Journal of Comparative Physiology B

, Volume 188, Issue 4, pp 599–610 | Cite as

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
Original Paper


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.


Cryoprotectants Urea Ice-nucleating protein Freeze tolerance Nanorana pleskei 



Antifreeze proteins


Freezing point


Supercooling point


Equilibrium freezing point


Differential scanning calorimetry


Thermal hysteresis activities


Ice-nucleating activities


Ice-nucleating proteins


Crystallization temperature


Trimethylamine oxide


Body mass


Snout-vent length



The authors greatly appreciate Dr. Yin Qi from Chinese Academy of Sciences Chengdu Institute of Biology for species identification and providing information on sampling site. We thank Prof. Ji Ma from Xinjiang University for technical assistance with differential scanning calorimetry assays. This work was supported by the National Natural Science Foundation of China (no. 31472005).


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Yonggang Niu
    • 1
  • Jianjun Wang
    • 2
  • Shengkang Men
    • 1
  • Yaofeng Zhao
    • 1
  • Songsong Lu
    • 1
  • Xiaolong Tang
    • 1
  • Qiang Chen
    • 1
  1. 1.Institute of Biochemistry and Molecular Biology, School of Life SciencesLanzhou UniversityLanzhouChina
  2. 2.Institute of ChemistryChinese Academy of SciencesBeijingChina

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