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

Yonggang Niu, Jianjun Wang, Shengkang Men, Yaofeng Zhao, Songsong Lu, Xiaolong Tang, Qiang Chen

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.