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.

Anhydrobiosis can be described as an adaptation to lack of water that enables some organisms, including tardigrades, to survive extreme conditions, even some that do not exist on Earth. The cellular mechanisms underlying anhydrobiosis are still not completely explained including the putative contribution of mitochondrial proteins. Since mitochondrial alternative oxidase (AOX), described as a drought response element in plants, was recently proposed for various invertebrates including tardigrades, we investigated whether AOX is involved in successful anhydrobiosis of tardigrades. Milnesium inceptum was used as a model for the study. We confirmed functionality of M. inceptum AOX and estimated its contribution to the tardigrade revival after anhydrobiosis of different durations. We observed that AOX activity was particularly important for M. inceptum revival after the long-term tun stage but did not affect the rehydration stage specifically. The results may contribute to our understanding and then application of anhydrobiosis underlying mechanisms.

Anhydrobiosis is a desiccation tolerance that denotes the ability to survive almost complete dehydration without sustaining damage. The knowledge on the survival capacity of various tardigrade species in anhydrobiosis is still very limited. Our research compares anhydrobiotic capacities of four tardigrade species from different genera, i.e. Echiniscus testudo, Paramacrobiotus experimentalis, Pseudohexapodibius degenerans and Macrobiotus pseudohufelandi, whose feeding behavior and occupied habitats are different. Additionally, in the case of Ech. testudo, we analyzed two populations: one urban and one from a natural habitat. The observed tardigrade species displayed clear differences in their anhydrobiotic capacity, which appear to be determined by the habitat rather than nutritional behavior of species sharing the same habitat type. The results also indicate that the longer the state of anhydrobiosis lasts, the more time the animals need to return to activity.

Anhydrobiosis can be described as an adaptation to lack of water. This adaptation provides some organisms including tardigrades with a set of capabilities allowing them to survive extreme conditions that even do not exist on Earth. However, the underlying cellular mechanisms are still not explained. Available data assumes important contribution of mitochondrial proteins. Since mitochondrial alternative oxidase (AOX) described as a drought response element has recently been proposed for various invertebrates including tardigrades, we have decided to check if AOX is involved in successful anhydrobiosis of tardigrades. Milnesium inceptum was used as a model for the study. We confirmed functionality of M. inceptum AOX and estimated its activity contribution to anhydrobiosis of different duration. We observed that AOX activity was particularly important for M. inceptum revival after longer-term anhydrobiosis but did not affect rehydration stage. The results may contribute to explanation and then application of anhydrobiosis underlying mechanisms.

The slime mold Dictyostelium discoideum’s life cycle includes different unicellular and multicellular stages that provide a convenient model for research concerning intracellular and intercellular mechanisms influencing mitochondria’s structure and function. We aim to determine the differences between the mitochondria isolated from the slime mold regarding its early developmental stages induced by starvation, namely the unicellular (U), aggregation (A) and streams (S) stages, at the bioenergetic and proteome levels. We measured the oxygen consumption of intact cells using the Clarke electrode and observed a distinct decrease in mitochondrial coupling capacity for stage S cells and a decrease in mitochondrial coupling efficiency for stage A and S cells. We also found changes in spare respiratory capacity. We performed a wide comparative proteomic study. During the transition from the unicellular stage to the multicellular stage, important proteomic differences occurred in stages A and S relating to the proteins of the main mitochondrial functional groups, showing characteristic tendencies that could be associated with their ongoing adaptation to starvation following cell reprogramming during the switch to gluconeogenesis. We suggest that the main mitochondrial processes are downregulated during the early developmental stages, although this needs to be verified by extending analogous studies to the next slime mold life cycle stages.