Strategies of hypoxia and anoxia tolerance in cardiomyocytes from the overwintering common frog, Rana temporaria

Using ventricular cardiomyocytes of the common frog, Rana temporaria, we investigated the metabolic strategies employed by the heart to tolerate 4 mo of hypoxic submergence (overwintering) as well as acute bouts of anoxia. In contrast to what is observed for the whole animal, there was no change in oxygen consumption in cardiomyocytes isolated from normoxic frogs compared with those isolated from 4-mo hypoxic animals. Furthermore, cells from both normoxic and hypoxic frogs were able to completely recover oxygen consumption following 30 min of acute anoxia. From estimates of ATP turnover, it appears that frog cardiomyocytes are capable of a profound, completely reversible metabolic depression, such that ATP turnover is reduced by >90% of control levels during anoxia but completely recovers with reoxygenation. Moreover, this phenomenon is also observed in frogs that have been subjected to 4 mo of extended hypoxia. We found a significant increase in the stress protein, hsp70, after 1 mo of hypoxic submergence, which may contribute to the heart's remarkable hypoxia and anoxia tolerance and may act to defend metabolism during the overwintering period.     

Frogs and turtles: different ectotherm overwintering strategies

The ability of frogs and turtles to overwinter and to survive hypoxia and anoxia has long been a topic of interest. While data remains scant, the emerging picture shows fundamentally different approaches to overwintering in these two groups of ectotherms. Frogs are far more limited by availability of oxygen than are turtles, even at near-freezing ambient temperatures. The reasons for this probably involve the vastly greater cutaneous permeability of the former. With their extreme tolerance of anoxia and profound suppression of metabolism, overwintering in turtles should not be viewed as simply prolonged diving but rather as ectotherm hibernation. Their incredible diving capabilities are merely a spin-off of a successful overwintering strategy. The following discussion reviews the major physiological mechanisms involved in the overwintering strategies of these two ectotherm groups.     

The effect of overwintering temperature on the body energy reserves and phenoloxidase activity of bumblebee Bombus lucorum queens

Warming winters and changes in species composition related to the estimated global warming may cause a threat to bumblebees adapted to cold winters. During the overwintering period, their intermediary and respiratory metabolism decreases but metabolism remains responsive to temperature. The effect of temperature on diapause survival, phenoloxidase (PO) activity, and energy expenditure of the white-tailed bumblebee (Bombus lucorum) after a 4-month diapause were studied by manipulating the diapause temperature. Two overwintering temperatures were used, cold (1.8 °C) and warm (9 °C). Body fat content was used as an estimate of the remaining energy resources and PO activity as an immune function parameter of overwintering bumblebee queens. The baseline levels of PO activity were used to measure the differences in B. lucorum queen responses after overwintering in either temperature. We found a 0.4 g pre-diapause threshold weight of survival in B. lucorum. Large queens had more fat left and a higher PO activity compared to small ones after overwintering in warm conditions, but in the cold there was no effect of size on the remaining fat in the fat body of queens or their PO activity. The observed difference in energy usage appears to relate to normal size-dependent metabolism and variation in energy allocation between basic metabolism and immune functions.     

Behavioural adaptations of Rana temporaria to cold climates

Environmental conditions at the edge of a species’ ecological optimum can exert great ecological or evolutionary pressure at local populations. For ectotherms like amphibians temperature is one of the most important abiotic factors of their environment as it influences directly their metabolism and sets limits to their distribution. Amphibians have evolved three ways to cope with sub-zero temperatures: freeze tolerance, freeze protection, freeze avoidance. The aim of this study was to assess which strategy common frogs at mid and high elevation use to survive and thrive in cold climates. In particular we (1) tested for the presence of physiological freeze protection, (2) evaluated autumnal activity and overwintering behaviour with respect to freeze avoidance and (3) assessed the importance of different high-elevation microhabitats for behavioural thermoregulation. Common frogs did not exhibit any signs of freeze protection when experiencing temperatures around 0 °C. Instead they retreated to open water for protection and overwintering. High elevation common frogs remained active for around the same period of time than their conspecifics at lower elevation. Our results suggest that at mid and high elevation common frogs use freeze avoidance alone to survive temperatures below 0 °C. The availability of warm microhabitats, such as rock or pasture, provides high elevation frogs with the opportunity of behavioural thermoregulation and thus allows them to remain active at temperatures at which common frogs at lower elevation cease activity.     

The aerobic physiology of the air-breathing blue gourami, Trichogaster trichopterus, necessitates behavioural regulation of breath-hold limits during hypoxic stress and predatory challenge

Physiological characteristics of the blood oxygen transport system and muscle metabolism indicate a high dependence on aerobic pathways in the blue gourami, Trichogaster trichopterus. Haemoglobin concentration and haematocrit were modest and the blood oxygen affinity (P50=2.31 kPa at pH 7.4 and 28 degrees C) and its sensitivity to pH (Bohr factor, phi=-0.34) favour oxygen unloading at a relatively high oxygen pressure (PO2). The intracellular buffering capacity (44.0 slykes) and lactate dehydrogenase (LDH) activity (154.3 iu g(-1)) do not support exceptional anaerobic capabilities. Air-breathing frequency in the blue gourami is expected to increase when aquatic oxygen tensions decline. Under threat of predation, however, this behaviour must be modified at a potential cost to aerobic metabolism. We therefore tested the hypothesis that metabolic responses to predatory challenge and aquatic hypoxia are subject to behavioural modulation. Computer-generated visual stimuli consistently reduced air-breathing frequency at 19.95, 6.65 and 3.33 kPa PO2. Bi-directional rates of spontaneous activity were similarly reduced. The metabolic cost of this behaviour was estimated and positively correlated with PO2 but not with visual stimulation thus indicating down-regulation of spontaneous activity rather than breath-holding behaviour. Neither PO2 nor visual stimulation resulted in significant change to muscle lactate and ATP concentrations and confirm that aerobic breath-hold limits were maintained following behavioural modulation of metabolic demands.     

Control of carbohydrate metabolism in an anoxia-tolerant nervous system

Anoxia-tolerant animal models are crucial to understand protective mechanisms during low oxygen excursions. As glycogen is the main fermentable fuel supporting energy production during oxygen tension reduction, understanding glycogen metabolism can provide important insights about processes involved in anoxia survival. In this report we studied carbohydrate metabolism regulation in the central nervous system (CNS) of an anoxia-tolerant land snail during experimental anoxia exposure and subsequent reoxygenation. Glucose uptake, glycogen synthesis from glucose, and the key enzymes of glycogen metabolism, glycogen synthase (GS) and glycogen phosphorylase (GP), were analyzed. When exposed to anoxia, the nervous ganglia of the snail achieved a sustained glucose uptake and glycogen synthesis levels, which seems important to maintain neural homeostasis. However, the activities of GS and GP were reduced, indicating a possible metabolic depression in the CNS. During the aerobic recovery period, the enzyme activities returned to basal values. The possible strategies used by Megalobulimus abbreviatus CNS to survive anoxia are discussed.     

Anoxic Responses of Seedling Roots of Rice Cultivars Varying in Tolerance to Submergence

Differences in tolerance to submergence and anoxia exhibited by cultivar-specific rice (Oryza sativa L.) extend to the primary root tips and axes of 3-day-old seedlings. This paper considers the physiological mechanisms which might account for rice root intolerance to anoxia, particularly those implicated in pH regulation and sugar metabolism in relation to hypoxic acclimation. Hypoxic treatment and the presence of glucose during anoxia did not permit root tips and axes of intolerant cultivars to survive 24-h anoxia. The absence of typical glycolytic and fermentative enzyme induction together with no improvement of ethanol production and energy status during anoxia suggest that intolerant cultivars are not capable of hypoxic acclimation at the level of energy and sugar metabolism. However, root tip survival was enhanced in buffered medium after hypoxic treatment, suggesting a relationship between hypoxic treatment and improved pH regulation.     

Metabolic and physiological responses in tissues of the long-lived bivalve Arctica islandica to oxygen deficiency

In Arctica islandica, a long lifespan is associated with low metabolic activity, and with a pronounced tolerance to low environmental oxygen. In order to study metabolic and physiological responses to low oxygen conditions vs. no oxygen in mantle, gill, adductor muscle and hemocytes of the ocean quahog, specimens from the German Bight were maintained for 3.5 days under normoxia (21 kPa=controls), hypoxia (2 kPa) or anoxia (0 kPa). Tissue levels of anaerobic metabolites octopine, lactate and succinate as well as specific activities of octopine dehydrogenase (ODH) and lactate dehydrogenase (LDH) were unaffected by hypoxic incubation, suggesting that the metabolism of A. islandica remains fully aerobic down to environmental oxygen levels of 2 kPa. PO(2)-dependent respiration rates of isolated gills indicated the onset of metabolic rate depression (MRD) below 5 kPa in A. islandica, while anaerobiosis was switched on in bivalve tissues only at anoxia. Tissue-specific levels of glutathione (GSH), a scavenger of reactive oxygen species (ROS), indicate no anticipatory antioxidant response takes place under experimental hypoxia and anoxia exposure. Highest specific ODH activity and a mean ODH/LDH ratio of 95 in the adductor muscle contrasted with maximal specific LDH activity and a mean ODH/LDH ratio of 0.3 in hemocytes. These differences in anaerobic enzyme activity patterns indicate that LDH and ODH play specific roles in different tissues of A. islandica which are likely to economize metabolism during anoxia and reoxygenation.     

The physiology of hibernation in Canadian leopard frogs (Rana pipiens) and bullfrogs (Rana catesbeiana)

Canadian northern leopard frogs (Rana pipiens) and bullfrogs (Rana catesbeiana) were acclimated to 3 degrees C and submerged in anoxic (0-5 mmHg) and normoxic (Po(2) approximately 158 mmHg) water. Periodic measurements of blood Po(2), Pco(2), and pH were made on samples taken anaerobically from subsets of each species. Blood plasma was analyzed for [Na(+)], [K(+)], [Cl(-)], [lactate], [glucose], total calcium, total magnesium, and osmolality. Blood hematocrit was determined, and plasma bicarbonate concentration was calculated. Both species died within 4 d of anoxic submergence. Anoxia intolerance would rule out hibernation in mud, which is anoxic. Both species survived long periods of normoxic submergence (R. pipiens, 125 d; R. catesbeiana, 150 d) with minimal changes in acid-base and ionic status. We conclude that ranid frogs require a hibernaculum where the water has a high enough Po(2) to drive cutaneous diffusion, allowing the frogs to extract enough O(2) to maintain aerobic metabolism, but that an ability to tolerate anoxia for several days may still be ecologically meaningful.     

Metabolic adaptation to prolonged anoxia in leaves of American cranberry (Vaccinium macrocarpon)

The indigenous North American Cranberry ( Vaccinium macrocarpon ), when cultivated in specially constructed cranberry bogs, is normally flooded in winter to prevent frost injury. This protection under ice can give rise to prolonged periods of anoxia, which depending on the state of the vines and environmental conditions, can cause severe oxygen-deprivation injury. An experimental study of the tolerance of cranberry vines to controlled total anoxia reveals that mature dark-green perennating leaves with high carbohydrate levels are able to survive prolonged periods of total oxygen-deprivation while younger newly formed leaves are readily damaged. During the anoxic treatment the mature leaves exhibit a marked downregulation of metabolism. Carbohydrate consumption and energy metabolism stabilize at low levels soon after the switch from aerobic to anaerobic pathways. Pathways such as TCA cycle or photosynthesis, which are non-operating during the anoxia treatment, are severely affected but still measurable after 28 days anoxia. In the post-anoxic period the perennating leaves rapidly re-establish their capacity for aerobic respiration and photosynthesis.     

The diving paradox: new insights into the role of the dive response in air-breathing vertebrates

When aquatic reptiles, birds and mammals submerge, they typically exhibit a dive response in which breathing ceases, heart rate slows, and blood flow to peripheral tissues is reduced. The profound dive response that occurs during forced submergence sequesters blood oxygen for the brain and heart while allowing peripheral tissues to become anaerobic, thus protecting the animal from immediate asphyxiation. However, the decrease in peripheral blood flow is in direct conflict with the exercise response necessary for supporting muscle metabolism during submerged swimming. In free diving animals, a dive response still occurs, but it is less intense than during forced submergence, and whole-body metabolism remains aerobic. If blood oxygen is not sequestered for brain and heart metabolism during normal diving, then what is the purpose of the dive response? Here, we show that its primary role may be to regulate the degree of hypoxia in skeletal muscle so that blood and muscle oxygen stores can be efficiently used. Paradoxically, the muscles of diving vertebrates must become hypoxic to maximize aerobic dive duration. At the same time, morphological and enzymatic adaptations enhance intracellular oxygen diffusion at low partial pressures of oxygen. Optimizing the use of blood and muscle oxygen stores allows aquatic, air-breathing vertebrates to exercise for prolonged periods while holding their breath.     

ECOLOGY AND PHYSIOLOGY OF HIBERNATION AND OVERWINTERING AMONG FRESHWATER FISHES, TURTLES, AND SNAKES

1. Freshwater fishes are the most northerly of freshwater ectotherms, followed by frogs. North American freshwater snakes, turtles, and salamanders do not range farther north than southernmost Canada. 2. Freezing and desiccation are the main challenges during terrestrial hibernation of ectotherms. Oxygen depletion, water balance, and ionic balance are the major problems for air breathing ectotherms that hibernate underwater. 3. The importance of accumulation of energy stores for overwintering among fishes depends upon the length and severity of the winters, whether or not there is springtime reproduction, body size, latitude, and the availability and use of food during overwintering. 4. Fishes can decrease energy demands during the winter by reductions in activity, metabolic depression, and entrance in semi-torpidity. 5. Adaptations for coping with hypoxia and anoxia among overwintering freshwater fishes may include metabolic depression, a decrease in blood O₂ affinity, microhabitat selection, air breathing, short-distance migration, biochemical modifications aimed at adjusting glycolytic rates, and alcoholic fermentation. 6. Freshwater turtles have a worldwide northern limit of approximately 50° N, which means that some species spend about half of their lives hibernating. 7. Aquatic turtles normally hibernate underwater, although occasionally they hibernate on land. In water they usually hibernate in a hypoxic or anoxic (mud) environment and in relatively shallow water. Wintertime movements of unknown frequency occur in some species. 8. The hatchlings of many turtle species can overwinter in the nest. Among northern species this behaviour is most common among painted turtles, whose hatchlings can withstand freezing. 9. Mortality among adult turtles is probably highest during the hibernation cycle. 10. Temperature appears to the most important cue for entry and exit from hibernation among freshwater turtles. 11. Little is known of the energetics of overwintering turtles. Energy stores for overwintering may be more important at lower latitudes than at higher ones, due to the higher metabolic rates of overwintering, but non-feeding, southern turtles. 12. The ability of turtles to tolerate submergence is a function of temperature, degree of water oxygenation, latitude of origin, efficacy of extrapulmonary respiratory pathways, and metabolic rate. 13. For turtles that hibernate in an anoxic hibernaculum, and for those without sufficient extrapulmonary uptake of O₂ to allow metabolism to be completely aerobic, the most important physiological perturbation is an acidosis developed from a continuing production of lactate. If sufficient O₂ can be obtained, the most likely factors limiting hibernation time are water balance and ion balance. 14. Mechanisms of turtles for coping with acidosis include metabolic depression, integumental CO₂ loss, bicarbonate buffering, and changes in ion concentrations that minimize the decrease in SID (strong ion difference). The most important among the latter are a decrease in plasma [Cl^-] and large increases in plasma calcium and magnesium. 15. Turtles are unique among reptiles in their ability to maintain both cardiovascular and nervous system function during prolonged anoxia. 16. Turtles gain weight from water uptake during submerged hibernation, but apparently maintain some kidney function; however, osmoregulation is one of the least known areas of the physiology of hibernation. 17. Recovery of turtles upon emergence commences with a rapid hyperventilatory compensation of pH, followed by a slower adjustment of ion levels. Basking speeds recovery greatly. 18. While hibernation of turtles in the northern parts of their ranges is most likely very stressful physiologically, northern range limits are more likely to be determined by reproductive restraints than by the rigors of extended hibernation. 19. The superior ability of turtles to tolerate anoxia may be more the result of an annual hibernation than of their diving habits during active periods of the year. 20. Freshwater snakes usually hibernate on land. However, they appear to be capable of aquatic hibernation and may not do so because of the risk of death from anoxia. 21. Some species of terrestrial snakes are known to hibernate underwater, and are able to do so in the laboratory for months. In the field, this behaviour is considered opportunistic, as there is no evidence to suggest that any snakes can tolerate extended anoxia.     

Blood oxygen transport in common map turtles during simulated hibernation

We assessed the effects of cold and submergence on blood oxygen transport in common map turtles (Graptemys geographica). Winter animals were acclimated for 6-7 wk to one of three conditions at 3 degrees C: air breathing (AB-3 degrees C), normoxic submergence (NS-3 degrees C), and hypoxic (PO2=49 Torr) submergence (HS-3 degrees C). NS-3 degrees C turtles exhibited a respiratory alkalosis (pH 8.07; PCO2=7.9 Torr; [lactate]=2.2 mM) relative to AB-3 degrees C animals (pH 7.89; PCO2=13.4 Torr; [lactate]=1.1 mM). HS-3 degrees C animals experienced a profound metabolic acidosis (pH 7.30; PCO2=7.9 Torr; [lactate]=81 mM). NS-3 degrees C turtles exhibited an increased blood O2 capacity; however, isoelectric focusing revealed no seasonal changes in the isohemoglobin (isoHb) profile. Blood O2 affinity was significantly increased by cold acclimation; half-saturation pressures (P50's) for air-breathing turtles at 3 degrees and 22 degrees C were 6.5 and 18.8 Torr, respectively. P50's for winter animals submerged in normoxic and hypoxic water were 5.2 and 6.5 Torr, respectively. CO2 Bohr slopes (Delta logP50/Delta pH) were -0.15, -0.16, and -0.07 for AB-3 degrees C, NS-3 degrees C, and HS-3 degrees C turtles, respectively; the corresponding value for AB-22 degrees C was -0.37. The O2 equilibrium curve (O2EC) shape was similar for AB-3 degrees C and NS-3 degrees C turtles; Hill plot n coefficients ranged from 1.8 to 2.0. The O2EC shape for HS-3 degrees C turtles was anomalous, exhibiting high O2 affinity below P50 and a right-shifted segment above half-saturation. We suggest that increases in Hb-O2 affinity and O2 capacity enhance extrapulmonary O2 uptake by turtles overwintering in normoxic water. The anomalous O2EC shape and reduced CO2 Bohr effect of HS-3 degrees C turtles may also promote some aerobic metabolism in hypoxic water.     

Molecular control of cold acclimation in trees

Frost tolerance is an acquired characteristic of plants that is induced in response to environmental cues preceding the onset of freezing temperatures and activation of a cold acclimation program. In addition to transient acclimation to low non-freezing temperatures and enhancing survival to short frost episodes during the growth season, perennial woody plants need additionally to survive the cold winter months. Trees have evolved a complex dynamic process controlling the development of dormancy and freezing tolerance that secures accurate initiation and termination of the overwintering process. Although the phenology of overwintering has been known for decades, only recently has there been progress in elucidating the molecular mechanisms of dormancy and freezing tolerance development in perennial plants. Current molecular and genomic studies indicate that herbaceous annual and woody perennial plants share similar cold acclimation mechanisms. Both the signal processes controlling cold acclimation and the cold-regulated target genes appear to be shared by herbaceous and woody plants. However, the dormancy development during overwintering brings new players in the molecular control of seasonal cold acclimation of woody perennials.     

越冬期不同阶段二点委夜蛾越冬幼虫耐寒性变化

【目的】二点委夜蛾Athetis lepigone (Moschler)是我国夏玉米苗期的新害虫,随着耕作制度的改变,二点委夜蛾的发生危害区域和面积逐渐扩大。本研究探讨二点委夜蛾的抗寒能力,为揭示抗寒机理提供理论基础。【方法】分别在越冬期的3个不同阶段,即越冬初期（2012年11月7日）、越冬期（2012年1月20日）和越冬末期（2013年3月5日）,对二点委夜蛾老熟幼虫的体重、过冷却点（supercooling point, SCP）、结冰点（freezing point, FP）、含水量、脂肪和糖原含量进行测定。【结果】过冷却点在这3个时期有显著差异,最低值（-23.16±0.38℃）出现在1月份,最高值（-16.24±1.24℃）出现在越冬初期,结冰点变化趋势与过冷却点一致。通过定量检测发现,虫体鲜重与过冷却点无相关性（r=0.17, P=0.12）;脂肪含量在越冬初期含量最高,而在越冬末期最低;糖原含量在越冬期含量最低;自由水的含量随着过冷却能力升高而降低,随其降低而升高,而结合水含量恰好相反。【结论】二点委夜蛾的抗寒性在越冬期不同阶段出现明显的变化,即随着冬季低温的到来,其抗寒能力逐渐增强,冬季过后又随气温的回升,其抗寒力逐渐减弱。     

Anoxia blocks thermotolerance and the induction of rapid cold hardening in the flesh fly, Sarcophaga crassipalpis

Abstract. Anoxia induced by nitrogen or carbon dioxide, or hypoxic/hypobaric conditions generated by a partial vacuum sensitizes red-eye pharate adults of Sarcophaga crassipalpis Macquart to a high temperature exposure that is normally nonlethal (40C for 2–3 h). Thermotolerance induced by a2h exposure to 40C (under aerobic conditions) doubles the pharate adults' tolerance to 45C but provides no protection against a combined exposure to 45C and anoxia, and only modest protection against a combined exposure to 40C and anoxia. Under aerobic conditions, exposing pharate adults to 0C for 2 h increases their tolerance to -10C (rapid cold hardening). Rapid cold hardening at 0C is not induced under anoxia. These results imply that tolerance to high temperatures and rapid cold hardening are dependent on aerobic processes and suggest that certain forms of temperature stress can be further exacerbated with anoxia.     

Differences in the altered energy metabolism of hemorrhagic shock and hypoxemia.

The effect of hemorrhagic shock, hypoxemia, and anoxia on the levels of adenine and pyridine nucleotides of liver and kidney was assessed. ATP levels in liver and kidney of animals in shock or animals subjected to 7 min of anoxia decreased by 85 and 73%, respectively. Under hypoxic conditions (arterial PO2 AT 18 MMHg), the decrease was only 62 and 48% in liver and kidney, respectively. Tissue NAD levels decreased and NADH levels increased during shock but were found to be essentially unaltered during experimental hypoxemia. Thus, shock produced greater alterations in adenine and pyridine nucleotides than did hypoxemia alone, indicating that stagnant hypoxemia due to shock is more deleterious to energy metabolism than is severe hypoxemia with an otherwise normal circulation. The results also suggest that if an anterial PO2 OF 18 MMHg represents the initial stages of tissue hypoxia, then tissue ATP levels are a more sensitive indicator of this than NAD levels.     

Metabolic Acclimation to Anoxia Induced by Low (2-4 kPa Partial Pressure) Oxygen Pretreatment (Hypoxia) in Root Tips of Zea mays

Young intact plants of maize (Zea mays L. cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretreatment) for 18 hours before excision of the 5 millimeter root apex and treatment with strictly anaerobic conditions (anoxia). Hypoxic acclimation gave rise to larger amounts of ATP, to larger ATP/ADP and adenylate energy charge ratios, and to higher rates of ethanol production when excised root tips were subsequently made anaerobic, compared with root tips transferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was associated with increased activity of alcohol dehydrogenase (ADH), and induction of ADH-2 isozymes. Roots of Adh1⁻ mutant plants lacked constitutive ADH and only slowly produced ethanol when made anaerobic. Those that were hypoxically pretreated acclimated to anoxia with induction of ADH2 and a higher energy metabolism, and a rate of ethanol production comparable to that of nonmutants. All these responses were insensitive to the presence or absence of NO₃⁻. Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NO₃⁻ to NO₂⁻. These results indicate that nitrate reductase does not compete effectively with ADH for NADH, or contribute to energy metabolism during anaerobic respiration in this tissue through nitrate reduction. Unacclimated root tips of wild type and Adhl⁻ mutants appeared not to survive more than 8 to 9 hours in strict anoxia; when hypoxically pretreated they tolerated periods under anoxia in excess of 22 hours.