put in a 10mm Petri dish. Each dish was then screened to find tardigrades. A glass pipette was used to transfer each tardigrade found in a sample into a well in the 24-well plate. The plate was kept in a dark, cool place because tardigrades are most active at 0-30 ℃ (Flinn Scientific, 2016). The plate was checked and about 50% of the water was changed every 1-2 days. More algae were added as needed.
Furthermore, I collected 30 samples of moss from two different substrates but screened a total of 42 samples. Out of the 42 samples, 9 of them contained tardigrades. A total of 28 tardigrades were collected and only one of them reproduced and produced two progeny, bringing the total to 30 tardigrades. Unfortunately, I lost 20 of them by dropping the 24-well plate. This was just one of the struggles that could happen at home or in a lab setting. The rest of the tardigrades were put in tubes and frozen. Before losing or freezing the tardigrades, they were all alive and active in the wells for the first week they were put in. After that, most of them seemed dead or were no longer visible.
Nevertheless, there were some minor challenges with this procedure. The tardigrades had to be followed individually and this was more difficult and less efficient than rearing groups together. Each tardigrade may have entered cryptobiosis, decreasing its visibility in a well, making it hard to keep track of them. One study actually showed that rearing tardigrades in groups resulted in more reproduction. Keeping them in small wells meant less space and food, hence could be the reason for reduced reproduction (Altiero and Rebecchi, 2001). In addition, the constant wet, artificial environment is not an optimal environment. A study showed that tardigrades prefer mosses that frequently dried out (Suzuki, 2003). Therefore, it is challenging to keep them alive in such conditions and might have been the reason some of the tardigrades died in the 24-well plates.
To conclude, raising tardigrades proved to be less challenging than I anticipated. It was relatively easy to find and move tardigrades. The main challenge was keeping them alive but I hope it will be more straightforward in a laboratory with more resources.
Fig. 2. Number of samples by substrate. The number of moss/lichen/algae samples collected by each collector classified by substrate.
Fig. 3. Proportion of tardigrades in different substrates. The proportion of tardigrades, out of the total amount of tardigrades found, in each substrate. The best substrate to find tardigrades is a log.