Biochemical Response to Freezing in the Siberian Salamander Salamandrella keyserlingii

Abstract

The Siberian salamander Salamandrella keyserlingii Dybowski, 1870 is a unique amphibian that is capable to survive long-term freezing at -55 °C. Nothing is known on the biochemical basis of this remarkable freezing tolerance, except for the fact that it uses glycerol as a low molecular weight cryoprotectant. We used ¹H-NMR analysis to study quantitative changes of multiple metabolites in liver and hindlimb muscle of S. keyserlingii in response to freezing. For the majority of molecules we observed significant changes in concentrations. Glycerol content in frozen organs was as high as 2% w/w, which confirms its role as a cryoprotectant. No other putative cryoprotectants were detected. Freezing resulted in ischemia manifested as increased concentrations of glycolysis products: lactate and alanine. Unexpectedly, we detected no increase in concentrations of succinate, which accumulates under ischemia in various tetrapods. Freezing proved to be a dramatic stress with reduced adenosine phosphate pool and high levels of nucleotide degradation products (hypoxanthine, β-alanine, and β-aminoisobutyrate). There was also significant increase in the concentrations of choline and glycerophosphocholine, which may be interpreted as the degradation of biomembranes. Thus, we found that freezing results not only in macroscopical damage due to ice formation, but also to degradation of DNA and biomembranes.

Keywords: Salamandrella keyserlingii; Siberian salamander; cryoprotectants; freeze tolerance; freezing; glycolysis.

Figure 1

(A) Scores plot of the principal component analysis (PCA) of liver metabolomic profiles of frozen (blue) and control (yellow) individuals of S. keyserlingii. The data are range scaled. Colored ovals indicate 95% confidence regions. Variance explained by the first (PC1) and second (PC2) principal components is indicated on the axis of scores plot. (B) Loadings plot for the same data. (C) Volcano plot for frozen and control liver samples.

Figure 2

(A) Scores plot of the principal component analysis (PCA) of hindlimb muscle metabolomic profiles of frozen (blue) and control (yellow) individuals of S. keyserlingii. The data are range scaled. Colored ovals indicate 95% confidence regions. Variance explained by the first (PC1) and second (PC2) principal components is indicated on the axis of scores plot. (B) Loadings plot for the same data. (C) Volcano plot for frozen and control muscle samples.

Figure 3

The concentrations of substances with cryoprotectant properties. Green columns, control; red, frozen; ** Welch test p < 0.01; *** p < 0.001; circles, individual data points; bar, SE. Glyc, glycerol; Glu, glucose; Myo I; myo-inositol.

Figure 4

(A) concentrations of glycolysis end products; (B) Krebs cycle intermediates. Green columns, control; red, frozen; * Welch test p < 0.05; ** p < 0.01; *** p < 0.001; circles, individual data points; bar, SE.

Figure 5

(A) products of nucleotide degradation: β-Ala, β-alanine; HPX, hypoxanthine; β-AIB, β-aminoisobutyrate. (B) Products of biomembrane degradation: GPC, glycerophosphocholine. Green columns, control; red, frozen; ** Welch test p < 0.01; *** p < 0.001; circles, individual data points; bar, SE.

Figure 6

Concentrations of amino acids: (A) liver; (B) muscles. Green columns, control; red, frozen; * Welch test p < 0.05; ** p < 0.01; *** p < 0.001; circles, individual data points; bar, SE.