Investigations on cryobiology of Anatolian mountain frogs

This study investigated strategies that enable Anatolian mountain frogs (R. macrocnemis and R. holtzi) to survive the winter period when they are exposed to freezing temperatures. In this context, frogs were subjected to conditions of freezing, anoxia, and dehydration and examined in terms of the levels of glucose, as an important cryoprotectant, in the blood, skeletal muscle, and liver tissues, the amount of water in muscle and liver tissues, the changes in body weight, and the transcript expression levels of the amphibian-specific antifreeze proteins (AFPs) FR10, Li16, and FR47. During freezing, the blood glucose level of R. macrocnemis increased 3.3-fold (1.35 ± 0.25 - 4.45 ± 0.51 μmol mL-1) and that of R. holtzi 4.5-fold (1.90 ± 0.25 - 8.67 ± 2.22 μmol mL-1). The liver tissue glucose level increased 6.7-fold (5.66 ± 0.15 - 38.27 ± 8.53 μmol g-1) in R. macrocnemis and about 6-fold (2.25 ± 0.46 - 13.36 ± 1.32 μmol g-1) in R. holtzi. The liver tissue glucose level increased in both species in response to anoxia and dehydration stresses. For both species, the glucose levels in muscle tissue increased more with the dehydration treatment than with the freezing and anoxia treatments. Significant changes were noted in the gene expression levels of some AFPs under stress and normalization conditions. In the liver tissue of R. macrocnemis, freezing stress increased the transcript level of the li16 gene approximately 3.5-fold, and anoxia increased the fr47 expression by 2.7-fold. The fr10 gene was upregulated under all conditions (freezing, anoxia, and dehydration) in the liver tissue of R. holtzi. Consequently, the results of this thesis study showed that Anatolian mountain frogs have the ability to survive in freezing temperatures and are freeze tolerant. Moreover, it was noted that in their strategies for this process, they use glucose, one of the important cryoprotectants, and that the frog-specific AFPs play a role in freezing and its associated stresses. Focusing on the bioinformatics of genomic, transcriptomic, and proteomic research on AFPs could provide clarification of the mechanisms by which AFPs bind to and control ice crystals. In addition, further research on cryoprotectants would provide a better understanding of the cryobiology of Anatolian mountain frogs.