Ice formation in the freeze-tolerant eutardigrades Adorybiotus coronifer and Amphibolus nebulosus studied by differential scanning calorimetry

This paper describes ice formation in two freeze-tolerant eutardigrades Adorybiotus coronifer and Amphibolus nebulosus, both commonly found in Arctic areas, based on a study by differential scanning calorimetry, and results are compared with similar investigations on freeze-tolerant insects. Under Arctic conditions A. coronifer typically inhabits drought-resistant mosses and overwinters in a frozen or dry state. A. nebulosus inhabits moist mosses or algae and overwinters as frozen cysts or, in few cases, as eggs or adults. Both species show a moderate capacity for supercooling, with crystallization temperatures (T_c) of -6° to -7°C. No differences in T_c were observed between summer- and winter-acclimatized A. coronifer or between active and encysted A. nebulosus. Distinct differences in the heat stability of their ice-nucleating activity indicate that the compounds responsible for nucleation are chemically different in the two species. Ice formation progresses rapidly in both species, and crystallization of water probably ceases within 1 min of the nucleation. Ice constitutes 80–90% of total body water, even at temperatures just below T_c, in both species. Winter-acclimatized A. coronifer build up about 10% less ice than summer-acclimatized animals, and a similar reduction was induced by cold-acclimation of summer animals in the laboratory. No winter reduction of the melting point could be detected in A. coronifer, indicating little, if any, accumulation of low molecular weight cryoprotectives. We suggest that the reduction in ice content is functionally related to increased amounts of water kept unfrozen due to interactions with macromolecules.