Extreme freeze-tolerance in cryophilic tardigrades relies on controlled ice formation but does not involve significant change in transcription.

Subzero temperatures are among the most significant factors defining the distribution of organisms, yet, certain taxa have evolved to overcome this barrier. The microscopic tardigrades are among the most freeze-tolerant animals, with selected species reported to survive milli-Kelvin temperatures. Here, we estimate survival of fully hydrated eutardigrades of the species Ramazzottius varieornatus following exposures to -20 °C and  -80 °C as well as -196 °C with or without initial cooling to -80 °C. The tardigrades easily survive these temperatures, yet with a significant decrease in viability following rapid cooling by direct exposure to -196 °C. Hence, post-freeze recovery of R. varieornatus seems to rely on cooling rate and thus controlled ice formation. Cryophilic organisms are renowned for having cold-active enzymes that secure appropriate reaction rates at low temperatures. Hence, extreme freeze-tolerance in R. varieornatus could potentially involve syntheses of cryoprotectants and de novo transcription. We therefore generated a reference transcriptome for this cryophilic R. varieornatus population and explored for differential gene expression patterns following cooling to -80 °C as compared to active 5 °C controls. Specifically, we tested for fast transcription potentially occurring within 25 min of cooling from room temperature to a supercooling point of ca. -20 °C, at which the tardigrades presumably freeze and enter into the ametabolic state of cryobiosis. Our analyses revealed no evidence for differential gene expression. We, therefore, conclude that extreme freeze-tolerance in R. varieornatus relies on controlled extracellular freezing with any freeze-tolerance related genes being constitutively expressed.