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Frogs and turtles: Different ectotherm overwintering strategies
Camp. Biochem. Physiol. Printed in Great Britain
Vol. 86A.
No. 4, pp. 609-615,
1987 0
MINI
0300-9629/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
REVIEW
FROGS AND TURTLES: DIFFERENT ECTOTHERM OVERWINTERING STRATEGIES DAVID G. PENNEY
Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA (Receiued 18 July 1986) Abstract-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 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 of oxygen
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.
INTRODUCTION
sence of oxygen. Let us now consider some of the specific ways in which frogs and turtles differ in their overwintering strategies. Because most of the work in this area has been carried out on Ranids among the anurans, and Chrysemys (Pseudemys) among the turtles, this review is primarily relevant to them, but should also have general applicability to all ectotherms.
At temperate to near arctic latitudes freshwater frogs and turtles, where present, disappear from the landscape during winter. Where do they go and how do they remain alive? While these questions have been posed before, little published natural history is available for this period, although unpublished anecdotes abound. In the absence of adequate field studies, some insight into the probable strategies used by these ectotherms may be gained from laboratory submersion or “diving” studies. The problem of cold and/or freezing tolerance is not considered in this brief review. Five general mechanisms are available to animals to allow survival while diving:
FROGS
(1) by making use of stored oxygen brought from the surface, (2) by absorbing oxygen from the water, (3) by a metabolic shutdown to a lower basal rate, (4) by developing an oxygen debt in the form of lactic acid, which must be repaid when oxygen is again available, and (5) by shifting to an anaerobic metabolic pathway which does not involve an oxygen debt. Each of the above will enter into the subsequent discussion. If diving occurs in water devoid of oxygen the second alternative is of course eliminated. Several important generalizations emerge when we compare the diving tolerance of selected vertebrates (Table 1): First, tolerance increases from homeotherms to ectotherms, even recognizing the amazing adaptive mechanisms of seals and related species. Secondly, it increases enormously as environmental temperature is lowered in ectotherms due to the energy sparing effect of lowered metabolic rate. Finally, turtles show clear superiority over all other air breathing vertebrates including anurans as long duration divers when submersion occurs in the ab609
Early reports placed overwintering frogs under rocks or in the mud at the bottom. Stories also abound of netting frogs from the mud of frozen ponds, lakes, etc. Such areas would almost certainly be in a reduced state, i.e. lacking in oxygen. In 1972, Emery et al. reported seeing “hibernating” frogs resting in small pits at the surface of the mud at the bottom of an ice-covered pond in Ontario. Water temperature was 2S”C and dissolved oxygen was 7 ppm. More recently, Cunjak (1986), found frogs during winter under rocks and among rubble in a well-oxygenated southern Ontario stream. When disturbed they swam away slowly. David Bradford (1983) studied Rana muscosa who overwinter for 6-9 months in ice-covered lakes and streams at high elevation in California and Nevada. Winterkill he found, was associated with oxygen depletion which occurs most rapidly in shallow lakes. However, tadpoles of this species survive for months in nearly anoxic conditions when shallow lakes are frozen to the bottom, suggesting far greater anaerobic tolerance in the larvae. The latter also appeared to have other advantages; a lower critical oxygen tension and a reduced consumption of energy and oxygen at low PO,. Adults appeared not to lower their standard metabolic rate by more than 5% as a consequence of prolonged exposure to low temperature, or seasonal effects.
610
DAVID G. PENNEY
Table 1. Diving tolerance of selected vertebrates AnimaVnrouo Human Weddell seal Ram pip&m Rnnn pipiens Ran0 pipienc Bullfrog (R. catesbeiana) Ram rnu~c~m Alligatormiss. Chrysemyspicta Ps&emys scrgta Pseudemysscripta Chrysemyspicia
Body temp. (“C)
0, in water
Tolerance time
31 34-37 20 5 5
+ + +i+I_
46min 73 min 8.3 hr 62.1 hr 12&168 hr
4 o-4 21-25
+ + +
40 + days 69 mo. 120fmin 2 days 30 hr 2 wks 155168idays
z;
7 -
14-18 3
-
Ref. Kooyman el al. (1980) Hu~hinson & Dady (1964) Hutchinson & Dady (1964) Christiansen Br Penney (1973) Lillo (1980) Bradford (1983) Andersen (19611 Musacchia (1959) Penney (1974) Robin ef at. (1964) Ultsch & Jackson (t982a)
( + ) = present, ( - ) = not present, ( + / - ) = some present, low and declining. ? = not specified.
Gn the other hand, Jones (1967), in brief diving experiments at 17”C, has shown that oxygen consumption and heart rate of frogs fall in air saturated water. This agrees with the earlier observations of Leivestad (1960) who found a decrease in heat production in the toad of the same order as the fall in oxygen utilization during a dive. Gatz and Piiper (1979) found that metabolism was reduced to 30% of normal during severe hypoxia in the salamander, ~e~~og~~~~ focus. As with diving physiology in general, early studies of anurans (and turtles) dealt heavily with the obvious and easily studied bradycardic response, a component of relatively minor overall importance. Others have observed that locomotor activity increases below the critical PO, in diving frogs, which in 12. mucos~ up to 25 g body wt is about 30 mmHg (Bradford, 1983); while it may be more than double this value in bullfrogs. This may be a behavior which functions to seek areas of higher O2 content as in fish (Petrosky and Magnuson, 1973). Recently, Loveridge and Withers (1981) have found rates of oxygen ~nsumption in the dormant cocooned African bullfrog, P. uhpersus, about l/4 those of the same frogs while resting. Although these frogs were not submerged the physiological state may he similar to diving. Bradford (1983) computed the energetic cost of winter dormancy, making the assumption that stored fat is the primary aerobic energy source. Estimates of body fat content from species closely related to R. mucosa show that sufficient fuel is on board in adults in autumn for at least 8 months in both sexes, and with enough remaining in females for egg production in the spring/summer. In a second paper, Bradford (1984) confirmed that body weight increases with dormancy in adult R. mucosa due to edema, even though fat content and the digestive tract are declining in mass. This produced a fall in extracellular osmolality due to dilution as water was taken up. The same is reported in hypoxic (Hutchinson and Dady, 1964) and anoxic (Christiansen and Penney, 1973) frogs. This is probably because the energy available to transport ions falls with declining environmental temperature or depressed oxidative metabolism, while ion diffusion rate remains relatively constant. Thus increased body fluid volume should act to retard rising lactate concentration, if and when frogs encounter anoxia, and
thus extend survival assuming glycogen is not depieted first. Thus it is clear that frogs can overwinter under ice when dissolved oxygen is available-however, what if it is not? In 1967, Rose and Drotman showed that anaerobic glycolysis is important to submerged R. pipiens. They found glycogen depletion and lactate accumulation during anoxia and noted that treatment with iodoacetate reduced tolerance to nitrogen anoxia. Under anoxic conditions at 3°C the survival time of R. pipiens as judged by persistence of cardiac activity is between 120 and 168 hr (Christiansen and Penney, 1973). In such frogs, cardiac glycogen content falls to 1.9% of that of aerobic controls, while whole body glycogen declines 25%. As glycogen is mobilized, blood and lymph glucose increase, presumably due to hepatic giycogenolysis. Blood lactate rises 33-fold, while lymph lactate rises 56-fold. The latter, coupled with large increases in lymph volume, suggests that lymph acts as a major repository of lactate in the cold anoxic frog. Christiansen and Penney (1973) found that less than 5% of the total lactic acid produced is recovered in the water in which the animal is immersed, indicating that loss of lactate via the skin or kidney is very slow and does not constitute an important mechanism to relieve metabolic acidosis as in some anaerobic parasites, In a related situation, exercise, Quinn and Burggren (1983) found that only 7% of the total lactate produced was eliminated by routes which include gills, skin and urine in bullfrog tadpoles. Laboratory studies thus preclude the possibility of anurans overwintering in complete anoxia, even with metabolic suppression in near-freezing water. Glycogen depletion, lactate ambulation, and acidosis limit survival under such conditions to a few days or a week at most. This conclusion flies in the face of authoritative statements that frogs overwinter in the mud; e.g. “. . dissolved oxygen content. . . must be extremely low since the animal (R. pipiens) is usually located in or below the reducing zone” (Hutchinson and Dady, 1964). Although there is the possiblity that alternate anaerobic end-products such as alanine and succinate are produced, only negligible increases in these substances were reported in a frog and a salamander by Bennett (1978), and no one has described as in anoxic
611
Frog/turtle overwintering Table 2. The diving Common to all *bradycardia & arrhythmias *cutaneous vasodilation *pulmonary blood flow reduction ‘major part of CO, lost through skin Normoxia *some deer. in consumption rate *oxidation of fat *CO, formed; lost through skin *lowered activity ‘edema *dew. hematocrit & blood osmolality (deer. blood viscosity?) *tolerated for many months Hypoxia *incr. 0, capacity through polycythemia ‘deer. P, through Bohr shift *activity increases below critical PO, deer. = decreased,
frog in the cold
Anoxia *deer. metabolic rate ‘glycogenolysis *lactate accumulation *deer. pH *acid buffered by HCO, in blood (also CaCO, in endolymphatic sacs?) ‘deer. blood pC0, (respiratory alkalosis partly compensates metabolic acidosis *water uptake--edema; incr. lymph holding lactate ‘deer., blood volume, hemoconcentration, incr. hematocrit & incr. RBC volume ‘inactivation of hemoglobin (methemoglobin) *tolerated for a few days to one week maximally
incr. = increased.
goldfish, ethanol and CO, release (Shoubridge and Hochachka, 1980). Almost certainly, anaerobic mechanisms are adequate in anurans only for brief periods of anoxic survival, and movement to areas of high oxygen content when available are required for survival. Overwintering must be carried out largely aerobically. In adult bullfrogs made chronically hypoxic, 0, capacity doubles through polycythemia, and P,, decreases by 11 mmHg due to a Bohr effect and not to organic phosphates (Pinder and Burggren, 1983). This contrasts with Bradford’s observation (1984) that hematocrit falls during overwintering in frogs obtaining adequate oxygen, largely due to hemodilution. Others have reported in frogs that the number of erythrocytes, hemoglobin concentration, and blood volume vary seasonally, being lowest during the winter months (in Jones, 1968). Cutaneous vasodilation in anurans first noted nearly two centuries ago, was studied in some detail by Poczopko (1960). It enhances gas exchange, especially oxygen uptake. Buccal pumping of water observed in many species of anurans presumably serves a similar function (Hutchinson and Whitford, 1966). Cutaneous vasodilation occurs in frogs in hypoxic and anoxic water, whereby the belly and legs take on a distinctly pink tinge (Christiansen and Penney, 1973). The opposite response, cutaneous vasoconstriction, is seen in bullfrogs upon emersion into the air (Burggren and Moalli, 1984), a mechanism presumably tied to water conservation. Based on the meager information available, we might summarize diving and overwintering in frogs in the cold in the following way (Table 2). Several responses are common to submersion in anoxic, normoxic, and hypoxic water: bradycardia and cardiac arrhythmias, decrease in pulmonary blood flow, cutaneous vasodilation, rapid cutaneous CO, loss, and some degree of water uptake. Under normoxic conditions some decrease in rate of oxygen consumption and locomotor activity occurs, but complete agreement does not exist as to the degree of the former. If a special physiological state with a lower metabolism than would be expected from the effect of temperature alone does not exist,
anurans may not be said to hibernate. Respiratory acidosis fails to develop due to efficient cutaneous CO, exchange. The principal metabolic fuel is fat; while metabolic suppression due to low temperature and diving combined suffice to match oxygen needs and cutaneous oxygen uptake. There may also be some decrease in blood hematocrit and osmolality due to water uptake and ion loss. In anoxia there is an even larger decrease in metabolic rate, although this condition is not sustainable for long and is almost certainly not the strategy pursued throughout overwintering. Breakdown of glycogen to lactic acid is the principal energy source; pH falls, but metabolic acidosis is countered by respiratory alkalosis, and by buffering from plasma bicarbonate which is of relatively low concentration in anurans, and possibly also by calcium carbonate stored in the endolymphatic sacs (Simkiss, 1968). Lactate is held in high concentration in the lymph. Blood volume decreases and hematocrit increases, probably due to plasma volume loss to the lymph. Hemoglobin is inactivated to methemoglobin (personal observations). Under mildly oxygen depleted conditions, increases in oxygen-carrying capacity occur through polycythemia, and P,, decreases through a Bohr shift; responses paralleling those seen in hypoxic terrestrial vertebrates. Locomotor activity increases below the critical PO,, facilitating the search for water of higher oxygen content. TURTLES
Years ago it was shown that freshwater turtles are much more tolerant of anoxia than are other reptiles (Belkin, 1963). The major reasons suggested to account for this were the ability to produce lactic acid, thus providing sufficient energy yield for essential body functions, and their high body fluid buffering capacity. Although in the present view this remains true, additional mechanisms recently have also been shown to play important roles. The glycogen content of turtle [i.e. Pseudemys (Chrysemys) scripta elegans] heart and liver are exceedingly high among vertebrates, 327 and 735 pmol/g wet wt, respectively (Penney, 1974).
DAVID G. PENNEY
612
When such turtles non-voluntarily dive for 24 hr in deoxygenated water at 2O”C, glycogen is 95% depleted in the heart, 83% in liver. At the same time, tissue lactate increases over 8-fold in heart (to 85~mol~g) and liver. Blood lactate rises 37-fold, to 37.7 mMolar. Blood glucose level increases S-fold; arising presumably from glycogenolysis, with substrate proceeding glycolytically to lactate. Belkin (1963) was also the first to ask whether turtles might not have a more exotic metabolism mechanism able to utilize hydrogen acceptors other than pyruvate. Succinate, an end-product of anaerobic metabolism in certain parasitic worms (Saz and Lescure, 1969) and in some marine molluscs (Simpson and Awapara, 1966) had not been considered. For example, among vertebrates, succinate is reported to accumulate in anoxic rat liver (Hoberman and Prosky, 1967). Liver, but not heart, of anoxic Pseudemys at 20°C accumulates 1.54 @mot/g of succinic acid (Penney, 1974). On a molar ratio basis, therefore, succinate accumulates to the extent of about l/30 that of lactate under such conditions. Interestingly, Hochachka et al. (1975) saw a lo-fold increase in blood succinate in the sea turtle Chelonia my&s, following a two-hour forced dive at 25°C. Enzyme studies suggest that in Pseudemys liver but not in heart, phosphoenolpyruvate carboxykinase (PEPCK) has the capability to sucessfully compete with pyruvate kinase (PK) for a common pool of PEP (Penney and Kornecki, 1973). PEPCK in turtle liver has an activity of 2.0 pmol/min/g wet wt, and K, of 35pmolar, while PK’s activity and K, are 13.4 and 214, respectively. On the other hand, the activities of these two enzymes in heart are 1.6 and 52, respectively, much less favorable for oxaloacetate (OAA) synthesis. Thus the pathway from the branchpoint involving carboxylation of PEP to OAA and subsequent double reduction to succinate operates to some extent in anoxic turtle liver. Although lactate is probably by far the major reduction product resulting from glycogen degradation in the turtle, the formation of succinate may have metabolic advantages. For one, an additional yield of ATP should be obtained as a result of reduction of fumarate to succinate, if turtle liver resembles Ascaris in this regard (Saz and Lescure, 1969). Secondly, succinate accumulation may prove less toxic to cellular metabolism than lactate. Finally, it may engage in an interorgan metabolic cycle (Hochachka and Murphy, 1979). Whether succinate actually is a significant anaerTable 3. Chr~sem~s pictab&ii submerged in 3°C water for up to 120 davs Parameter art. PO, (mmHg) art. PC4 (mmHg) art. pH art. HCO; (mMf art. lactate (mMf art. Ca” (mEq/L) Hct. (%) body wt (%.)
Anoxia
Normoxia/hypoxia (4 mo.)
M1.4 (4 mo.) elevated, Later falls 8.0 -9 7.3 (4 mo.) 40 -+ 7 (4 ma.) --t 140 -+ 85 25-+lS -45.4
art. = arterial, Hct. = hematocrit ratio, w. = weight. Source: Ultsch & Jackson (1982a).
0.5-I .o constant 8.0 -t 7.6 40+ 18 -+ 63 -+ 28 28 -+ 35 +11.9
obic end-product during long dives in turtles is still not clear. Professor G. Ultsch failed to detect succinate in turtles during long-term submersion (personal communication). The fact that succinate and possibly other metabolites may accumulate during anoxia with possibly higher energetic yields than lactate, gives pause to estimates of anaerobic metabolism based on lactate alone, as have recently been made by Herbert and Jackson (1985). Loss of lactate by excretion (Jackson and Silverblatt, 1974) or diffusion, or inhomogeneity in the body due to organ compartmentalization (Jackson and Heisler, 1984) produces similar problems for that approach when only plasma lactate is considered. In the 1960’s Donald Jackson (1968) convincingly showed that turtles drastically reduce their rate of metabolism during diving at 24”C, as evidenced by reduced heat production. He found an 80% decline in dives lasting 2-4 hr and >95% in dives lasting up to 7 hr. Since lung oxygen stores were exhausted in the first hour and the surrounding water was devoid of oxygen, later energy release was entirely by anaerobic means. Notwithstanding the reservations voiced above, plasma lactate accumulation rates during anoxic submergence now suggest metabolic depression to 0.6% of the aerobic rate at 20°C (Herbert and Jackson, 1985). In particular, a very high Q,, from IO down to 3°C (ca 13.3) is responsible for a large fall in metabolic rate as the lower temperature is approached (Herbert and Jackson, 1985). Until a few years ago it was widely believed that turtles other than kinosteroids could not make use of dissolved oxygen in the water due to the inability for si~ifi~nt cutaneous uptake due to their thick leathery skin, although it was realized that the critical PO, of turtles was low (e.g. 12-30 mmHg). Ultsch and Jackson (1982a) showed that arterial PO, was significantly elevated, albeit still very low by most standards, in Chrysemys submerged for 4-6 months at 3°C in air equilibrated water as opposed to those in water devoid of oxygen (Table 3). Furthermore, arterial PCOZ rose less, pH fell less, plasma bicarbonate fell less, and plasma lactate rose far less in those submerged in water containing oxygen. For example, plasma lactate in some turtles in the deoxygenated water rose to 200 mM, while the maximum in those in aerated water was less than 100 mM. Thus the turtles were able to extract dissolved oxygen from the water and support a significant portion of their vastly reduced metabolic requirements by aerobic metabolism. It is unknown what portion of extrapulmonary gas exchange in this state occurred through the skin and what portion via buccal membranes. Their data indicate that although overwinte~ng turtles can remain alive for up to 6 months at near freezing temperatures, even while totally anoxic and severely acidotic, the acid-base status and probably the recovery potential are improved if dissolved oxygen is available for extrapulmonary uptake. In fact, Herbert and Jackson (1985) found that at 3°C anaerobic metabolic rate is lower than the rate that can be supported by cutaneous respiration in aerated water, although metabolic rate may not remain constant in the presence of dissolved oxygen. Even at a PO* of 1 mmHg, turtle hemoglobin at 3°C is 7-8% saturated with oxygen. Maginniss et al.
Frog/turtle overwintering
(1983) report that the P,, in uivo under those conditions in Chrysemysis 4.5 mmHg, because turtle hemoglobin has a high temperature sensitivity. Cold diving also has a profound influence on oxygen dissociation curve shape: The curve exhibits high 0, affinity below the P,. Reductions in erythrocyte organic phosphate concentrations resulting from diving may be largely responsible. Although 7-8% hemoglobin saturation is low, it represents an O2 concentration 40-fold that of dissolved oxygen at a PO2 of 1 mmHg. Combined with temperature and anoxic suppression of metabolism, the oxygen delivery which might be achieved at this level of hemoglobin saturation is adequate for significant oxidative metabolism. The studies of Jackson and Ultsch (1982) also show that turtles are tolerant of profound plasma ion shifts and large increases in osmolality. For example, during prolonged submersion Cl- declines modestly, while Ca*+ increases over 30-fold to as much as 120 m,/L. Mg*+ increases over lo-fold, and K+ increases to 9 mM, a level toxic to the human heart. Plasma Na+ concentration remains stable. Loss of K+ and the presumed gain of Na+ by muscle and other tissues may reflect a partial failure of the Nat/K+ exchange pump. Nevertheless the ion changes serve to balance increasing lactate, but as a result plasma osmolality increases 65%, even though body weight increases up to 12% through water uptake, and even more if loss of weight to oxidation of body fuels is taken into account. The Ca2+ and Mg*+ are presumably mobilized from skeletal elements and the shell, not due to release from muscle cells (Jackson and Heisler, 1983); however, the Ca*+ :Mg*+ ratio is about SO:1 in shell and skeleton while these ions enter the plasma in a ratio of 3:1, suggesting preferential release of Mg*+. In a succeeding experiment by Ultsch and Jackson (1982b), turtles were first submerged in deoxygenated water for 3 wks, then changed to aerated water for 3 wks in order to first simulate anoxic diving or burying in mud, and were later changed to conditions of available dissolved oxygen. In the presence of oxygen, arterial PO2 and pH rose, and lactate stabilized but did not fall. Thus, while correction to pre-dive conditions did not occur, sufficient oxygen was taken up for a maintenance level of oxidative metabolism. The fact that large amounts of CO, generated from oxidative metabolism and through bicarbonate buffering of lactic acid indicates that turtles lose significant CO, through the skin. Thus the cold diving turtle, like predominantly skin-breathing salamanders at higher temperatures can to some degree control their blood pH using the largely passive extrapulmonary gas exchange mode. Recently, Ultsch, Jackson and their collaborators (Ultsch et al., 1984; Jackson et al., 1984) examined the anoxic diving tolerance of 4 turtle species: Chrysemys picta, Chelydra serpentina, Sternotherus odor atus and Trionyx spiniferus. Sternotherus and Trionyx
showed the fastest rates of lactate accumulation, the poorest anoxic survival, and presumably had the highest rates of extra-pulmonary CO2 loss. The latter is consistent with the known ease of oxygen uptake by these species in aerated water. Thus despite their
613 Table 4. The diving turtle in the cold
*Tolerance of anaerobiosis of days to months (incr. with deer. temp.) *Deer. pulmonary blood flow; initially peripheral vasoconstriction? ‘Bradycardia (variable) & cardiac arrhythmias *Greatly deer. metabolic rate & activity lGlycogenolysis & glycolysis *Tolerance of high plasma lactate cont. (i.e. 2OOmM) & low pH (severe metabolic acidosis) ltJnusttally high normal extracellular bicarbonate cont. *Mobilization of Ca*+ & Mg from skeleton & shell for buffering *Reduction in Cl- and bicarbonate cones.; Na+ cow. stable ‘Extrapulmonary loss of CO, to water *Tolerance of profound ion shifts & large incr. in plasma osmolality *Oxygen uptake from water, when possible ‘Very low hemoglobin P, (44.5 mmHg) at 3°C *Incomplete torpor *Collapsed lung in anoxia ‘Extravascular lactate storage (urine, pericard. fluid); excretion? *Unorthodox anaerobic end-products (succinate)? *Depressed tendency for blood clotting? cont. = concentration.
higher’ rates of CO, generation, they experienced less hypercapnic acidosis. Because pH depression is known to inhibit glycolysis, relative lack of CO2 retention in these turtles may be the very reason they accumulated lactate more rapidly and showed less tolerance. In the same way, the lower anoxia tolerance of anurans probably derives largely from their efficient cutaneous gas exchange. Regardless of the perceived advantages of dissolved oxygen for overwintering derived from laboratory studies, freshwater turtles are frequently found buried in mud (with or without overlying water) without apparent access to oxygen. Thus, in some species (e.g. Chrysemys, Chelydra) extrapulmonary O2 uptake may not be essential for overwintering, while in others it probably is (e.g. Sternotherus, Trionyx). However, in high latitudes with very long winters even the more anoxia resistant turtles probably depend upon some oxidative metabolism for part or all of the winter in order to survive. Turtles frequently observed swimming under the ice in winter may be seeking water of higher O2 content, whereas if this occurs in late winter they may be moving to shallower water in order to gauge the proper time of emergence. It has even been suggested that the northern limits of many turtles may not be determined by the rigor of overwintering, but rather by the period available for reproduction. Thus the turtle in the cold has a number of characteristics which favor its great submersion tolerance of days to months (Table 4). Peripheral vasoconstriction, decreased pulmonary blood flow (White and Ross, 1966) and bradycardia (Belkin, 1964) all act to conserve on-board oxygen for the heart and brain, at least initially during the dive as in diving mammals. Depressed metabolic rate which intensifies with low ambient temperature, prolonged submersion, and anoxia is vitally essential and is the centerpiece of the response. Glycogenolysis occurs, with glucose release and lactate production, and small amounts of succinate. Torpor, however, is incomplete, as turtles are known to move about during overwintering. Turtles show remarkable tolerance of both high lactate concentrations, low pH, profound ion shifts and large increases in plasma osmolality. However,
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DAVID G. PENNEY
the slower the rate of body lactate rise, the greater the period of submersion tolerated. Lactate and other anaerobic end-products are possibly to some extent stored in the urine and pericardial fluid (Jackson and Heisler, 1984) or excreted. Jackson and Silverblatt (1974), for example, saw significant lactate in the urinary bladder of turtles submerged at 24°C. In addition, water uptake during long continued submersion may serve to further dilute lactate, and other metabolic end-products. Turtles normally have unusually high extracellular bicarbonate concentrations, as compared to amphibians and fish. This, and skeletal element Ca*+ and Mg*+, provide the major buffering capacity for metabolic acidosis, while other ions are also adjusted up or down in concentration. CO, generated by oxidative metabolism and through acid buffering is lost through extrapulmonary routes. When available and where possible, oxygen is taken up from the water by the same extra-pulmonary routes. Since turtle hemoglobin has this a very low P,, at near freezing temperatures, assists tissue oxygen delivery. The lungs of anoxic or nearly anoxic turtles, are collapsed. Unlike diving mammals, in turtles on long dives or overwintering lactate is not confined to the peripheral organs ready to flood out into circulation following emergence, with oxygen reserved for the heart and brain. Instead, complete or nearly complete anoxia exists throughout the body and all organs are using anaerobic metabolism, even the heart and brain. It now appears that no exotic mechanisms need be invoked to permit such incredible diving feats, just optimal use of those present in other vertebrates. Finally, overwintering in turtles should not be viewed as simply prolonged diving-with the profound suppression of metabolism it must be viewed as ectotherm hibernation! In turn, their incredible diving capabilities are merely a spin-off of a successful overwintering strategy. CONCLUSIONS
We might contrast the major features of the overwintering/diving strategies of frogs and turtles in the following way (Table 5): (1) Frogs use cutaneous obligatory for submersion duration. Fat is probably Table
5. Maior
features
0, uptake and in fact it is of more than a few days the major metabolic fuel.
of the overwintering/diving frogs and turtles
strategies
of
Frogs
*Use cutaneous 0, uptake, turtles to a much smaller extent; varies with species ‘More readily lose CO, through skin. Thus, respiratory alkalosis can partially compensate metabolic acidosis in frogs, not in turtles ‘Extreme cutaneous permeability may limit ability to lower metabolism like turtles because of need to maintain ionic & osmotic balance; also, lost CO, & smaller pH fall inhibits metabolism (i.e. glycolysis) less TUdt-S ‘For a given temperature under anaerobiosis, metabolism is lowered more than in frogs (>95% vs 3&70%, respectively) *Heart and liver contain more glycogen than in frog *Tolerate much larger increases in lactate than frogs (> 200 mM vs 35 mM. respectively) *Have greater ootential buffering _.capacity: plasma HCO, , shell C&6, & M&O,
Turtles to a much smaller extent respire through the skin, but this varies with species and ambient temperature. (2) Frogs more readily lose CO2 through the skin. Thus respiratory alkalosis can partially compensate metabolic acidosis in frogs. Although this also occurs in turtles, the process is much slower and is of little value in rapid lactate acidosis at higher temperature. (3) The extreme cutaneous permeability of frogs may limit their ability to lower metabolism like turtles because of the need to maintain ionic and osmotic balance. Also, the lost CO, and smaller pH fall may serve to inhibit glycolysis less, allowing lactate to rise rapidly and consequently decrease anoxia tolerance. (4) In the turtle, for a given temperature under anaerobiosis, metabolism is lowered much more than in the frog (> 95% vs 3&70%, respectively). (5) The turtle heart and liver contain more glycogen than in the frog. Under strictly anoxic conditions this is the primary fuel, whereas with oxygen availability fat is also presumably metabolized. (6) Turtles tolerate much larger increases in lactate than do frogs (> 200 vs 35 mM, respectively). (7) And finally, turtles have far greater potential buffering capacity, including plasma bicarbonate and shell calcium carbonate and magnesium carbonate. Note: This review developed from the author’s presentation at a symposium, Physiology and Ecology of Ectotherm Hibernation, organized by G. R. Ultsch, at the American Society of Ichthyologists and Herpetologists meeting, 19 June 1986, at the University of Victoria, Victoria, BC, Canada.
REFERENCES
Andersen H. T. (1961) Physiological adjustments to prolonged diving in the American alligator, Alligator mississippiensis. Acta Physiol. Stand. 53, 2345. Belkin D. A. (1963) Anoxia: Tolerance in reptiles. Science 139, 492493. Belkin D. A. (1964) Variations in heart rate during voluntary diving in the turtle Pseudemys concinna. Copeia, 2. Bennett A. F. (1978) Activity metabolism of the lower vertebrates. Ann. Rec. Physiol. 40, 447469. Bradford D. F. (1983) Winterkill, oxygen relations, and energy metabolism of a submerged dormant amphibian. Rana muscosa. Ecology 64, 1171-l 183. Bradford D. F. (1984) Water and osmotic balance m overwintering tadpoles and frogs. Rana muscosa. Physiol. Zool. 57, 474480. Burggren W. and Moalli R. (1984) ‘Active’ regulation of cutaneous gas exchange by capillary recruitment in amphibians: experimental evidence and a revised model for skin respiration. Respir. Physiol. 55, 379-392. Christiansen J. and Penney D. (1973) Anaerobic glycolysis and lactic acid accumulation in cold submerged Rana pipiens. J. camp. Physiol. 87, 237-245. Cunjak R. A. (1986) Winter habitat of northern leopard frogs, Rana pipiens, in a southern Ontario stream. Can. J. Zool. 64, 255-257. Emery A. R., Berst A. H. and Kodaira K. (1972) Underice observations of wintering sites of leopard frogs. Copein, 1. Gatz R. N. and Piiper J. (1979) Anaerobic energy metabolism during severe hypoxia in the lungless salamander Desmognathus fuscus (Plethodontidae). Respir. Physiol. 38, 377-384.
Frog/turtle Herbert C. V. and Jackson D. C. (1985) Temperature effects on the responses to prolonged submergence in the turtle Chrysemys picta bellii. Metabolic rate, blood acid-base and ionic changes, and cardiovascular function in aerated and anoxic water. Physiol. Zool. 58, 670-681. Hochachka P. W. and Murphy B. (1979) Metabolic Status during diving and recovery in marine mammals. In Environmenral Physiology III, 20, 2533287. (Edited by Robertshaw D.), Univ. Park Press, Baltimore. Hochachka P. W., Owen T. G., Allen J. F. and Whittow G. C. (1975) Multiple end products of anaerobiosis in diving vertebrates. Comp. Biochem. Physiol. SOB, 17-22. Hoberman J. D. and Prosky L. (1967) The evidence of reduction of fumarate to succinate in perfused rat liver under conditions of reduced 0, tension. Biochim. biophys. Acta 148, 392-399. Hutchinson V. H. and Dady M. J. (1964) The viability of Rana pipiens and Bufo terrestris submerged at different temperatures. Herpt. 20, 1499162. Hutchinson V. H. and Whitford W. G. (1966) Survival and underwater buccal movements in submerged anurans. Herpt. 22, 122-127. Jackson D. C. (1968) Metabolic depression and oxygen depletion in the diving turtle. J. appl. Physiol. 24, 5033509. Jackson D. C. and Heisler N. (1984) The contribution of the alkaline pericardial fluid of freshwater turtles to acid buffering during prolonged anoxia. J. exp. Biol. 109, 55562. Jackson D. C., Herbert C. V. and Ultsch G. R. (1984) The comparative physiology of diving in North American freshwater turtles. II. Plasma ion balance during prolonged anoxia. Physiol. Zool. 57, 632-O. Jackson D. C. and Silverblatt H. (1974) Respiration and acid-base status of turtles following experimental dives. Am. .I. Physiol. 226, 9033909. Jackson D. C. and Ultsch G. R. (1982) Long-term submergence at 3°C of the turtle, Chrysemys picta belli, in normoxic and severely hypoxic water. II. Extracellular ionic responses to extreme lactic acidosis. J. exp. Biol. 96, 2943. Jones D. R. (1967) Oxygen consumption and heart rate of several species of anuran amphibia during submergence. Comp. Biochem. Physiol. 20, 691-707. Jones D. R. (1968) Specific and seasonal variations in development of diving bradycardia in anuran amphibia. Camp. Biochem. Physiol. 25. 821-834. Kooyman G. L. (1981) Physiology of diving in marine mammals. Ann. Rev. Physiol. 43, 343-356. Leivestad H. (1960) The effect of prolonged submersion on the metabolism and the heart rate in the toad Bufo bufo. Arbok. Univ. Bergen 5, I-15. Lillo R. S. (1980) Heart rate and blood pressure in bullfrogs during prolonged maintenance in water at low temperature. Comp. Biochem. Physiol. 65A, 251-253. Loveridge J. P. and Withers P. C. (1981) Metabolism and water-balance of active and cacooned african bullfrogs Pyxicephalus adsversus. Phvsiol. Zool. 54. 2033214. Maginniss L. A., Tapper S. S-and Miller L. S. (1983) Effect of chronic cold and submergence on blood oxygen transport in the turtle. Chrysemys picta. Respir. Physiol. 53, 15529.
overwintering
615
Musacchia X. J. (1959) The viability of Chrysemys picta submerged at various temperatures. Physiol. Zoo/. 32, 47750. Penney D. G. (1974) Effects of prolonged diving anoxia on the turtle, Pseudemys scripta elegans. Camp. Biochem. Physiol. 47A, 933394 1. Penney D. G. and Kornecki E. H. (1973) Activities, intracellular localization and kinetic properties of phosphenolpyruvate carboxykinase, pyruvate kinase and malate dehydrogenase in turtle (Pseudemys scripfa elegans) liver, heart and skeletal muscle. Camp. Biochem. Physiol. 46B, 405415. Pinder A. and Burggren W. (1983) Respiration during chronic hypoxia and hyperoxia in larval and adult bullfrogs (Rana catesbeiana) II. Changes in respiratory properties of whole blood. J. exp. Biol. 105, 2055213. Poczopko P. (1959) The changes of the circulation of blood in Rana esculenta L. while diving. Zool. Pol. 10, 2943. Petrosky B. R. and Magnuson J. J. (1973) Behavioral responses of northern pike, yellow perch and bluegill to oxygen concentrations under simulated winterkill conditions. Copeia, 124133. Quinn D. and Burggren W. (1983) Lactate production, tissue distribution, and elimination following exhaustive exercise in larval and adult bullfrogs Rana catesbeiana. Physiol. Zool. 56, 5977613. Robin E. D., Vester J. W., Murdaugh H. V. and Millen J. E. (1964) Prolonged anaerobiosis in a vertebrate: Anaerobic metabolism in the freshwater turtle. J. cell. camp. Physiol. 63, 287~-297. Rose F. L. and Drotman R. B. (1967) Anaerobiosis in a frog, Rana pipiens. J. exp. Zool. 166, 427432. Saz H. J. and Lescure 0. L. (1969) The functions of phosphoenolpyruvate carboxykinase and malic enzyme in the anaerobic function of succinate by Ascaris lumbricoides. Comp. Biochem. Physiol. 30, 49-60. Shoubridge E. A. and Hochachka P. W. (1980) Ethanol: Novel end product of vertebrate anaerobic metabolism. Science 209, 308-309. Simkiss K. (1968) Calcium and carbonate metabolism in the frog (Rana temporaria) during respiratory acidosis, Am. J. Physiol. 214, 6277634. Simpson J. W. and Awapara J. (1966) The pathway of glucose degradation in some invertebrates. Comp. Biothem. Physiol. 18, 537-548. Ultsch G. R., Herbert C. V. and Jackson D. C. (1984) The comparative physiology of diving in North American freshwater turtles. I. Submergerice tolerance, gas exchange, and acid-base balance. Physiol. Zool. 51, 62CM3 1. Ultsch G. R. and Jackson D. C. (1982a) Long-term submergence at 3°C of the turtle, Chrysemys picta belli, in normoxic and severely hypoxic water. I. Survival, gas exchange and acid-base status. J. exp. Biol. 96, 11-28. Ultsch G. R. and Jackson D. C. (1982b) Long-term submergence at 3°C of the turtle, Chrysemys picta belli, in normoxic and severely hypoxic water. III. Effects of changes in ambient PO, and subsequent air breathing. J. exp. Biol. 97, 87-99. White F. N. and Ross G. (1966) Circulatory changes during experimental diving in the turtle. Am. J. Physiol. 211, 15-18.
Vol. 86A.
No. 4, pp. 609-615,
1987 0
MINI
0300-9629/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
REVIEW
FROGS AND TURTLES: DIFFERENT ECTOTHERM OVERWINTERING STRATEGIES DAVID G. PENNEY
Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA (Receiued 18 July 1986) Abstract-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 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 of oxygen
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.
INTRODUCTION
sence of oxygen. Let us now consider some of the specific ways in which frogs and turtles differ in their overwintering strategies. Because most of the work in this area has been carried out on Ranids among the anurans, and Chrysemys (Pseudemys) among the turtles, this review is primarily relevant to them, but should also have general applicability to all ectotherms.
At temperate to near arctic latitudes freshwater frogs and turtles, where present, disappear from the landscape during winter. Where do they go and how do they remain alive? While these questions have been posed before, little published natural history is available for this period, although unpublished anecdotes abound. In the absence of adequate field studies, some insight into the probable strategies used by these ectotherms may be gained from laboratory submersion or “diving” studies. The problem of cold and/or freezing tolerance is not considered in this brief review. Five general mechanisms are available to animals to allow survival while diving:
FROGS
(1) by making use of stored oxygen brought from the surface, (2) by absorbing oxygen from the water, (3) by a metabolic shutdown to a lower basal rate, (4) by developing an oxygen debt in the form of lactic acid, which must be repaid when oxygen is again available, and (5) by shifting to an anaerobic metabolic pathway which does not involve an oxygen debt. Each of the above will enter into the subsequent discussion. If diving occurs in water devoid of oxygen the second alternative is of course eliminated. Several important generalizations emerge when we compare the diving tolerance of selected vertebrates (Table 1): First, tolerance increases from homeotherms to ectotherms, even recognizing the amazing adaptive mechanisms of seals and related species. Secondly, it increases enormously as environmental temperature is lowered in ectotherms due to the energy sparing effect of lowered metabolic rate. Finally, turtles show clear superiority over all other air breathing vertebrates including anurans as long duration divers when submersion occurs in the ab609
Early reports placed overwintering frogs under rocks or in the mud at the bottom. Stories also abound of netting frogs from the mud of frozen ponds, lakes, etc. Such areas would almost certainly be in a reduced state, i.e. lacking in oxygen. In 1972, Emery et al. reported seeing “hibernating” frogs resting in small pits at the surface of the mud at the bottom of an ice-covered pond in Ontario. Water temperature was 2S”C and dissolved oxygen was 7 ppm. More recently, Cunjak (1986), found frogs during winter under rocks and among rubble in a well-oxygenated southern Ontario stream. When disturbed they swam away slowly. David Bradford (1983) studied Rana muscosa who overwinter for 6-9 months in ice-covered lakes and streams at high elevation in California and Nevada. Winterkill he found, was associated with oxygen depletion which occurs most rapidly in shallow lakes. However, tadpoles of this species survive for months in nearly anoxic conditions when shallow lakes are frozen to the bottom, suggesting far greater anaerobic tolerance in the larvae. The latter also appeared to have other advantages; a lower critical oxygen tension and a reduced consumption of energy and oxygen at low PO,. Adults appeared not to lower their standard metabolic rate by more than 5% as a consequence of prolonged exposure to low temperature, or seasonal effects.
610
DAVID G. PENNEY
Table 1. Diving tolerance of selected vertebrates AnimaVnrouo Human Weddell seal Ram pip&m Rnnn pipiens Ran0 pipienc Bullfrog (R. catesbeiana) Ram rnu~c~m Alligatormiss. Chrysemyspicta Ps&emys scrgta Pseudemysscripta Chrysemyspicia
Body temp. (“C)
0, in water
Tolerance time
31 34-37 20 5 5
+ + +i+I_
46min 73 min 8.3 hr 62.1 hr 12&168 hr
4 o-4 21-25
+ + +
40 + days 69 mo. 120fmin 2 days 30 hr 2 wks 155168idays
z;
7 -
14-18 3
-
Ref. Kooyman el al. (1980) Hu~hinson & Dady (1964) Hutchinson & Dady (1964) Christiansen Br Penney (1973) Lillo (1980) Bradford (1983) Andersen (19611 Musacchia (1959) Penney (1974) Robin ef at. (1964) Ultsch & Jackson (t982a)
( + ) = present, ( - ) = not present, ( + / - ) = some present, low and declining. ? = not specified.
Gn the other hand, Jones (1967), in brief diving experiments at 17”C, has shown that oxygen consumption and heart rate of frogs fall in air saturated water. This agrees with the earlier observations of Leivestad (1960) who found a decrease in heat production in the toad of the same order as the fall in oxygen utilization during a dive. Gatz and Piiper (1979) found that metabolism was reduced to 30% of normal during severe hypoxia in the salamander, ~e~~og~~~~ focus. As with diving physiology in general, early studies of anurans (and turtles) dealt heavily with the obvious and easily studied bradycardic response, a component of relatively minor overall importance. Others have observed that locomotor activity increases below the critical PO, in diving frogs, which in 12. mucos~ up to 25 g body wt is about 30 mmHg (Bradford, 1983); while it may be more than double this value in bullfrogs. This may be a behavior which functions to seek areas of higher O2 content as in fish (Petrosky and Magnuson, 1973). Recently, Loveridge and Withers (1981) have found rates of oxygen ~nsumption in the dormant cocooned African bullfrog, P. uhpersus, about l/4 those of the same frogs while resting. Although these frogs were not submerged the physiological state may he similar to diving. Bradford (1983) computed the energetic cost of winter dormancy, making the assumption that stored fat is the primary aerobic energy source. Estimates of body fat content from species closely related to R. mucosa show that sufficient fuel is on board in adults in autumn for at least 8 months in both sexes, and with enough remaining in females for egg production in the spring/summer. In a second paper, Bradford (1984) confirmed that body weight increases with dormancy in adult R. mucosa due to edema, even though fat content and the digestive tract are declining in mass. This produced a fall in extracellular osmolality due to dilution as water was taken up. The same is reported in hypoxic (Hutchinson and Dady, 1964) and anoxic (Christiansen and Penney, 1973) frogs. This is probably because the energy available to transport ions falls with declining environmental temperature or depressed oxidative metabolism, while ion diffusion rate remains relatively constant. Thus increased body fluid volume should act to retard rising lactate concentration, if and when frogs encounter anoxia, and
thus extend survival assuming glycogen is not depieted first. Thus it is clear that frogs can overwinter under ice when dissolved oxygen is available-however, what if it is not? In 1967, Rose and Drotman showed that anaerobic glycolysis is important to submerged R. pipiens. They found glycogen depletion and lactate accumulation during anoxia and noted that treatment with iodoacetate reduced tolerance to nitrogen anoxia. Under anoxic conditions at 3°C the survival time of R. pipiens as judged by persistence of cardiac activity is between 120 and 168 hr (Christiansen and Penney, 1973). In such frogs, cardiac glycogen content falls to 1.9% of that of aerobic controls, while whole body glycogen declines 25%. As glycogen is mobilized, blood and lymph glucose increase, presumably due to hepatic giycogenolysis. Blood lactate rises 33-fold, while lymph lactate rises 56-fold. The latter, coupled with large increases in lymph volume, suggests that lymph acts as a major repository of lactate in the cold anoxic frog. Christiansen and Penney (1973) found that less than 5% of the total lactic acid produced is recovered in the water in which the animal is immersed, indicating that loss of lactate via the skin or kidney is very slow and does not constitute an important mechanism to relieve metabolic acidosis as in some anaerobic parasites, In a related situation, exercise, Quinn and Burggren (1983) found that only 7% of the total lactate produced was eliminated by routes which include gills, skin and urine in bullfrog tadpoles. Laboratory studies thus preclude the possibility of anurans overwintering in complete anoxia, even with metabolic suppression in near-freezing water. Glycogen depletion, lactate ambulation, and acidosis limit survival under such conditions to a few days or a week at most. This conclusion flies in the face of authoritative statements that frogs overwinter in the mud; e.g. “. . dissolved oxygen content. . . must be extremely low since the animal (R. pipiens) is usually located in or below the reducing zone” (Hutchinson and Dady, 1964). Although there is the possiblity that alternate anaerobic end-products such as alanine and succinate are produced, only negligible increases in these substances were reported in a frog and a salamander by Bennett (1978), and no one has described as in anoxic
611
Frog/turtle overwintering Table 2. The diving Common to all *bradycardia & arrhythmias *cutaneous vasodilation *pulmonary blood flow reduction ‘major part of CO, lost through skin Normoxia *some deer. in consumption rate *oxidation of fat *CO, formed; lost through skin *lowered activity ‘edema *dew. hematocrit & blood osmolality (deer. blood viscosity?) *tolerated for many months Hypoxia *incr. 0, capacity through polycythemia ‘deer. P, through Bohr shift *activity increases below critical PO, deer. = decreased,
frog in the cold
Anoxia *deer. metabolic rate ‘glycogenolysis *lactate accumulation *deer. pH *acid buffered by HCO, in blood (also CaCO, in endolymphatic sacs?) ‘deer. blood pC0, (respiratory alkalosis partly compensates metabolic acidosis *water uptake--edema; incr. lymph holding lactate ‘deer., blood volume, hemoconcentration, incr. hematocrit & incr. RBC volume ‘inactivation of hemoglobin (methemoglobin) *tolerated for a few days to one week maximally
incr. = increased.
goldfish, ethanol and CO, release (Shoubridge and Hochachka, 1980). Almost certainly, anaerobic mechanisms are adequate in anurans only for brief periods of anoxic survival, and movement to areas of high oxygen content when available are required for survival. Overwintering must be carried out largely aerobically. In adult bullfrogs made chronically hypoxic, 0, capacity doubles through polycythemia, and P,, decreases by 11 mmHg due to a Bohr effect and not to organic phosphates (Pinder and Burggren, 1983). This contrasts with Bradford’s observation (1984) that hematocrit falls during overwintering in frogs obtaining adequate oxygen, largely due to hemodilution. Others have reported in frogs that the number of erythrocytes, hemoglobin concentration, and blood volume vary seasonally, being lowest during the winter months (in Jones, 1968). Cutaneous vasodilation in anurans first noted nearly two centuries ago, was studied in some detail by Poczopko (1960). It enhances gas exchange, especially oxygen uptake. Buccal pumping of water observed in many species of anurans presumably serves a similar function (Hutchinson and Whitford, 1966). Cutaneous vasodilation occurs in frogs in hypoxic and anoxic water, whereby the belly and legs take on a distinctly pink tinge (Christiansen and Penney, 1973). The opposite response, cutaneous vasoconstriction, is seen in bullfrogs upon emersion into the air (Burggren and Moalli, 1984), a mechanism presumably tied to water conservation. Based on the meager information available, we might summarize diving and overwintering in frogs in the cold in the following way (Table 2). Several responses are common to submersion in anoxic, normoxic, and hypoxic water: bradycardia and cardiac arrhythmias, decrease in pulmonary blood flow, cutaneous vasodilation, rapid cutaneous CO, loss, and some degree of water uptake. Under normoxic conditions some decrease in rate of oxygen consumption and locomotor activity occurs, but complete agreement does not exist as to the degree of the former. If a special physiological state with a lower metabolism than would be expected from the effect of temperature alone does not exist,
anurans may not be said to hibernate. Respiratory acidosis fails to develop due to efficient cutaneous CO, exchange. The principal metabolic fuel is fat; while metabolic suppression due to low temperature and diving combined suffice to match oxygen needs and cutaneous oxygen uptake. There may also be some decrease in blood hematocrit and osmolality due to water uptake and ion loss. In anoxia there is an even larger decrease in metabolic rate, although this condition is not sustainable for long and is almost certainly not the strategy pursued throughout overwintering. Breakdown of glycogen to lactic acid is the principal energy source; pH falls, but metabolic acidosis is countered by respiratory alkalosis, and by buffering from plasma bicarbonate which is of relatively low concentration in anurans, and possibly also by calcium carbonate stored in the endolymphatic sacs (Simkiss, 1968). Lactate is held in high concentration in the lymph. Blood volume decreases and hematocrit increases, probably due to plasma volume loss to the lymph. Hemoglobin is inactivated to methemoglobin (personal observations). Under mildly oxygen depleted conditions, increases in oxygen-carrying capacity occur through polycythemia, and P,, decreases through a Bohr shift; responses paralleling those seen in hypoxic terrestrial vertebrates. Locomotor activity increases below the critical PO,, facilitating the search for water of higher oxygen content. TURTLES
Years ago it was shown that freshwater turtles are much more tolerant of anoxia than are other reptiles (Belkin, 1963). The major reasons suggested to account for this were the ability to produce lactic acid, thus providing sufficient energy yield for essential body functions, and their high body fluid buffering capacity. Although in the present view this remains true, additional mechanisms recently have also been shown to play important roles. The glycogen content of turtle [i.e. Pseudemys (Chrysemys) scripta elegans] heart and liver are exceedingly high among vertebrates, 327 and 735 pmol/g wet wt, respectively (Penney, 1974).
DAVID G. PENNEY
612
When such turtles non-voluntarily dive for 24 hr in deoxygenated water at 2O”C, glycogen is 95% depleted in the heart, 83% in liver. At the same time, tissue lactate increases over 8-fold in heart (to 85~mol~g) and liver. Blood lactate rises 37-fold, to 37.7 mMolar. Blood glucose level increases S-fold; arising presumably from glycogenolysis, with substrate proceeding glycolytically to lactate. Belkin (1963) was also the first to ask whether turtles might not have a more exotic metabolism mechanism able to utilize hydrogen acceptors other than pyruvate. Succinate, an end-product of anaerobic metabolism in certain parasitic worms (Saz and Lescure, 1969) and in some marine molluscs (Simpson and Awapara, 1966) had not been considered. For example, among vertebrates, succinate is reported to accumulate in anoxic rat liver (Hoberman and Prosky, 1967). Liver, but not heart, of anoxic Pseudemys at 20°C accumulates 1.54 @mot/g of succinic acid (Penney, 1974). On a molar ratio basis, therefore, succinate accumulates to the extent of about l/30 that of lactate under such conditions. Interestingly, Hochachka et al. (1975) saw a lo-fold increase in blood succinate in the sea turtle Chelonia my&s, following a two-hour forced dive at 25°C. Enzyme studies suggest that in Pseudemys liver but not in heart, phosphoenolpyruvate carboxykinase (PEPCK) has the capability to sucessfully compete with pyruvate kinase (PK) for a common pool of PEP (Penney and Kornecki, 1973). PEPCK in turtle liver has an activity of 2.0 pmol/min/g wet wt, and K, of 35pmolar, while PK’s activity and K, are 13.4 and 214, respectively. On the other hand, the activities of these two enzymes in heart are 1.6 and 52, respectively, much less favorable for oxaloacetate (OAA) synthesis. Thus the pathway from the branchpoint involving carboxylation of PEP to OAA and subsequent double reduction to succinate operates to some extent in anoxic turtle liver. Although lactate is probably by far the major reduction product resulting from glycogen degradation in the turtle, the formation of succinate may have metabolic advantages. For one, an additional yield of ATP should be obtained as a result of reduction of fumarate to succinate, if turtle liver resembles Ascaris in this regard (Saz and Lescure, 1969). Secondly, succinate accumulation may prove less toxic to cellular metabolism than lactate. Finally, it may engage in an interorgan metabolic cycle (Hochachka and Murphy, 1979). Whether succinate actually is a significant anaerTable 3. Chr~sem~s pictab&ii submerged in 3°C water for up to 120 davs Parameter art. PO, (mmHg) art. PC4 (mmHg) art. pH art. HCO; (mMf art. lactate (mMf art. Ca” (mEq/L) Hct. (%) body wt (%.)
Anoxia
Normoxia/hypoxia (4 mo.)
M1.4 (4 mo.) elevated, Later falls 8.0 -9 7.3 (4 mo.) 40 -+ 7 (4 ma.) --t 140 -+ 85 25-+lS -45.4
art. = arterial, Hct. = hematocrit ratio, w. = weight. Source: Ultsch & Jackson (1982a).
0.5-I .o constant 8.0 -t 7.6 40+ 18 -+ 63 -+ 28 28 -+ 35 +11.9
obic end-product during long dives in turtles is still not clear. Professor G. Ultsch failed to detect succinate in turtles during long-term submersion (personal communication). The fact that succinate and possibly other metabolites may accumulate during anoxia with possibly higher energetic yields than lactate, gives pause to estimates of anaerobic metabolism based on lactate alone, as have recently been made by Herbert and Jackson (1985). Loss of lactate by excretion (Jackson and Silverblatt, 1974) or diffusion, or inhomogeneity in the body due to organ compartmentalization (Jackson and Heisler, 1984) produces similar problems for that approach when only plasma lactate is considered. In the 1960’s Donald Jackson (1968) convincingly showed that turtles drastically reduce their rate of metabolism during diving at 24”C, as evidenced by reduced heat production. He found an 80% decline in dives lasting 2-4 hr and >95% in dives lasting up to 7 hr. Since lung oxygen stores were exhausted in the first hour and the surrounding water was devoid of oxygen, later energy release was entirely by anaerobic means. Notwithstanding the reservations voiced above, plasma lactate accumulation rates during anoxic submergence now suggest metabolic depression to 0.6% of the aerobic rate at 20°C (Herbert and Jackson, 1985). In particular, a very high Q,, from IO down to 3°C (ca 13.3) is responsible for a large fall in metabolic rate as the lower temperature is approached (Herbert and Jackson, 1985). Until a few years ago it was widely believed that turtles other than kinosteroids could not make use of dissolved oxygen in the water due to the inability for si~ifi~nt cutaneous uptake due to their thick leathery skin, although it was realized that the critical PO, of turtles was low (e.g. 12-30 mmHg). Ultsch and Jackson (1982a) showed that arterial PO, was significantly elevated, albeit still very low by most standards, in Chrysemys submerged for 4-6 months at 3°C in air equilibrated water as opposed to those in water devoid of oxygen (Table 3). Furthermore, arterial PCOZ rose less, pH fell less, plasma bicarbonate fell less, and plasma lactate rose far less in those submerged in water containing oxygen. For example, plasma lactate in some turtles in the deoxygenated water rose to 200 mM, while the maximum in those in aerated water was less than 100 mM. Thus the turtles were able to extract dissolved oxygen from the water and support a significant portion of their vastly reduced metabolic requirements by aerobic metabolism. It is unknown what portion of extrapulmonary gas exchange in this state occurred through the skin and what portion via buccal membranes. Their data indicate that although overwinte~ng turtles can remain alive for up to 6 months at near freezing temperatures, even while totally anoxic and severely acidotic, the acid-base status and probably the recovery potential are improved if dissolved oxygen is available for extrapulmonary uptake. In fact, Herbert and Jackson (1985) found that at 3°C anaerobic metabolic rate is lower than the rate that can be supported by cutaneous respiration in aerated water, although metabolic rate may not remain constant in the presence of dissolved oxygen. Even at a PO* of 1 mmHg, turtle hemoglobin at 3°C is 7-8% saturated with oxygen. Maginniss et al.
Frog/turtle overwintering
(1983) report that the P,, in uivo under those conditions in Chrysemysis 4.5 mmHg, because turtle hemoglobin has a high temperature sensitivity. Cold diving also has a profound influence on oxygen dissociation curve shape: The curve exhibits high 0, affinity below the P,. Reductions in erythrocyte organic phosphate concentrations resulting from diving may be largely responsible. Although 7-8% hemoglobin saturation is low, it represents an O2 concentration 40-fold that of dissolved oxygen at a PO2 of 1 mmHg. Combined with temperature and anoxic suppression of metabolism, the oxygen delivery which might be achieved at this level of hemoglobin saturation is adequate for significant oxidative metabolism. The studies of Jackson and Ultsch (1982) also show that turtles are tolerant of profound plasma ion shifts and large increases in osmolality. For example, during prolonged submersion Cl- declines modestly, while Ca*+ increases over 30-fold to as much as 120 m,/L. Mg*+ increases over lo-fold, and K+ increases to 9 mM, a level toxic to the human heart. Plasma Na+ concentration remains stable. Loss of K+ and the presumed gain of Na+ by muscle and other tissues may reflect a partial failure of the Nat/K+ exchange pump. Nevertheless the ion changes serve to balance increasing lactate, but as a result plasma osmolality increases 65%, even though body weight increases up to 12% through water uptake, and even more if loss of weight to oxidation of body fuels is taken into account. The Ca2+ and Mg*+ are presumably mobilized from skeletal elements and the shell, not due to release from muscle cells (Jackson and Heisler, 1983); however, the Ca*+ :Mg*+ ratio is about SO:1 in shell and skeleton while these ions enter the plasma in a ratio of 3:1, suggesting preferential release of Mg*+. In a succeeding experiment by Ultsch and Jackson (1982b), turtles were first submerged in deoxygenated water for 3 wks, then changed to aerated water for 3 wks in order to first simulate anoxic diving or burying in mud, and were later changed to conditions of available dissolved oxygen. In the presence of oxygen, arterial PO2 and pH rose, and lactate stabilized but did not fall. Thus, while correction to pre-dive conditions did not occur, sufficient oxygen was taken up for a maintenance level of oxidative metabolism. The fact that large amounts of CO, generated from oxidative metabolism and through bicarbonate buffering of lactic acid indicates that turtles lose significant CO, through the skin. Thus the cold diving turtle, like predominantly skin-breathing salamanders at higher temperatures can to some degree control their blood pH using the largely passive extrapulmonary gas exchange mode. Recently, Ultsch, Jackson and their collaborators (Ultsch et al., 1984; Jackson et al., 1984) examined the anoxic diving tolerance of 4 turtle species: Chrysemys picta, Chelydra serpentina, Sternotherus odor atus and Trionyx spiniferus. Sternotherus and Trionyx
showed the fastest rates of lactate accumulation, the poorest anoxic survival, and presumably had the highest rates of extra-pulmonary CO2 loss. The latter is consistent with the known ease of oxygen uptake by these species in aerated water. Thus despite their
613 Table 4. The diving turtle in the cold
*Tolerance of anaerobiosis of days to months (incr. with deer. temp.) *Deer. pulmonary blood flow; initially peripheral vasoconstriction? ‘Bradycardia (variable) & cardiac arrhythmias *Greatly deer. metabolic rate & activity lGlycogenolysis & glycolysis *Tolerance of high plasma lactate cont. (i.e. 2OOmM) & low pH (severe metabolic acidosis) ltJnusttally high normal extracellular bicarbonate cont. *Mobilization of Ca*+ & Mg from skeleton & shell for buffering *Reduction in Cl- and bicarbonate cones.; Na+ cow. stable ‘Extrapulmonary loss of CO, to water *Tolerance of profound ion shifts & large incr. in plasma osmolality *Oxygen uptake from water, when possible ‘Very low hemoglobin P, (44.5 mmHg) at 3°C *Incomplete torpor *Collapsed lung in anoxia ‘Extravascular lactate storage (urine, pericard. fluid); excretion? *Unorthodox anaerobic end-products (succinate)? *Depressed tendency for blood clotting? cont. = concentration.
higher’ rates of CO, generation, they experienced less hypercapnic acidosis. Because pH depression is known to inhibit glycolysis, relative lack of CO2 retention in these turtles may be the very reason they accumulated lactate more rapidly and showed less tolerance. In the same way, the lower anoxia tolerance of anurans probably derives largely from their efficient cutaneous gas exchange. Regardless of the perceived advantages of dissolved oxygen for overwintering derived from laboratory studies, freshwater turtles are frequently found buried in mud (with or without overlying water) without apparent access to oxygen. Thus, in some species (e.g. Chrysemys, Chelydra) extrapulmonary O2 uptake may not be essential for overwintering, while in others it probably is (e.g. Sternotherus, Trionyx). However, in high latitudes with very long winters even the more anoxia resistant turtles probably depend upon some oxidative metabolism for part or all of the winter in order to survive. Turtles frequently observed swimming under the ice in winter may be seeking water of higher O2 content, whereas if this occurs in late winter they may be moving to shallower water in order to gauge the proper time of emergence. It has even been suggested that the northern limits of many turtles may not be determined by the rigor of overwintering, but rather by the period available for reproduction. Thus the turtle in the cold has a number of characteristics which favor its great submersion tolerance of days to months (Table 4). Peripheral vasoconstriction, decreased pulmonary blood flow (White and Ross, 1966) and bradycardia (Belkin, 1964) all act to conserve on-board oxygen for the heart and brain, at least initially during the dive as in diving mammals. Depressed metabolic rate which intensifies with low ambient temperature, prolonged submersion, and anoxia is vitally essential and is the centerpiece of the response. Glycogenolysis occurs, with glucose release and lactate production, and small amounts of succinate. Torpor, however, is incomplete, as turtles are known to move about during overwintering. Turtles show remarkable tolerance of both high lactate concentrations, low pH, profound ion shifts and large increases in plasma osmolality. However,
614
DAVID G. PENNEY
the slower the rate of body lactate rise, the greater the period of submersion tolerated. Lactate and other anaerobic end-products are possibly to some extent stored in the urine and pericardial fluid (Jackson and Heisler, 1984) or excreted. Jackson and Silverblatt (1974), for example, saw significant lactate in the urinary bladder of turtles submerged at 24°C. In addition, water uptake during long continued submersion may serve to further dilute lactate, and other metabolic end-products. Turtles normally have unusually high extracellular bicarbonate concentrations, as compared to amphibians and fish. This, and skeletal element Ca*+ and Mg*+, provide the major buffering capacity for metabolic acidosis, while other ions are also adjusted up or down in concentration. CO, generated by oxidative metabolism and through acid buffering is lost through extrapulmonary routes. When available and where possible, oxygen is taken up from the water by the same extra-pulmonary routes. Since turtle hemoglobin has this a very low P,, at near freezing temperatures, assists tissue oxygen delivery. The lungs of anoxic or nearly anoxic turtles, are collapsed. Unlike diving mammals, in turtles on long dives or overwintering lactate is not confined to the peripheral organs ready to flood out into circulation following emergence, with oxygen reserved for the heart and brain. Instead, complete or nearly complete anoxia exists throughout the body and all organs are using anaerobic metabolism, even the heart and brain. It now appears that no exotic mechanisms need be invoked to permit such incredible diving feats, just optimal use of those present in other vertebrates. Finally, overwintering in turtles should not be viewed as simply prolonged diving-with the profound suppression of metabolism it must be viewed as ectotherm hibernation! In turn, their incredible diving capabilities are merely a spin-off of a successful overwintering strategy. CONCLUSIONS
We might contrast the major features of the overwintering/diving strategies of frogs and turtles in the following way (Table 5): (1) Frogs use cutaneous obligatory for submersion duration. Fat is probably Table
5. Maior
features
0, uptake and in fact it is of more than a few days the major metabolic fuel.
of the overwintering/diving frogs and turtles
strategies
of
Frogs
*Use cutaneous 0, uptake, turtles to a much smaller extent; varies with species ‘More readily lose CO, through skin. Thus, respiratory alkalosis can partially compensate metabolic acidosis in frogs, not in turtles ‘Extreme cutaneous permeability may limit ability to lower metabolism like turtles because of need to maintain ionic & osmotic balance; also, lost CO, & smaller pH fall inhibits metabolism (i.e. glycolysis) less TUdt-S ‘For a given temperature under anaerobiosis, metabolism is lowered more than in frogs (>95% vs 3&70%, respectively) *Heart and liver contain more glycogen than in frog *Tolerate much larger increases in lactate than frogs (> 200 mM vs 35 mM. respectively) *Have greater ootential buffering _.capacity: plasma HCO, , shell C&6, & M&O,
Turtles to a much smaller extent respire through the skin, but this varies with species and ambient temperature. (2) Frogs more readily lose CO2 through the skin. Thus respiratory alkalosis can partially compensate metabolic acidosis in frogs. Although this also occurs in turtles, the process is much slower and is of little value in rapid lactate acidosis at higher temperature. (3) The extreme cutaneous permeability of frogs may limit their ability to lower metabolism like turtles because of the need to maintain ionic and osmotic balance. Also, the lost CO, and smaller pH fall may serve to inhibit glycolysis less, allowing lactate to rise rapidly and consequently decrease anoxia tolerance. (4) In the turtle, for a given temperature under anaerobiosis, metabolism is lowered much more than in the frog (> 95% vs 3&70%, respectively). (5) The turtle heart and liver contain more glycogen than in the frog. Under strictly anoxic conditions this is the primary fuel, whereas with oxygen availability fat is also presumably metabolized. (6) Turtles tolerate much larger increases in lactate than do frogs (> 200 vs 35 mM, respectively). (7) And finally, turtles have far greater potential buffering capacity, including plasma bicarbonate and shell calcium carbonate and magnesium carbonate. Note: This review developed from the author’s presentation at a symposium, Physiology and Ecology of Ectotherm Hibernation, organized by G. R. Ultsch, at the American Society of Ichthyologists and Herpetologists meeting, 19 June 1986, at the University of Victoria, Victoria, BC, Canada.
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