Protein biosynthesis responses occurring during the postthaw period (after 12 h freezing at −1.4°C), dehydration (to 27 or 40% of total body water lost), or rehydration (after the loss of 40% of body water) were monitored in tissues of spring-collected wood frogs (Rana sylvatica) after intraperitoneal injection of35S-labeled methionine + cysteine. All six organs tested accumulated radiolabeled amino acids and organs of both thawing and rehydrating frogs held at 3–5°C showed a linear increase in amino acid incorporation into the acid-precipitable protein fraction over time. By contrast, dehydrating animals showed little or no increment in protein bound radioactivity over the course of the stress, a result that may be indicative of metabolic suppression in organs when dehydration became severe. Isoelectrofocusing (IEF) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were used to characterize the proteins synthesized by liver under each experimental state. IEF revealed both new peaks of35S-labeled proteins and enhanced labeling of others in extracts from experimental animals, compared with controls. In particular, new synthesis of proteins with isoelectric points of about 6.0 was prominent and labeled proteins in this IEF peak persisted at 5, 10, or 24 h postinjection, becoming proportionally more important over time. SDS–PAGE analysis of the pI6.0 peaks from thawed, dehydrated, and rehydrated frogs revealed the presence of one major low molecular weight protein in each case with molecular masses of 15, 13, and 21 kDa, respectively. These data indicate that the biochemical adaptations supporting freeze tolerance and dehydration tolerance in anurans include the stress-induced biosynthesis of a suite of proteins including the novel synthesis of selected specific proteins. These proteins may represent stress-related (or shock) proteins or may have specific roles in metabolic adaptation in each state such as in water and ionic balance or cell volume regulation.

The effects of freezing on organ metabolism were monitored over a 36-h time period at -4 C in spring-collected specimens of a freeze-tolerant frog, Pseudacris crucifer. Within 2 min after nucleation, glycogenolysis in liver was activated as indicated by elevated levels of hexose phosphates; and levels of the cryoprotectant, glucose, rose quickly thereafter. Freezing stimulated a 3.3-fold increase in liver glycogen phosphorylase a activity, and a rapid phase of liver glucose production was maintained for at least the first hour of freezing (21 mu mol . g wet mass(-1) . h(-1)), followed by a steady rate of glucose increase of about 3 mu mol . gwm(-1) h(-1). Final liver glucose levels reached 141 mu mol . gwm(-1) after 36 h. Changes in the concentrations of hexose phosphates and fructose-1,6-bisphosphate in liver indicated that glucose production was promoted by an inhibitory block on glycolysis at the phosphofructokinase reaction. Other organs accumulated the glucose exported by liver and cryoprotectant accumulation by heart paralleled the rise in liver glucose levels. Liver energetics were not disrupted during early freezing but with prolonged freezing (4-36 h), ATP content fell, ADP and AMP increased, and energy charge, [ATP + ADP/2]/[ATP + ADP + AMP], was reduced from 0.90 in controls to 0.57 after 36 h freezing. Liver metabolism during freezing was supported by fermentative reactions with anaerobic glycolysis leading to lactate and alanine accumulation; concomitantly, levels of fermentable-free amino acids (aspartate and glutamate) decreased. Changes in free amino acid patterns in skeletal muscle and heart also indicated a reliance on amino acid fermentation during freezing, but additional increases in the levels of several other amino acids suggested that freezing might also elevate proteolysis. The results show a role for amino acids in anuran freezing survival and demonstrate that the biochemical mechanisms involved in controlling liver glucose output during freezing, previously described only for the wood frog Rana sylvatica, are probably general mechanisms for regulating cryoprotectant pools in all freeze-tolerant anurans.

1. The metabolic responses to freezing at -8 degrees C for up to 72 h vs 72 h anoxia exposure under N-2 gas at 5 degrees C were compared in foot muscle of the intertidal gastropod, Littorina littorea.2. Freezing resulted in the accumulation of D-lactate and succinate (net increases of 1.1 and 3.9 mu mol/g wet weight, respectively, compared with 5 degrees C-acclimated controls) with an opposite decrease in the fermentative substrate, L-aspartate, whereas, by contrast, anoxia resulted in only a small, 0.5 mu mol/gww, accumulation of D-lactate.3. Neither freezing nor anoxia exposure had a significant effect on muscle adenylate energy charge, suggesting that strong metabolic arrest mechanisms lowered energy demand to a level that could be met by fermentative metabolism alone.4. Activation of glycogenolysis was implied by elevated glucose-6-phosphate levels in muscle under both stresses but only during freezing did glucose accumulate (a net of 1.2 mu mol/gww); changes in the levels of other glycolytic intermediates indicated regulatory control of glycolysis at pyruvate kinase under both stresses.5. No carbohydrate cryoprotectants were accumulated during freezing and neither stress changed the composition or size of the free amino acid pool (approx. 100 mu mol/gww total amino acids composed of 77% taurine and 12% alanine).6. The data show that the metabolic responses to freezing and anoxia share some elements, indicating the importance of a good anaerobic capacity to freezing survival, but other aspects of metabolic response to freezing differ from anoxia and suggest factors that may be specifically important to cryoprotection.

The effects of 2 or 12 h freezing at −6 °C or anoxia exposure at 5 °C on the metabolic responses of gill, mantle, adductor muscle, and hepatopancreas were characterized in the freeze tolerant, intertidal ribbed mussel Geukensia demissus (Dillwyn). In general, freezing and anoxia elicited different metabolic responses and these also differed in a tissue-specific manner. Fermentative end products accumulated in all tissues except gill under both stresses. In adductor muscle, lactate was produced in both cases. In mantle and hepatopancreas, only succinate accumulated in anoxia, correlated with an opposite decrease in aspartate, and changes in glycolytic intermediates were consistent with anoxia-induced metabolic arrest. During freezing, however, both lactate and succinate accumulated (as well as alanine in hepatopancreas) with net end product accumulation 3–4-fold greater than during anoxia and changes in glycolytic intermediates were consistent with the derivation of these products from carbohydrate catabolism. Other differences in response to freezing versus anoxia included the accumulation of glucose in all tissues and a reduction of energy charge in mantle during freezing, but not in anoxia. Taurine and glycine comprised about 80–85% of the total free amino acid pools in all tissues; in gill levels of both amino acids fell significantly during freezing or anoxia exposures whereas in mantle a significant decrease in these amino acids occurred during anoxia only. The data show both a broader range of metabolic responses, and greater net changes in metabolite levels during freezing compared with anoxia. This suggests (1) that metabolic responses to freezing include both cryoprotective and ischemia-protective changes, and (2) that tissue mechanisms of metabolic rate depression are rapidly induced during anoxia exposure but develop more slowly during freezing.

Cellular responses to dehydration were analyzed in six organs of leopard frogs Rana pipiens. Frogs at 5 degrees C endured the loss of up to 50% of their total body water content but water contents of individual organs were strongly defended. Skeletal muscle water content was strongly affected by dehydration, dropping from 80.7% of wet mass in controls to 67.2% in frogs that had lost 50% of their total body water. However, water contents of internal organs dropped by only 3-8% of their wet masses. Water contents of all organs except skeletal muscle were fully restored by 24h of rehydration in water at 5 degrees C. Dehydration had no consistent effect on the protein content of five organs but in a sixth, the kidney, protein levels were elevated (by 60-72%) at the higher levels of dehydration and during rehydration. Dehydration led to a rapid increase in glucose concentration in the liver; compared with control values of 13 +/- 2 nmol mg-1 protein, levels were doubled by 12.2% dehydration and continued to increase to a maximum of 307 +/- 44 nmol mg-1 protein (20 mumol g-1 wet mass) in 50% dehydrated frogs. Glucose accumulation was supported by a decrease in liver glycogen content and a parallel rise in glucose 6-phosphate levels, but not in the levels of other glycolytic intermediates, confirming that glycogenolytic flux was being directed into glucose synthesis. Blood glucose levels also increased as a function of increasing dehydration, reaching values 13.8 times higher than controls, but only the kidney and brain showed a significant accumulation of glucose over the course of dehydration.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolic responses by the liver to the evaporative loss of up to 60% of total body water were quantified in spring-collected wood frogs, Rana sylvatica, a freeze-tolerant species. Dehydration stimulated rapid hyperglycemia, liver glucose levels rising 3.8-fold to 90 nmol/mg protein (9.9 μmol/g wet mass) by the time that 10% of total body water had been lost. Glucose accumulation occurred at the expense of liver glycogen reserves, which fell over the course of dehydration, and was supported by a 5.8-fold increase in the activity of glycogen phosphorylase a in the liver, made up of increases in both the total phosphorylase activity expressed and the percentage of the enzyme in the active form. Analysis of changes in the levels of glycolytic intermediates in the liver over the course of dehydration showed sharp increases in glucose-6-phosphate and fructose-6-phosphate during the period of active glucose synthesis but no change in the levels of fructose-1,6-bisphosphate or triose phosphates. This indicated that an inhibitory block on glycolysis at the phosphofructokinase reaction helped to promote the diversion of glycogenolysis into glucose export. When water loss exceeded 10%, cellular energetics were affected; ATP levels fell progressively between 25 and 60% dehydration, but a concomitant drop in the total adenylate pool held the energy charge stable at 0.7–0.8 up to 35% dehydration. At extreme dehydration (50 and 60%), metabolic indicators of hypoxia stress appeared in the liver: lactate accumulated and the energy charge fell. The data show that a primary response to whole-body dehydration in wood frogs is the activation of liver glucose synthesis and this suggests that the production of glucose as a cryoprotectant during freezing in this species is probably derived from a pre-existing amphibian volume-regulatory response to dehydration.

Larvae of Osmoderma eremicola (Knoch)(Coleoptera: Scarabaeidae) were found to be freeze tolerant. Early (1–2.5 cm) and late (3–5 cm) larval stages survived freezing to −8.3°C for 96 h with 64% of body water as ice. Glycogen phosphorylase activity in the fat body was increased 6–7-fold during freezing, and glycogen stores were depleted in fat body (from 237 ± 1 to 57 ± 7 μmol/g fresh weight) and body wall (89 ± 4 to 40 ± 1 μmol/g fresh weight). However, no glycogen derived cryoprotectants (glycerol, sorbitol, glucose, fructose) were found to accumulate in tissues or haemolymph during freezing or acclimation at 9,0 and −5°C. Osmolatity (∼450 mOsmol) of haemolymph was constant during freezing and acclimation from 9.to −5°C, also suggesting the absence of low molecular weight cryoprotectants. Haemolymph trehalose levels never rose above 2.7 mM as detected by NMR. Total protein levels in fat body and body wall remained unchanged during acclimation. HPLC analysis showed increases in amino acid levels by 10–15 μmol/g fresh weight in the body wall of both early and late instar larvae during freezing to −8.3°C for 96 h, with consistent increases in glycine (7.4–8.7 μmol/g fresh weight), alanine (4.2–5.8 μmol/g fresh weight), glutamate (2.4 μmol/g fresh weight), and valine (0.8–1.6 μmol/g fresh weight). Smaller increases in glutamic acid (by 1 μmol/g fresh weight) and alanine (by 3.5 μmol/g fresh weight) were also seen in late instar fat body, whereas glycine levels remained unchanged and valine decreased slightly (by 1.4 μmol/g fresh weight) in this tissue. Proline levels, detected by NMR, ranged from 10 to 18 μmol/g fresh weight in fat body and 40–70 μmol/g fresh weight in haemolymph, making it the most abundant amino acid in fat body.

Wood frogs, Rana sylvatica, tolerate the loss of 50-60% of total body water during experimental dehydration. The rate of water loss for unprotected frogs is the same whether animals are frozen (at -2 degrees C) or unfrozen (at 1 degrees C) but is greatly reduced when frogs are frozen under a protective layer of moss. Dehydrational death could occur in as little as 7-9 days for unprotected animals; this indicates the importance for winter survival of selecting well-protected and damp hibernation sites. Prior dehydration affected the cooling and freezing properties of frogs, reducing supercooling point and the amount of ice formed after 24 h at -2 degrees C and acting synergistically with freezing exposure in stimulating cryoprotectant synthesis. Analysis of the effects of controlled dehydration at 5 degrees C showed that changes in body water content alone (without freezing) stimulated liver glycogenolysis and the export of high concentrations of glucose into blood and other organs. Autumn-collected frogs dehydrated to 50% of total body water lost showed glucose levels of 165-1,409 nmol/mg protein in different organs, increases of 9- to 313-fold compared with control values and reaching final levels very similar to those induced by freezing exposure. The data support the proposal that various adaptations for natural freeze tolerance may have been derived from preexisting mechanisms for dealing with water stress in amphibians and that cell volume change may be one of the signals involved in triggering and sustaining molecular adaptations (e.g., cryoprotectant output) that support freezing survival.

Adult eastern box turtles Terrapene carolina carolina from Ohio readily recovered after 44 h of freezing exposure at -2 C. During thawing at 25 C, motor responses resumed in a sequence of increasing complexity with a reflex twitch in response to poking seen after 1.2 +/- 0.5 h, coordinated retraction of the limbs after 2.0 +/- 0.18 h, and voluntary locomotion after 2.7 +/- 0.4 h. Turtles dissected immediately after freezing exposure had ice in body cavities and surrounding skeletal muscles in limbs. Analysis of putative cryoprotectants in serum and seven organs showed that all organs accumulated glucose during freezing. Net glucose accumulations of 6-20 mumol/g wet weight represented 4-22-fold increases; the highest glucose levels were in liver, heart, and serum. Liver glycogenolysis was identified as the source of the glucose; this was supported by both metabolite (a 62.5-fold increase in the precursor glucose-6-phosphate) and enzyme (the percentage of glycogen phosphorylase in the active a form rose from 21.3-57.5%) responses to freezing. Freezing exposure also caused an increase in lactate levels in serum and four organs, but glycerol (<2 mumol/g), sorbitol (<0.3 mumol/g), and free amino acid levels in organs were unchanged. Both the low levels of putative cryoprotectants and measurements of serum osmolality, 244 +/- 4.3 and 315 +/- 15.4 mOsmol/l for control and freezing exposed turtles, indicated that box turtles can endure freezing without an accumulation of large pools of low molecular weight cryoprotectant.

Cold hardiness was evaluated for the red-sided garter snake, Thamnophis sirtalis parietalis. Snakes collected in the autumn near communal den sites showed an ability to supercool (Supercooling point, SCP = −5.5 °C). However, by midwinter, supercooling capacity was reduced and snakes cooled only to −0.8 to −1.2 °C before freezing. Survival of freezing and body ice contents were determined over a time course of freezing exposure at −2.5 °C. Snakes recovered fully after freezing exposures of 3 h or less that produced ice contents of up to 40% of total body water. After longer periods and with ice contents of over 50%, survival was reduced. Only 50% of snakes survived 10 h of freezing and no snakes recovered after 24 or 48 h with a maximal ice content of 70% of body water. Putative cryoprotectants were assessed in seven organs (liver, kidney, muscle, intestine, brain, lung, heart) as well as the eggs after 5 h of freezing at −2.5 °C. Glucose content increased 4-fold in liver, and lactate rose by 50% in heart, but levels of these and other possible cryoprotectants did not increase in other organs during freezing. However, a high free amino acid pool, including 14–24 μmol/g wet weight taurine, was present in the organs. The data suggest that long-term freezing survival is not part of the winter hibernation strategy of this species, but tolerance of brief freezing exposures may be adaptive in dealing with overnight frosts when the animals are active above ground.

Hatchling red-eared turtles Trachemys (= Pseudemys) scripta elegans (Wied) from a Louisiana population display a significant ability to withstand the freezing of extracellular body fluids. All animals survived at least 2 h of freezing at -2.5 or -4 degrees C. At -2.5 degrees C, survival declined to 50% after 6 h of freezing and no animals recovered after 24 h or longer, when mean ice content reached 54.7 +/- 1.4% of total body water. At -4 degrees C, all turtles recovered from 4 h of freezing exposure with a mean ice content of 49.6 +/- 2.4%, but survival dropped sharply thereafter with no animals recovering after 8 h, when ice content had reached 64.5 +/- 0.7%. Survival times were substantially shorter and percentage ice values greater than comparable values for hatchling painted turtles (Chrysemys picta (Schneider)) from northern populations subjected to identical freezing exposures. The ability to synthesize cryoprotectants in response to freezing was poorly developed in T. s. elegans; maximal accumulation of glucose was only 3.2 mumol g-1 wet mass in liver. Lactate content increased two- to threefold in oxygen-sensitive organs (heart and brain) during freezing, but levels of lactate and other putative cryoprotectants were unchanged in other organs. Total free amino acid content rose significantly in liver, muscle and blood during freezing; increased taurine concentration was primarily responsible for the changes in liver and blood. The capacity for freezing survival by T. s. elegans hatchlings from southern populations would be of limited use for hibernation in a cold climate, but the metabolic responses to freezing displayed by these animals might be enhanced by northern populations to increase their freeze tolerance.

Hatchlings of both the Midland (Chrysemys picta marginata) and Western (C. picta bellii) subspecies of the painted turtle tolerate the freezing of extracellular body fluids while overwintering in terrestrial nests. Fall-collected hatchlings survived 3 days of continuous freezing at -2.5 degrees C, with ice contents of 43.5 +/- 1.0% of total body water (SE; n = 24) for C. picta marginata and 46.5 +/- 0.8% (n = 32) for C. picta bellii. Survival times dropped to 4-5 h when temperature was lowered to -4 degrees C, correlated with ice contents of greater than or equal to 50%. However, C. picta marginata tested immediately after excavation from nests in the spring showed greater freeze tolerance, with survival extending to 11 days at -2.5 degrees C and a higher mean ice content of 50.2 +/- 1.2% (n = 6). Spring hatchlings also had high supercooling points, -1.07 +/- 0.13 degrees C (n = 8), that dropped within 3 days to -4.83 +/- 0.83 degrees C (n = 4), suggesting a breakdown of endogenous ice-nucleating agents when hibernation ended. A search for possible cryoprotectants showed that both subspecies accumulated glucose and lactate in liver during freezing (net increase = 3-13 mumols/g wet wt); both also maintained large free amino acid pools in organs, with taurine making up 21-47% of the total.

Organ-specific metabolic responses to freezing were monitored over the course of 4 h exposure to freezing at −4 °C for autumn-collected hatchlings of midland (Chrysemys picta marginata) and western (Chysemys picta bellii) painted turtles. Both subspecies accumulated glucose and lactate in seven organs during freezing for possible use as cryoprotectants. Overall levels of both were higher in C. p. bellii than in C. p. marginata, with glucose ranging up to 16 μmol/g wet weight and lactate up to 25 μmol/g in C. p. bellii organs. Glucose accumulation by C. p. bellii organs appeared to be initiated during prefreeze cooling, whereas glucose production in C. p. marginata was triggered by the initiation of freezing; this difference may explain the higher overall accumulations of both glucose and lactate in C. p. bellii organs. Glucose content in most organs declined over the course of the 4-h freezing exposure, suggesting that the main function of the sugar may be as the substrate for lactate production. Analysis of changes in the levels of glycolytic intermediates in liver revealed an accumulation of glucose-6-phosphate, indicating activation of liver glycogenolysis during freezing. An increase in liver fructose-6-phosphate and a decrease in fructose-1,6-bisphosphate concentrations over the course of freezing were consistent with an inhibition of liver phosphofructokinase that would support glucose export to other organs.

Five hours of freezing exposure at -2.5-degrees-C produced organ-specific changes in adenylates and energy charge, glycolytic intermediates, fermentative end products, glucose, fructose-2,6-bisphosphate, and the activity of glycogen phosphorylase in seven organs of garter snakes. Significant cryoprotectant synthesis was lacking, but the liver showed an activation of glycogenolysis and a 4-fold increase in glucose during freezing. Oxygen-sensitive organs, heart and brain, accumulated lactate during freezing exposure.

Metabolic responses to dormancy and anoxia were assessed in foot muscle and he-patopancreas of the land snail Otala lactea (Pulmonata, Helicidae). In both states cellular energetics (arginine phosphate content, adenylate energy charge) were maintained at a high level, fueled during estivation by aerobic metabolism and during anoxia by carbohydrate fermentation. D-lactate was the major product of anaerobiosis in hepatopancreas, but in foot muscle D-lactate, L-alanine, and succi-nate accumulated (in net amounts of 13.5, 3.5, and 1.8 μmolg⁻¹ wet weight, respec-tively). Changes in the concentrations of glycolytic intermediates were compared for both short-and long-term stress: 2 and 14 h of exposure to N₂ gas atmosphere, 3 and 22 d of dormancy at 22° C. Both stresses appeared to include glycolytic activation in the short term in foot muscle, with crossover analyses indicating regulatory control at the phosphofructokinase, aldolase, and pyruvate kinase loci. Over the long term, however, this was reversed and a glycolytic rate depression was observed aspart of the overall metabolic rate depression of these states. In foot muscle, inhibition at phosphofructokinase and pyruvate kinase was apparent during anoxia, whereas aldolase and pyruvate kinase were the key sites of inhibitory control during estivation. In hepatopancreas, phosphofructokinase was the primary locus for inhibitory control during both prolonged anoxia and estivation. These data suggest that common molecular mechanisms underlie glycolytic rate depression during dormancy and anaerobiosis.

The metabolic responses to repeated cycles of temperature change, alternating 24 h at −16 and +3°C, were compared for a freeze-tolerant (Eurosta solidaginis) vs a freeze-avoiding (Epiblema scudderiana) insect. The two species differed most strongly in the response by cellular energetics. ATP content and energy charge were depressed in E. scudderiana larvae with each −16°C exposure but rebounded with each return to +3°C; after 12 such cycles, final energy status at +3°C was not significantly different than control values. By contrast, E. solidaginis larvae maintained a high energy charge (at the expense of a decrease in the total adenylate pool) over the first two cycles of freeze/thaw only. Subsequently energy stress was cumulative, with no recovery in the +3°C half of each cycle, and energy charge fell to 0.70–0.75. Both species showed a rise in glucose-6-P with each cold exposure, indicative of cold shock activation of glycogen phosphorylase. E. scudderiana showed no net increase in cryoprotectant content over the experimental course but sorbitol levels rose 4-fold in E. solidaginis. Alanine content increased significantly in E. solidaginis over the 12 cycles but no glycolytic end products accumulated in E. scudderiana. The data indicate that the freeze-avoiding specie is better able to maintain cellular homeostasis in the face of rapid and wide variations in environmental temperature than is the freeze-tolerant specie and that this may represent a worthwhile advantage of the freeze-avoidance strategy of cold hardiness for some species.

Hatchlings of the painted turtle (Chrysemys picta marginata) are unique as the only reptile and highest vertebrate life form known to tolerate the natural freezing of extracellular body fluids during winter hibernation. Turtles survived frequent exposures to temperatures as low as -6 degrees C to -8 degrees C in their shallow terrestrial nests over the 1987-1988 winter. Hatchlings collected in April 1988 had a mean supercooling point of -3.28 +/- 0.24 degrees C and survived 24 hr of freezing at -4 degrees C with 53.4% +/- 1.98% of total body water as ice. Recovery appeared complete after 20 hr of thawing at 3 degrees C. However, freezing at -10.9 degrees C, resulting in 67% ice, was lethal. A survey of possible cryoprotectants revealed a 2- to 3-fold increase in glucose content of liver and blood and a 3-fold increase in blood glycerol in response to freezing. Although quantitatively low, these responses by spring turtles strongly indicate that these may be the winter-active cryoprotectants. The total amino acid pool of blood also increased 2.25-fold in freezing-exposed turtles, and taurine accounted for 52% of the increase. Most organs accumulated high concentrations of lactate during freezing, a response to the ischemic state imposed by extracellular freezing. Changes in glycogen phosphorylase activity and levels of glucose 6-phosphate and fructose 2,6-bisphosphate were also consistent with a dependence on anaerobic glycolysis during freezing. Studies of the molecular mechanisms of natural freeze tolerance in these turtles may identify protective strategies that can be used in mammalian organ cryopreservation technology.