Geographic variation in energy storage and physiological responses to freezing in the gray treefrogsHyla versicolorandH. chrysoscelis

Abstract

SUMMARYThe physiological responses supporting freeze tolerance in anurans are well known, but the evolution of this trait remains little studied. This is the first common-garden study of geographic variation in cryoprotective responses to freezing and the degree of freeze tolerance. We studied the gray treefrogs(Hyla versicolor and H. chrysoscelis) from sympatric sites in Minnesota, Indiana and Missouri. Patterns in the literature suggest that northern frogs produce more cryoprotectants upon freezing, but we found no geographic variation in cryoprotective responses or degree of freeze tolerance. The concentration of glucose produced upon freezing was higher than previously reported for this species (liver: 475 μmol g–1dry mass). Unfrozen frogs had high levels of glycerol (liver: approx. 150μmol g–1 dry mass), and did not produce more upon freezing. Liver glycogen content (concentration multiplied by liver mass) was highest in frogs from Minnesota and Missouri, and was stored in preference to lipids in Minnesota frogs, possibly to provide energy for the longer northern winters. Minnesota frogs accumulated more ice (53.4±1.8%) after freezing to–2.5°C than Indiana frogs (45.5±3.3%). The two species differed in body size but not in any of the physiological parameters measured. We conclude that these populations show no adaptive variation in freeze tolerance and that comparing published studies may be misleading because of different acclimation and feeding regimes.

Glycerol production in rainbow smelt (Osmerus mordax) may be triggered by low temperature alone and is associated with the activation of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase

“…This anticipatory response is similar to activation of glycerol production in a number of insects (Storey and Storey, 1988) and in the gray treefrogs, Hyla veriscolor and H. chrysoscelis, where an increase in plasma and organ glycerol occurs in response to extended cold acclimation at temperatures above 0°C (Layne and Jones, 2001;Irwin and Lee, 2003). This situation differs from the accumulation of glucose in these frogs and other frog species that occurs only in response to freezing (Storey and Storey, 1992;Layne and Jones, 2001;Irwin and Lee, 2003).…”

Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog

“…Abundant glycogen may be advantageous in cold climates, as levels in Alaskan frogs were 2.2-fold greater than those in Ohioan frogs. Furthermore, the pre-hibernal supply of muscle glycogen in a Minnesotan population (~47.6°N) of the gray tree frog was nearly twice that of conspecifics from Missouri (~37.9°N) (Irwin and Lee, 2003). Muscle stores of this substrate probably contribute little to whole-animal metabolism in winter (Pasanen and Koskela, 1974;Tattersall and Ultsch, 2008), but may help fuel occasional movements to new hibernacula, which have been observed for R. sylvatica wintering in an outdoor enclosure (J.P.C.…”

“…Alaskan frogs accumulated less lactate than did Ohioan frogs, perhaps because they formed less ice (and better sustained tissue perfusion) and/or used oxygen reserves more efficiently. Similarly, gray tree frogs in a northern population accumulated less lactate with freezing than did more southerly conspecifics (Irwin and Lee, 2003). Mitigating hypoxic stress, perhaps by enhancing metabolic downregulation, or forming ice more slowly, may contribute to survival in extremely cold habitats.…”

“…Whereas winter Ohioan frogs retained a modicum of this adipose, Alaskan frogs expended their fat bodies whilst preparing for hibernation. Enhanced lipolysis in high-latitude populations of R. sylvatica, and also freezetolerant tree frogs (Irwin and Lee, 2003), likely improves survival by magnifying the glycogen reserve. Additionally, more of the energy in lipid, which cannot be oxidized in frozen (hypoxic) tissues, is converted to a more useful substrate; indeed, R. sylvatica undergo a seasonal downregulation of certain enzymes that would curtail lipid use in hibernation (Kiss et al, 2011).…”

Excretion and conservation of glycerol, and expression of aquaporins and glyceroporins, during cold acclimation in Cope's gray tree frogHyla chrysoscelis

“…It is likely that this glycerol is synthesized by the liver and eventually released to the circulation and distributed to tissues throughout the body. Glycerol may circulate at elevated concentration (and accumulate at high concentrations in tissues) in cold-acclimated organisms for weeks or months, as long as the animals remain cold (12). Although the function of this glycerol has not been definitively established and other frogs that tolerate freezing do so without glycerol, the presumption is that this solute acts as a cryoprotectant, as described above.…”