Cryptobiosis 300 Years on from van Leuwenhoek: What Have We Learned about Tardigrades?

Biochemical and ultrastructural advances in the latter part of the 20th C. have revolutionized our understanding of cryptobiosis since Anton van Leeuwenhoek gave the first formal description of the phenomenon at a Royal Society lecture in 1702. Keilin coined the term cryptobiosis in 1959 to describe the entry into a reversible ametabolic state and recognized that such ‘latent life’ could encompass processes induced by dehydration, cooling, and perhaps osmotic stress and anoxia. True cryptobiosis, as now understood, depends on the loss of a liquid water phase and can be induced by desiccation or freezing. Loss of ‘bulk’ or liquid water may result directly from evaporation, or arise through vitrification promoted by the formation of a carbohydrate matrix. The carbohydrates in question appear to be ubiquitous in cryptobiotes and serve multiple additional roles: as compatible intracellular osmolytes during desiccation or freeze-dehydration; as stabilizers of protein quaternary structure and lipid bilayer integrity with declining free water activity; and as supercoolants. Plants tend to rely on oligosaccharides such as stachyose and raffinose, while yeasts, spores, and metazoans depend primarily on disaccharides, particularly sucrose and trehalose, and on glycerol. As in many other anhydrobiotes, a metabolic preparatory stage in which these carbohydrates are synthesized from glycogen reserves appears essential for anhydrobiosis in tardigrades, and thus limits physiological tolerance of desiccation rate. Adaptive processes such as tun-formation in tardigrades and bdelloids, coiling in nematodes, and gradient-dependent changes in integumental permeability, retard water losses during preliminary desiccation and exert important influence on survival in xeric extremes. In tardigrades, cryobiosis, or cold-induced cryptobiosis, differs from anhydrobiosis in several important details. Tun formation is not essential for survival, and tolerance of cooling rate depends on the ability to inhibit intracellular freezing. Unlike many cold-tolerant arthropods, tardigrades are freeze-tolerant. Extracellular freezing is promoted by one or more ice-nucleating proteins in Adorybiotus coronifer, and occurs at high temperatures close to 0 °C. Tolerance of variable cooling rates to sub-freezing temperatures in this species does not seem to depend on trehalose synthesis, although a role of other possible intracellular cryoprotectants is likely. It is presently unclear whether cryobiotic tardigrades undergo cytoplasmic vitrification, or whether freeze-dehydration and colligative lowering of cytoplasmic water activity renders the remaining water unfreezable. The profound tolerance of environmental extremes displayed by cryptobiotic organisms apparently depends on the loss of a liquid water phase with accompanying metabolic depression as elegantly described by Clegg's vicinal network model. Thus protected from temperature- and solute-dependent effects on reaction kinetics, and (in part) from destructive free-radical oxidation, cryptobiotes can retain viability in a near-inert state for decades. Disruption of multi-subunit enzyme reactions accompanying loss of the vicinal water fraction may control the decline in metabolism, but also eliminates important free-radical scavenging pathways. Glycerol partly offsets this, acting as an antioxidant, but progressive free-radical oxidation in cryptobiotes may set the upper limit to longevity under aerobic conditions. While we can make many inferences of the physiology of cryptobiosis in tardigrades based on information gathered from other cryptobiotic organisms, specific studies on tardigrades are few, and encompass only a small number of species. This should prove a fruitful field for future research.