Respiratory adaptations in selected amphibians

Respiration Physiology (1967) 2, 247-260; North-Holland Publishing Company, Amsterdam

RESPIRATORY

ADAPTATIONS

IN SELECTED AMPHIJ31ANS1

CLAUDE LENFANT AND KJELL JOHANSEN’ Institute of Respiratory Physiology, Firland Sanatorium, and Departments of Physiology and Zoology, University of Washington, Seattle, Washington, U.S.A.

Abstract. In order to evaluate some of the respiratory adaptations concomitant with the transition from water breathing to air breathing, three amphibians were studied: the aquatic Necturus maculosus which possesses external gills and a poorly developed lung, Amphiuma tridactylum which has no gills but remains in water and depends on pulmonary breathing, and the bullfrog, Rana catesbeiana, which is somewhat terrestrial and uses pulmonary respiration. Respectively for Necturus, Amphiuma and bullfrog the oxygen capacity was 6.3, 7.6, and 8.0 vol %; the affinity for 0~ or PSOat the physiological Pco,, was 14.5,27 and 39 mm Hg; the Bohr effect or Alog Pso/ApH was -0.131, -0.205 and -0.288; the CO2 combining power at the physiological Pco, was 20.0, 31.2 and 30.4 vol % and the buffering capacity, or AHCO;/ApH, was - 8.0, -9.2, and- 16.4mM/l/pH. The respiratory characteristics of these animals kept at 20 “C was studied by in vivo blood gas measurements made at the same temperature. Pao, and Pace, of Necturus kept in water were 35 and 4.4 mm Hg respectively. When removed from water the exchange of blood gases was greatly impaired in spite of frequent air breathing. In free-breathing Amphiuma Pao, was 81 and Pace, 6 mm Hg. When prevented from air breathing PO, decreased and Pco, increased slowly suggesting that the animal uses its skin to exchange gases with water fairly effectively. In the bullfrog Pao, was 95 and Pace, 8 mm Hg. When the animal was immersed, PO, decreased and P co2 increased rapidly suggesting the absence of efficient skin respiration. These findings are regarded as expressing adaptive changes to meet an increased 02 availability of the external medium, and on elevated internal PCO, in the transition from aquatic to aerial respiration. Air breathing in amphibian Blood gases Gill respiration

Skin respiration Water respiration

The diverse adaptations amphibians show in their relation to environmental conditions are clearly reflected in their multiple ways of exchanging gases with the environment. Thus lungs, gills, skin and buccal cavity may all play a role in gas exchange in some species. In most cases, however, successful adaptation to special habitats has favored specialization of one or two modes of respiration. Accepted for publication 13 February 1967. l This study was supported by grant G.B. 358 from the National Science Foundation and grants H.E. 0845 and H.E. 01892 from the National Institutes of Health. 2 This work was carried out during the tenure of an established Investigatorship of the American Heart Association. 241

248

C. LENFANTANDK. JOHANSEN

A comparative study of respiratory behavior in amphibians promises to elucidate the basic differences between air and water breathers in dynamics of gas exchange and in adaptive changes of the respiratory properties of blood. For the present study, three species were selected having distinctly different associations with the external environment. The two urodeles, Necturus maculosus and Amphiuma tridactylum, are exclusively aquatic. The former is alleged to depend primarily on external gills for gas exchange while pulmonary respiration is considered relatively unimportant. The adult Amphiuma, on the other hand, has no gills and depends largely on lungs for gas exchange. Finally, the bullfrog, Rana catesbeiana, is a lung breather that spends much time out of water. Material and methods

Six specimens of each species were used in the present investigation. They were kept at 20-22 “C several days prior to the experiments which were performed at the same temperature. The animals were anesthetized by immersion in a solution of MS 22 (Sandoz). Samples of arterial blood were obtained by chronic cannulation of the coeliac artery or one of the mesenteric arteries using polyethylene catheters. The animals were allowed to recover from anesthesia before any samples were collected and they were unrestrained and free to move about during the experiments. Following the in vivo experiments, blood was withdrawn through the arterial catheter and used for the in vitro studies. Blood samples were analyzed for partial pressures of 0, and CO* using the Beckman Spinco gas analyzer with the O2 macro electrode in a special cuvette (.03 ml) and the Severinghaus electrode for PcoI. Calibration was done with known gas mixtures or blood equilibrated to known gas composition. For the in vitro studies blood pH was measured by means of a Beckman pH micro electrode and pH meter; blood gas contents were measured by gas chromatography (LENFANTand AUCUTT, 1966). All measurements were done at room temperature ranging between 20-22 “C. Oxyhemoglobin dissociation curves and CO, dissociation curves were established according to the description of LENFANTand JOHANSEN(1965). The O2 capacity was assessed by determination of the O2 content of blood equilibrated with room air at 20 “C. Affinity of the blood for oxygen is defined as l/P,, at a Pcol within the normal physiological range. The magnitude of the Bohr effect is expressed as Alog P,,/ApH and the Haldane effect as AC,o,/OZ capacity. AC,,, represents the difference in CO, combining power between reduced and oxygenated blood when Pcol is within the physiological range. The buffering capacity is expressed by AHCO;/ApH when AHCO; represents the change in bicarbonate concentration in millimoles/liter when pH changes by 1 unit. Results

RESPIRATORY PROPERTIES OF BLOOD Table 1 lists the mean values of the respiratory properties of blood from the three

RESPIRATION IN AMPHIBIANS

TABLE Comparative

1

values of respiratory

Hemoglobin, g % Hematocrit, % Oxygen capacity, vol % PSOat Pace, (see table 2) mm Hg Bohr effect, Alog Pso/ApH CO* combining power at Pace,, Vol % Haldane effect, ACCO,/OZ capacity Buffering capacity, mMol/l/pH

249

properties

of blood.

Necturus

Amphiuma

4.5 19.0 6.26 14.5 -0.131 20.0 - 0.320 -8.00

23.0 7.62 27.0 - 0.205 31.2 -0.131 -9.20

5.1

Bullfrog 5.7 23.5 8.02 39.0 - 0.288 30.4 - 0.280 - 16.40

studied. Fig. 1 shows the O,-Hb dissociation curves for the three species. Necturus blood exhibits the highest affinity for oxygen, the bullfrog the lowest. The reduction in affinity is accompanied by a more S-shaped lower limb of the curve and by increase of the Po, required to reach lOOo/osaturation (PI&. The Bohr effect is low in all species (table 1; fig. 2) showing a decline from the bullfrog to Necturus; Amphiuma again occupies an intermediate position. Fig. 3 depicts the CO, dissociation curves for oxygenated blood for the three species. They are characterized by a steep initial slope that changes rather abruptly to a more gradual slope. The CO, combining power of the blood in bullfrog and Amphiuma greatly surpasses that of Necturus. The Haldane effect (table 1) is low for all three species; it shows no apparent correlation with habitat or mode of breathing, being lower in Amphiuma and higher in Necturus and the bullfrog. species

20 Fig. 1. Oa-Hb dissociation

40 60 Pop mm Hg curves. Comparison

80 of the three species.

250

C. LENFANT

100 00

AND K. JOHANSEN

1

60-

$40t p 30-

200 Id

7.0

7. I

7.2

7.3

7.4

7.5

7.6

7.7

,

7.0

7.9

PH

Fig. 2. Comparison

of the Bohr effect in the three species.

I Oxygenated

I

IO

30

20 Pco.

Fig. 3. CO2 dissociation

blood)

40

mmN

curves. Comparison

of the three species.

RESPIRATION

7.3

7.4

251

IN AMPHIBIANS

7.5

7.6

7.7

7.8

PH

Fig. 4. Comparison

of the buffering capacity of the three species.

Fig. 4 shows a comparison of the buffering capacity of the blood in the three species. There is an explicit trend from a high buffering capacity in the bullfrog decreasing through Amphiuma to Necturus. In vivo

EXPERIMENTS

Table 2 lists the mean normal Pao, and Pace, of each species for fully awake and unrestrained animals. It also indicates the 0, partial pressure in the water and in the ambient air at the time of the blood sampling. The levels of Pa,, and Paco2 during normal conditions increase from Necturus to Amphiuma to bullfrog. The PO, increase was much greater between Necturus and Amphiuma than between Amphiuma and the bullfrog, whereas the Pco2 increase was of the same magnitude. Fig. 5 demonstrates the time course of the Pao2 changes for Necturus when the animal was initially in water with free access to air, then was placed in air for 25 min TABLE 2 Mean arterial blood PO, and PCO, in normal environment

PO, in water, mm Hg PO, in air, mm Hg Paoz, mm Hg *l SD Paco2, mm Hg +l SD SD = Standard deviation

Necturus

Amphiuma

BulIfroe

125 152 35.2

125 152 81.2

12.5 152 94.6

4.3

8.6

12.6

4.4 0.4

6.1 0.6

8.2 0.6

252

C. LENFANT

AND K. JOHANSEN

Time

Fig. 5. Changes

in minutes

in Pao, and Pace, of Necturus during and after exposure to air.

and subsequently returned to water. When transferred to air, the Pa,, dropped rapidly to very low values (Pa,, z 10 mm Hg) in spite of voluntary efforts to fill the lungs with air. Conversely, Pace, climbed rapidly to values approximately twice the normal. The recovery period after transfer back to aerated water was characterized by an increase in the respiratory efforts with quick waving motion of the external fan-like gills. This resulted in a rapid rise of Pa,, transiently reaching values in excess of the original level. The elimination of CO2 from the blood was, however, surprisingly slow and required more than 30 mm to reach normal levels. Amphiuma, being an aquatic lung-breather, was initially kept in water with free access to air, then prevented from surfacing for air and finally allowed to recover under the initial conditions (fig. 6). The rate of change in Pao, when the animals were prevented from breathing was very slow in comparison to that of Necturus in air.

RESPIRATION

253

IN AMPHIBIANS

AMPHIUMA-20%

00-

_A

\

8

L-

o

\\

\O

9

\O \

g 60-

0

o

2

o

o

\O

I

‘ox o’..

I

\ “0

40-

\

f’.d /

,

/

9’

\

Q

P

/

I

o 0

o

\ \

0.

I I

---_

20-

1 -0 /// 0-

/

\ 1 \ \ \ 0 \

xf’o

/ / / P

7--o--’ 0”

f

8

6 e

6 - fo

0

/

\

0

1

00

0a 9’

0

/

4’

/

9 \

_-

00

,/o-

\

O

\

I

6

\ I \

--ao----i~

o \ 0

5-

-Prevented

0

from

20

IO Time

surfacing---

30

400

IO

( 20

tn minutes

Fig. 6. Changes in Pao, and Pace, of Amphiuma while and after being prevented from breathing air.

The animals showed signs of restlessness but attempts to surface were sporadic and non-violent. PacOz showed a slow increase but again of far less magnitude than in Necturus when gill breathing was arrested. The recovery was rapid both for Pa,, and PaLcoI. The levels of Po, and Pco2 in arterial blood of resting bullfrogs breathing air are the highest compared to the levels of the other species. When the animals were immersed they showed early signs of distress and repeatedly attempted to surface. Fig. 7 shows that they were unable to maintain the gas tensions in the blood. Pao2 dropped rather precipitously reaching values of 25-30 mm Hg within 5 min from the onset of immersion. The simultaneous rise in Pa co2 was more gradual. The recovery after transfer to air was very rapid with normal levels re-established in less than four minutes.

254

C. LENFANT AND K.JOHANSEN

\\ ,,,_-:--

\ \

-_Eb--

I

“\

0

,‘\

“1:

20-

-

0

BULLFROG-20T

:

Immersed--RRec0very./ a, 0

12 j / ,

0 --a-_

8

0

I/

’

\o ' I 10 \ \ \ \ I \ \ 0 I \ \ "\" 0 \4 \ o o 0 \

40-

$

o

;a o/---9--/

\

I0 I

3 60a

0 0

10 I o

\

i

00-

0 0

Ilo

\

0

s$

~--------

/

\

w

_

/ \

/

/’ 4 0% 0

0 0 0

\

pLo

\

\

\

\ \

IO

‘b ‘.

-o-6-

0

0 0

5 Time

I”

IO minutes

5

IO

Fig.7.Changes in Pao, and Pace, of bullfrog during and after submersion.

The objectives of the present study were to assess whether adaptive trends in the respiratory properties of the blood could be correlated with aerial and aquatic respiration and to seek expressions of such trends in the in uivo levels of blood gases and in the mode of gas exchange. RESPIRATORY PROPERTIES OFBLOOD

It has been amply documented that respiratory properties of blood may show adaptive features correlated with environmental conditions and levels of metabolic activity as well as with ontogenetic development and age (BARCROFT, 1934). Such complexity of modifying factors demands extreme caution in interpretation of data. In the present context one must remember that Necturus as well as Amphiuma are permanent larvae. Necturus has retained long external gills and a branchial apparatus of typical larval character. Amphiuma, often called a “semilarval” animal, has lost its external gills and shows other evidence of partial metamorphosis. 1) The increase in oxygen capacity from Necturus to the bullfrog may reflect the

RESPIRATION IN AMPHIBIANS

255

neotenic character of Necturus or may be associated with greater dependence on aerial breathing in the bullfrog (MCCUTCHEON,1936). Results obtained by WOLVEKAMP and LODEWIJKS (1934) and LEFTWICHand BURKE(1964) in different species of adult frogs strongly suggest that terrestrial forms have a higher 0, capacity than the aquatic forms. 2) The afinity of the blood for O2 in the three species decreased with increasing dependence on air as a respiratory medium. Such correlations have been demonstrated for other transitional forms (MCCUTCHEONand HALL, 1937) and might reflect an adaptation to oxygen availability as the 0, supply in water is limited compared to that in air. The decrease of affinity for 0, may also be related to the higher metabolic requirements associated with terrestrial life (HELFF, 1927). A greater “unloading capacity” favored by a low affinity for O2 and a sigmoid shape of the 02-Hb dissociation curve, as in the bullfrog, helps to meet these requirements. MCCUTCHEONand HALL (1937) reported a similar change in shape of dissociation curves correlated with a decreased affinity in their study of eight species of adult amphibians. The low P 100 of Necturus results in a large O2 gradient across the respiratory surface which, combined with the steep 02-Hb dissociation curve, facilitates the diffusion of 0, and thus the “loading” of the blood with 0, even when PO, in the water is low (Necturus often lives in stagnant water). The large gradient in PO, will also facilitate loading during the long periods when the gills of Necturus remain motionless and stirring and hence ventilation is very low. In addition, oxygen affinity decreases from fetal to adult life (BARCROFT,1933). MCCUTCHEON(1936) demonstrated this shift in the developing bullfrog and explained the phenomenon as an adaptation to the change from the aquatic environment of the larval frog to the adult air-breathing conditions. In the present study, the permanent larval condition of Necturus and the semilarval condition of Amphiuma may modify the respiratory properties of the blood. A change in shape from a hyperbolic dissociation curve in Necturus to a sigmoid curve for the bullfrog is similar to the change noted by MCCUTCHEON(1936) comparing larval and adult bullfrog blood. 3) The Bohr effect shows no trend to decrease as the animal becomes more dependent on aerial respiration. Such a trend has been demonstrated by comparison of the Australian and South-American lungfish (LENFANT,JOHANSEN and GRIGG, 1966/67; JOHANSENand LENFANT,1967). The South-American lungfish shows a much smaller Bohr effect correlated with a markedly larger dependence on pulmonary breathing. Although such a trend is not apparent in the present comparison of amphibians, this may be related to the larval or semilarval conditions of Necturus and Amphiuma MCCUTCHEON(1936) showed that larval bullfrogs have a much smaller Bohr effect than adults and even show a reversal within the pH interval that shifts the adult O,-Hb affinity to the right. Also if high metabolic activity is a determining factor of the shape of the O,-Hb dissociation curve, an increased sensitivity to CO, (larger Bohr effect) would maintain a larger O2 diffusion gradient to the active tissues having high CO, tensions. More work is needed to resolve the various factors that may affect the affinity of amphibian blood for oxygen.

256

C. LENFANTANDK. JOHANSEN

4) The CO2 di~s~ciu~i~ncumes (fig. 3) are conspicuously steep, especially for the bullfrog and Amphiuma. These species also show far higher CO, combining powers. However, the measurement of the CO2 combining power reveals very large individual variations due to differences in hemoglobin content between specimens. This was emphasized by SCOTT(193 1) for Amphiuma and it probably explains the enormous differences observed in Rana catesbeiana by various authors. For instance WASTL and SELISKAR(1925) presented values of CO2 combining power considerably higher than the present results. No previous reports of CO, combining power in Necturus have come to our attention. 5) The bz&ring capacity showed a marked increase from Necturus to the bullfrogs with Amphiuma occupying an intermediate position. These differences are closely correlated with the relationships of the three species to water and air as respiratory media. The increase in buffer capacity with an increased importance of pulmonary breathing is also correlated with an increased level of arterial PcoZ (table 2). Differences in buffering capacity also correspond with differences in Hb content of the blood. A similar increase in buffer capacity correlated with dependence on air breathing and elevated arterial P,, was demonstrated in lungfishes by LENFANTet al. (1966) and JOHANSEN and LENFANT(1967). This result is reasonable in view of the higher metabolic requirements associated with a terrestrial existence since a higher metabolism will demand a higher buffering capacity against acid metabolites. In Vi00EXPERIMENTs This work provides information about the dynamics of gas exchange when the different species were prevented from using their primary mode of respiration and consequently had to depend on means of gas exchange that normally are of lesser importance. 1) Nectarus had the lowest levels of Pao, and Pac.% during normal conditions. However, the great affinity for O2 permits a high arterial blood 0, saturation (Sa,, > 90%) even at the low arterial Pol. Necturus then seems typical of a water breather adapted to tolerate hypoxic water. Normally the lungs are very rarely used whereas the long external gills are intermittently waved to stir the water. This waving increases the efficiency of gas exchange as indicated by the changes in the redness of the gill filaments. When brought out of water, the arterial PO, dropped precipitously in spite of increased attempts to gulp air. Thus the inefficiency of the lung was demonstrated. With the delicate external gills collapsed and ineffective in air the PCo2 rose to high levels. The recovery following return to water was slow in spite of very vigorous movements of the gills. The arterial Po, became overcompensated before returning to normal levels. The Pco, showed a surprisingly slow return to normal levels without overcom~nsation. A large CO, storage capacity of the blood, shown by the steep CO2 dissociation curve within the physiological range of PcoZ, and probably of the tissues, may be responsible for the slow Pcol recovery. However, one would predict a more rapid rate of COZ store adjustment since CO, is readily eliminated by water breathers. This problem deserves further study.

257

RESPIRATION IN AMPHIBIANS

2) Compared with Necturus, ~~~h~~~~ blood has a lower affinity for oxygen; but the higher arterial PO2in Amphiuma normally leads to the same degree of saturation (&to, >90%). When prevented from breathing air, the arterial Po, of Amphiuma declined and Paco2 increased. However, the rate of change was less than for Necturus in air. When an animal is prevented from breathing, Pcot increases proportionally to the increase in CO, storage, and the rate of change depends on the accumulation of COz in the body as a function of time. Although our experiments were not designed to measure the whole body CO, accumulation directly, it can be estimated indirectly from the change in COz content of the arterial blood if it is assumed that the ratio of CO2 accumulation in the blood to CO, accumulation in the body is constant throughout the experiment. Fig. 8 shows a plot of the COz content increase in blood as a function of duration of the “equivalent breath holding” (EBH, defined as air exposure for Necturus, absence of air breathing for Amphiuma, and immersion for bullfrog) for the three species. The area under each curve is proportional to the rate of CO2 accumulation in the body (voljtime). If it is assumed that the three species have the same CO2 production per unit of weight and an equal ratio of COz accumulation in blood to CO2 accumulation in tissues, such a representation permits us to assess the importance of the skin respiration in the three selected species. For instance, it can be seen that the COz accumulation of Amphiuma (area under the curve) is much smaller than that of Necturus, which indicates that Amphiuma has an important COz output through the skin during EBH. This mechanism would explain the slow rise in arterial P,,. The rapid return to the original values of P,,

IO Duration

20 of “equivalent

30 breoth

40

holding”(min.)

Fig. 8. Increase of the CO% content in the arterial blood of the three species during the Equivalent Breath Holding. Each curve is tbe average curve for each species. CO2 content was determined from the CO2 dissociation curves represented in fig. 3.

258

C. LENFANTANDK. JOHANSEN

and Po, when air-breathing was resumed stresses the contribution of the Amphiuma lung to the overall gas exchange under normal conditions. Since, in fig. 8, the curve for Amphiuma does not plateau off, Amphiuma can not depend on skin for the total gas exchange (unlike several species of lungless salamanders). The relative importance of the various respiratory surfaces in amphibians has been estimated from studies of their vascular anatomy. Such analyses on Amphiuma (CZOPEK, 1965) revealed a vascularization (expressed as total length of capillaries) more than twice as extensive in the lungs as in the skin. This may be correlated with the large size of the animal giving a small surface to volume ratio (SZARSKI,1964). WHITFORDand HUTCHISON(1963, 1965) have evaluated the relative importance of cutaneous and pulmonary gas exchange for a number of amphibians and have demonstrated how temperature and other environmental factors modify the relative importance of the various respiratory surfaces. They found that at 20 “C, 40% of To, and 88% of %02 are exchanged through the skin of lunged salamanders. 3) The bullfrog, with the highest arterial oxygen tensions during normal conditions and the lowest affinity of the Hb for oxygen, showed arterial 0, saturation values similar to the other two species. The present data suggest that a correlation exists between the affinity of the Hb for oxygen and the normal levels of arterial Po,. A high affinity seems to be matched with lower values of Pa,,. The earlier established increase in the levels of Paco2 as vertebrate animals become more dependent on pulmonary respiration was confirmed by the present results in which the bullfrog showed the highest Paco2 values. Although a direct relationship exists between Paco2 and the temperature of the animal (CAMPBELL,1926; HOWELL,BOUVEROT and RAHN, 1966), there are large individual variations in the PacoZ at any given temperature. When bull frogs were submerged, the PO, decreased and the Pco2 increased very rapidly. If the CO, production of bullfrog does not exceed that of the other species, these findings and fig. 8, which reveals that the body CO, accumulation was greatest in the bullfrog, demonstrate an inability of the skin to maintain the gas exchange in equilibrium at 20 “C. This inadequacy is amplified as the reduced oxygen uptake in submerged frogs and toads (POCZOPKO,1959) does not prevent the CO2 production from anaerobic metabolism. The importance of external factors such as temperature for the survival of submerged frogs was emphasized by SERFATYand GUENTAL(1943) and POCZOPKO(1959). Serfaty and Guental maintain that frogs (Rana esculenta) submerged in well aerated water at 15 “C can survive for 2-3 weeks and, at lower temperatures, gas exchange through the skin can sustain life through many months of hibernation. The extremely rapid return to the original Pa,, and Paco2 during the recovery period established that lung respiration is the essential means of gas exchange in bullfrog. The fact that the recovery is faster than in Amphiuma indicates that this means of respiration is more efficient in the bullfrog than in Amphiuma. The importance of skin respiration in the three species can be compared in fig. 8 which reveals that there is little difference between Necturus and the bullfrog as compared with Amphiuma. These findings seem to be contrary to current teaching

RESPIRATION IN AMPHIBIANS

259

that the skin of frogs is responsible for the major part of CO2 excretion (KROGH, 1904). In Necturus, skin and gill respiration cannot be distinguished by the methods employed since both involve aquatic gas exchange. It is, however, reasonable to assume that specialized gill respiration is far more important than skin respiration. The respective roles of the different gas exchangers can now be placed in a phylogenetic context. First, strictly aquatic species depended mostly on their gills for external respiration; then the skin respiration served an important role first as an adjunct to gill respiration and then in support of aerial respiration, Finally, after the lung had reached a higher degree of efficiency, exclusive terrestrial life became possible. References BARCRO~, J. (1933). The conditions of foetal respiration. Lancef 225: 1021-1027. BARCROFT,J. (1934). Features in the architecture of physiological function. Cambridge Univ. Press. CAMPBELL,J. A. (1926). The normal COZ and 0~ tensions in the tissues of various animals. J. Physiol. 61: 248-254. CZOPEK, J. (1965). Quantitative studies on the morphology of respiratory surfaces in amphibians. Acta Anat. 62: 296-323. HELFF, 0. M. (1927). The rate of 02 consumption in five species of Amblystoma larvae. J. Exptl. Zooi. 49: 353-361. HOWELL, B., P. BOWEROT and H. RAHN (1966). Effect of temperature on acid base balance in Bullfrog. Physiolo~~t 9: 206. JOHANSEN,K. and C. LENFANT(1967). Respiratory function in the Ruth-Ameri~n lung&h, Lepidosiren paradoxa (Fitz). f. Exp. Biof., in press. KROGH, A. (1904). On the cutaneous and pulmonary respiration of the frog. Skand. Arch. Physio!. 15: 382-419. LEFTWICH,F. B. and J. C. BURKE (1964). Blood oxygen capacity in ranid frogs. Am. Midl. Nat. 72: 241-248. LENFANT,C. and K. JOHANSEN(1965). Gas transport by hemocyanin-containing blood of the cephalopod Octopus dofleini. Am. J. Physioi. 209: 991-998. LENFANT,C. and C. AUCUTT (1966). Measurement of blood gases by gas chromatography. Respir. Physiol. 1: 398-407. LENFANT,C., K. JOHANSENand G. C. GRIGG (1966/67). Respiratory properties of blood and pattern of gas exchange in the lungfish, Neoceratodus forsteri (Krefft). Respir. Physiol. 2: I-21. M&UTC&~EON,F. H. (1936). Hemoglobin function during the life history of the bullfrog. J. Cell. Comp. Physiol. 8 : 63-8 1. MCCUT~HEON,F. H. and F. G. HALL (1937). Hemoglobin in the Amphibia. J. Celi. Comp. Physiol. 9: 191-197. POCZOPKO,P. (1959). Respiratory exchange in Rana esculenta L. in different respiratory media. zoo/. Polon. 10: 45-55. SCOTT,W. J. (1931). Oxygen and carbon dioxide transport by the blood of the urodele, Amphiuma tridactylumn. Biol. Bull. 61: 21 l-222. SERFATY,A. and J. GUENTAL(1943). La rbsistance de la grenouille 11l’asphyxie lors d’une immersion prolong&e. C. R. Sot. Biol. 137: 154-156. SZARSKI,H. (1964). The structure of respiratory organs in relation to body size in Amphibia. ,?%oZc&on 18: 118-126. WASTL, H. and A. SELISKAR(1925). Observations on the combination of CO2 in the blood of the bullfrog (Rana catesbeiana). J. Physiool. (Lotid.) 60: 264-268.

260 WHITFORD,

C. LENFANT

AND K. JOHANSEN

W. G. and V. H. HUTCJSISON(1963). Cutaneous and pulmonary gas exchange in the spotted salamander, Anabystonia maculatum. Biol. Bull. 124: 344-354. WHITFORD, W. G. and V. H. HUTCHISIN (1965). Gas exchange in Salamanders. Physiol. Zool. 38: 228-242. WOLVEKAMP, H. P. and J. M. LODEWIJKS(1934). Ueber die Sauerstoffbindung durch Hiimoglobin vom Frosch (Rana esculenta und Rana Umporania). Zeitsch. vergl. Physiol. 20: 382-387.