Urea and plasma ice-nucleating proteins promoted the modest freeze tolerance in Pleske's high altitude frog Nanorana pleskei.

The frog Nanorana pleskei (Dicroglossidae) is indigenous to the Qinghai-Tibetan Plateau. To identify its strategies in coping with the cold climate, we measured the hibernacula microhabitat temperature during winter. We also examined the freezing-induced and seasonal variation of several putative cryoprotectants in the heart, liver, brain, kidney and muscle, as well as ice-nucleating protein in plasma. Our results showed that N. pleskei survived exposure to temperatures as low as - 2.5 ± 0.40 °C during hibernation, which was lower than the body fluid freezing point (- 0.43 ± 0.01 °C). Experimental freezing results indicated that four of six specimens could survive 12 h of freezing at - 2 °C with 27.5 ± 2.5% of total body water as ice. Concomitantly, the water contents of all examined organs decreased after being frozen for 24 h at - 2 °C. The levels of urea in heart significantly increased from 71.05 ± 7.19 to 104.59 ± 10.11 µmol g-1, and in muscle increased from 72.23 ± 3.40 to 102.42 ± 6.24 µmol g-1 when exposed to freezing; other cryoprotectants (glucose, glycerol, and lactate) showed no significant increase in all examined tissues. In addition, urea levels were significantly higher in fall-collected frogs than summer-collected frogs in the tissues of heart, brain, kidney, and muscle. The results of differential scanning calorimetry indicated that the ice-nucleating protein was present only in cold-acclimated and fall-collected frogs' plasma. We concluded that the urea serves as a primary cryoprotectant and accumulates in anticipation of freezing in N. pleskei, coupling with the seasonal production of plasma ice-nucleating protein.