Andy Reuner

Freeze tolerance, supercooling points and ice formation: comparative studies on the subzero temperature survival of limno-terrestrial tardigrades

SUMMARY Many 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.

DNA damage in storage cells of anhydrobiotic tardigrades.

In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 +/- 1.98% DNA in tail, compared to 0.44 +/- 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 +/- 6.41% of DNA was found in the comet tail. After ten months, 23.66 +/- 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation.

Stress response in tardigrades: differential gene expression of molecular chaperones

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 (Mt-sHsp17.2 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.

Heat shock proteins (Hsp70) and water content in the estivating Mediterranean Grunt Snail (Cantareus apertus)

Pulmonate land snails often are able to estivate to survive dry hot seasons were water and food are scarce. The aperture of the shell is closed with an epiphragm, and metabolism is depressed to approximately one fourth of basal metabolism. We investigated a molecular aspect of estivation focussing on the heat shock protein 70 (Hsp70) stress response during estivation in the Mediterranean Grunt Snail Cantareus apertus. Sequences of a new inducible hsp70 and of actin are presented and expression of the hsp70 gene as well as Hsp70 protein content was measured in estivating animals. Both Hsp70 protein and mRNA do not show a significant change from the control, although there is a trend that hsp70 mRNA is less abundant in estivating specimens. After heat shock, the expression of hsp70 increased and a higher Hsp70 protein content was detected. Water relations were also investigated. After a period of 6 months in the dormant state, the snails contained 14% less water than active ones, implying a constricted protection against desiccation, compared to the desert snail Sphincterochila zonata, and a Mediterranean-type water economy.

Ice crystallization and freeze tolerance in embryonic stages of the tardigrade Milnesium tardigradum.

In tardigrades, tolerance to low temperature is well known and allows them to cope with subzero temperatures in their environment. Although the ability to tolerate freezing body water has been demonstrated in some tardigrades, freeze tolerance of embryonic stages has been little studied, although this has ecological significance. In this study, we evaluated the subzero temperature survival of five different developmental stages of the eutardigrade species Milnesium tardigradum after freezing to -30 degrees C. Embryos were exposed to five different cooling rates between room temperature and -30 degrees C at 1 degrees C/h, 3 degrees C/h, 5 degrees C/h, 7 degrees C/h, and 9 degrees C/h followed by a warming period at 10 degrees C/h. The results showed that the developmental stage and the cooling rate have a significant effect on the hatching rate. Less developed embryonic stages were more sensitive to freezing at higher freezing rates than more developed stages. Differential Scanning Calorimetry (DSC) was used to determine the temperature of crystallization (Tc) in single embryos of the different developmental stages and revealed no differences between the stages. Based on the calorimetric data, we also conclude that the ice nucleation is homogeneous in embryonic stages in tardigrades, as also recently shown for fully developed tardigrades, and not triggered by nucleating agents.