Multiple Freeze‐thaw Cycles in Cope’s Gray Treefrog Dryophytes chrysoscelis Reveal Novel Physiological, Biochemical, and Morphological Insights into Vertebrate Freeze Tolerance
Authors: Evans Elizabeth , Wascher Matthew , Goldstein David , Krane Carissa 
Published: May 2022

The freeze tolerant anuran Dryophytes chrysoscelis, Cope’s gray treefrog, mobilizes a complex cryoprotectant system that includes glycerol, glucose and urea to minimize damage induced by freezing and thawing of up to 65% of body water. It is hypothesized that, compared with a single freeze‐thaw cycle, repeated freeze‐thaw cycles in D. chrysoscelis will enhance cryoprotectant accumulation, glycogen depletion, and cryoinjury, while locomotor and morphological characteristics will be altered in association with a delay in thawing time. Wild‐caught male treefrogs were cold acclimated prior to freezing. Animals were chilled to ‐2.5°C over 5 days, inoculated with ice to induce freezing, held frozen for 24 hrs, then thawed at 5°C for 24 hrs (“single freeze‐thaw”) or refrozen and thawed twice more over a period of 4 days (“repeated freeze‐thaw”) (n=4‐6 per group). Cryoprotectant levels were measured in liver, muscle, and plasma. Glycogen was assayed in liver and muscle, and osmolality and hemoglobin (Hb; indicative of RBC hemolysis) in plasma. Body movements and skin color were documented by digital photography. Linear regression models of log‐transformed data were used to test for multiplicative differences among groups and variable relationships. Glycerol, the most abundant cryoprotectant in all tissues measured in both thawed groups (single and repeated), increased 112‐fold in single freeze‐thaw animals and 185‐fold in repeated freeze‐thaw, compared with cold acclimated controls (P = 0.001). Glucose increased in both thawed groups in liver and plasma but not in muscle, whereas urea did not change. Liver glycogen decreased by ~50% and ~25% compared with controls in single and repeatedly thawed groups, respectively (P < 0.001). Osmolality increased ~2‐fold in single and repeated freeze‐thaw groups compared with controls (P < 0.001). A linear regression model using plasma glycerol alone can explain 65% of variation in plasma osmolality; the model is only marginally enhanced by the addition of plasma urea and is not enhanced by the addition of plasma glucose. Plasma Hb significantly increased in the repeated freeze‐thaw group compared to the control (P < 0.05), indicating greater hemolysis with repeated freezing and thawing. Locomotor changes–the time for animals to lift their head, open their eyes, move a limb, and change overall body position–were significantly delayed in the second thaw compared with the first (P < 0.05). Skin color dynamically changes from blue to green to brown as frozen frogs thaw throughout all freeze‐thaw cycles. Repeated freezing and thawing of vertebrates is almost entirely unstudied. Our results demonstrate cryoinjury (hemolysis) and delayed return of locomotor function after repeated freeze‐thaw, despite progressive depletion of glycogen stores (a cryoprotectant source) and accumulation of glucose and glycerol and sustained levels of urea. Our ecologically relevant protocol, combined with an innovative and comprehensive analysis, provides novel insights into the complex and systemic nature of freeze tolerance in D. chrysoscelis.