Survival in microhabitats that experience extreme fluctuations in water availability and temperature requires special adaptations. To withstand such environmental conditions, tardigrades, as well as some nematodes and rotifers, enter a completely desiccated state known as anhydrobiosis. We examined the effects of high temperatures on fully desiccated (anhydrobiotic) tardigrades. Nine species from the classes Heterotardigrada and Eutardigrada were exposed to temperatures of up to 110 degrees C for 1 h. Exposure to temperatures of up to 80 degrees C resulted in a moderate decrease in survival. Exposure to temperatures above this resulted in a sharp decrease in survival, with no animals of the families Macrobiotidae and Echiniscidae surviving 100 degrees C. However, Milnesium tardigradum (Milnesidae) showed survival of >90% after exposure to 100 degrees C; temperatures above this resulted in a steep decrease in survival. Vitrification is assumed to play a major role in the survival of anhydrobiotic organisms during exposure to extreme temperatures, and consequently, the glass-transition temperature (T(g)) is critical to high-temperature tolerance. In this study, we provide the first evidence of the presence of a glass transition during heating in an anhydrobiotic tardigrade through the use of differential scanning calorimetry.

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SUMMARYMany limno-terrestrial tardigrades live in unstable habitats where they experience extreme environmental conditions such as drought, heat and subzero temperatures. Although their stress tolerance is often related only to the anhydrobiotic state, tardigrades can also be exposed to great daily temperature fluctuations without dehydration. Survival of subzero temperatures in an active state requires either the ability to tolerate the freezing of body water or mechanisms to decrease the freezing point. Considering freeze tolerance in tardigrades as a general feature, we studied the survival rate of nine tardigrade species originating from polar, temperate and tropical regions by cooling them at rates of 9, 7, 5, 3 and 1°C h -1 down to -30°C then returning them to room temperature at 10°C h -1 . The resulting moderate survival after fast and slow cooling rates and low survival after intermediate cooling rates may indicate the influence of a physical effect during fast cooling and the possibility that they are able to synthesize cryoprotectants during slow cooling. Differential scanning calorimetry of starved, fed and cold acclimatized individuals showed no intraspecific significant differences in supercooling points and ice formation. Although this might suggest that metabolic and biochemical preparation are non-essential prior to subzero temperature exposure, the increased survival rate with slower cooling rates gives evidence that tardigrades still use some kind of mechanism to protect their cellular structure from freezing injury without influencing the freezing temperature. These results expand our current understanding of freeze tolerance in tardigrades and will lead to a better understanding of their ability to survive subzero temperature conditions.

Semi-terrestrial tardigrades exhibit a remarkable tolerance to desiccation by entering a state called anhydrobiosis. In this state, they show a strong resistance against several kinds of physical extremes. Because of the probable importance of stress proteins during the phases of dehydration and rehydration, the relative abundance of transcripts coding for two α-crystallin heat-shock proteins (MtsHsp17.2 and Mt-sHsp19.5), as well for the heat-shock proteins Mt-sHsp10, Mt-Hsp60, Mt-Hsp70 and Mt-Hsp90, were analysed in active and anhydrobiotic tardigrades of the species Milnesium tardigradum. They were also analysed in the transitional stage (I) of dehydration, the transitional stage (II) of rehydration and in heat-shocked specimens. A variable pattern of expression was detected, with most candidates being downregulated. Gene transcripts of one Mt-hsp70 isoform in the transitional stage I and Mt-hsp90 in the anhydrobiotic stage were significantly upregulated. A high gene expression (778.6-fold) was found for the small α-crystallin heat-shock protein gene Mt-sHsp17.2 after heat shock. We discuss the limited role of the stress-gene expression in the transitional stages between the active and anhydrobiotic tardigrades and other mechanisms which allow tardigrades to survive desiccation.

The impact of starvation and anhydrobiosis on the number and size of the storage cells in the tardigrade species Milnesium tardigradum, Paramacrobiotus tonollii and Macrobiotus sapiens was investigated to gain more insight on the energetic side of anhydrobiosis. Storage cells are free floating cells within the body cavity of tardigrades and are presumed to store and release energy in form of glycogen, protein and fat to maintain a constant nutrient regime for the other tissues. The body size of the animals was not correlated with the size of the storage cells, however, M. tardigradum the largest species analysed also had the largest storage cells. A reduction in the size of the storage cells is apparent in all three species after seven days of starvation. A seven-day period of anhydrobiosis leads to a decrease in cell size in M. tardigradum but not in P. tonollii and M. sapiens. Although M. sapiens was raised on green algae, and M. tardigradum and P. tonollii were fed with rotifers and nematodes this difference in nourishment was not reflected in the response of the storage cells to anhydrobiosis.