J. Exp. Biol. (1970), 5a, 1-15 jWith 1 text-figures

Printed in Great Britain

THE RELATIVE EFFICIENCYOF GASEOUS EXCHANGE ACROSS THE LUNGS ANDGILLS OF AN AFRICAN LUNGFISH PROTOPTERUS

AETHIOPICUS

BY B. R. M C M A H O N

Department of Zoology, University of Bristol*

(Received 31 March 1969)

Of the three genera of lungfishes living today Neoceratodus, the Australian lungfish,is possibly the most primitive. The respiratory physiology of this animal has beenextensively studied in recent years (Grigg, 1965 a, b, c; Lenfant, Johansen & Grigg,1966; Johansen & Lenfant, 1967) and the conclusions these authors draw support theassumptions of the earlier workers in this field (Dean, 1906; Longman, 1928; Spencer,1891), who maintained that in this animal the lung was an accessory respiratory organused principally when the animal was in poorly aerated water. The gas exchangeoccurring over the gills is apparently sufficient to satisfy the requirements of the animalin well-aerated water. Earlier workers on the respiratory physiology of Lepidosiren,the South American lungfish, suggested that in this animal the lungs were the principalrespiratory organs (Carter & Beadle, 1930; Cunningham, 1934; Kerr, 1897, 1898).Sawaya (1946) found that the gills accounted for only 2% of the total oxygen uptake,and Johansen & Lenfant (1967) confirmed that the lungs were the principal site foroxygen exchange but thought that some carbon dioxide might be exchanged via theaquatic route. These conclusions show that these two forms are well adapted to theirpredominant habitat. Neoceratodus is usually found in permanent, well-aeratedwaters, which only rarely become stagnant (Grigg, 1965 c) and is only a facultative air-breathing form. Lepidosiren, on the other hand, is normally found in marshy waterswhich are not only often extremely hypoxic and hypercarbic, but are also liable todry out periodically. Lepidosiren is an obligatory air-breathing animal and can survivethe periods of drought by aestivating in a burrow in the mud (Kerr, 1898).

The habitat of Protopterus aethiopicus is similar to that of Lepidosiren, and, thoughit has long been assumed that the lungs were the principal respiratory organ, untilvery recently little experimental work has been carried out to verify this assumption.Prior to the completion of this manuscript, however, several workers have reportedstudies which verify this assumption. Jesse et al. (1968) suggest that both lungs andgills are important in respiration in juvenile Protopterus (species not certain) but reportno result from adult animals. Johansen & Lenfant (1968), however, demonstrate thatthe lung is the principal organ in oxygen uptake in Protopterus aethiopicus. In thisstudy experiments to elucidate and quantify the actual efficiency of lung and gill ingaseous exchange will be described and discussed.

• Present address: Assistant Professor of Biology, Department of Biology, The University of CalgaryAlberta, Canada.

! EXB 52

B. R. MCMAHON

METHODS

Recordings were made of aerial and aquatic respiratory frequencies, both in animalsimmersed in well-aerated water with access to air and in animals which were confinedeither under water or in moist air. The animals were confined in a Perspex observationtank which could be perfused with either warmed aerated water or warmed humidi-fied air. Ambient water was warmed by passage through a glass warming coil immersedin a thermostatically controlled water bath. The temperature in these experimentswas maintained at 24+ i°C.

Reservoir

Valve

Fig. 1. Diagram of the respirometer used in the determination of oxygen consumption andcarbon dioxide production via the aerial and aquatic routes. A-E, taps controlling flow of airor water.

Ventilation rates were recorded in three ways: (a) observed by the investigator andtransferred immediately to an event recorder; (b) by the electromyograms of therespiratory muscles picked up incidentally by ECG probes; (c) by the pressuresrecorded by means of cannulae implanted in buccal and/or opercular cavities(McMahon, 1969).

In all the experiments animals were allowed to acclimatize to the experimentalchamber for at least 4 hr. and usually over 20 hr. A record of the activity of the animalswas taken at all times, but in fact acclimatized animals usually remained at rest on thefloor of the chamber, moving only to take air. All recordings discussed here weretaken from animals at rest.

Rates of gas exchange were measured by respirometry. The simultaneous measure-ment of aerial and aquatic gas exchange poses problems in the lungfish, as streams ofrespiratory air and water must be kept separate to avoid interdiffusion of gases.

Gaseous exchange efficiency in Protopterus aethiopicus 3

Methods where the animal is partitioned using rubber membranes, as used by VanDam (1938) and Berg & Steen (1965), are not easily adapted for use with a free-swimming animal breathing air or water at will, and may also impair the respiratorymechanism (Piiper & Schumann, 1967). In these experiments the rates were measuredusing a specially designed respirometer in which air and water streams flowing pastthe animal were kept separate except at the moment of breathing. This apparatus isillustrated in Fig. 1.

The respirometer consisted of a Perspex tube flanged at both ends which could bemounted diagonally in a rigid frame. The animal was persuaded to swim into the tubeand the ends were sealed by Perspex discs equipped with ' O' ring seals. The discsheld a number of taps which controlled the flow of respiratory media. Once the animalwas installed in the respirometer a flow of water was allowed to pass through it. Thiswater was collected under paraffin in 1 1. samples during the experimental periodsand was preserved for subsequent analysis.

Air was introduced into the respirometer only during lung ventilation. (The animalalways lifted the head prior to lung ventilation, and on this 'signal' air was allowed intothe respirometer). Air was drawn in by lowering the water level in an accessory con-tainer outside the respirometer but connected to it (Fig. 1). As the water level fell inthis outer container, so it fell in the respirometer, drawing in air from the perfusingair stream and allowing the animal to ventilate the lung. Immediately after thelung ventilation the original water level in the outside container was restored and theexpired air was then forced out of the respirometer. This air passed into the perfusingair stream and was collected in a large bottle to await analysis. All gas and water inter-phases, with the exception of that in the respirometer itself, were protected withparaffin. The water samples were analysed for oxygen content using the Winkler methodand for carbon dioxide content by the nomograph method of Dye (1951). The gassamples were analysed for both oxygen and carbon dioxide content using a Scholander\ ml. Gas Analyser (Scholander, 1947). All experiments were carried out in a constanttemperature room (24 ± 1 ° C), thus ensuring that gas and water samples were at thesame temperature.

It was not possible to acclimatize the animals in the respirometer for long periodsbecause continual manipulation by the operator was necessary to maintain the animalsin the chamber. In fact, they were to some degree pre-acclimatized to the tubes (whichwere often inhabited by them when left in the 'home' tanks). This suggests that theanimals were not seriously disturbed by their close confinement during the experimen-tal periods. Once installed and settled into the chamber the animals very rarelyshowed any activity not associated with air breathing.

Samples of pulmonary gas, and both inhalant and exhalant branchial water wereobtained from chronically implanted cannulae. The pulmonary cannula was insertedinto the anterior median sac of the lung, and the other cannulae into the buccal andopercular cavities as in McMahon (1969). Samples of 0-5-1-o ml. of pulmonary gaswere withdrawn into a syringe, the dead space of which was filled with a solution whichwas non-absorbent for gases. A volume of gas equivalent to the cannula volume waswithdrawn and rejected before each sample. Samples were taken at intervals through-a number of air-breathing cycles and stored immersed in cooled, non-absorbentsolution for periods of 2-4 hr. to await analysis. Tests indicated that no significant

4 B. R. M C M A H O N

change occurred in samples stored in this way. In some of the experiments the POa ofthe samples was taken on withdrawal and this provided a check on the subsequentanalysis.

Water samples were withdrawn from the buccal and opercular cavities to determinethe extent of the gas exchange ocurring over the gill surface. During slow branchialirrigation (less than i/min.) very small samples (o-2-O'3 ml.) were taken into a syringeattached to the cannula. The samples were taken very slowly to avoid drawing waterover the gills artificially, and were withdrawn at varying intervals after the lastbranchial respiratory movement. During faster branchial irrigation larger samples(0-5-1'5 c.c.) were withdrawn over a period covering several branchial respiratorymovements (0-5-1 -5 min.). In all cases a volume equivalent to the cannula volume wastaken and discarded before a sample was taken. The samples were analysed for oxygenand carbon dioxide tension using an 'Eschweiler' gas analyser. The electrodes werecalibrated with gas mixtures of known oxygen and carbon dioxide tension severaltimes during each experiment. The oxygen electrode system was very stable and gaverepeatable results at 1 % level of accuracy. The carbon dioxide electrode, however, hada slow response time at this temperature and was liable to drift from the calibrationsettings. These factors may have introduced a level of inaccuracy (< 5 %) into theresults.

RESULTS

In the first series of experiments the frequencies of branchial irrigation and lungventilation were monitored in animals before, during and after confinement in eitherair or water. Even in the resting animals the rates were very irregular and for this reasonthe rates were usually recorded for periods of at least 30 min. and the results expressedas an average over this period.

The graphs in Fig. 2 show the results of typical experiments. Fig. 2 A shows theeffect of protracted confinement under water. Branchial and aerial respiratory rateswere both low before confinement. A considerable increase in activity was seenimmediately following confinement as the animals struggled to reach the surface,and reliable estimates of the resting branchial rate could not be made. After30-60 min., however, the animals' activity decreased and long periods were spentat rest at the bottom of the chamber. During the rest of the submergence theanimals were quiet but initially showed a marked increase in the rate of branchialirrigatory movements. This increase continued for the first 3-5 hr. of confinementand the branchial respiratory rate reached levels of 25-30 beats/min. This high levelwas maintained throughout the rest of the submergence.

Both pressure recordings from the buccal and opercular cavities and direct observa-tion of the confined animals indicated that the amplitude of the branchial movementsincreased together with the frequency of respiration. The increase in amplitude of therecorded pressure waveforms was not constant but decreased almost to the restinglevel at times. As no change in the observed amplitude of the breathing movementswas seen at this time the decrease in the pressures recorded was apparently due eitherto a change of gill resistance or a failure of the mouth or opercular flaps to close at thecorrect phase of the cycle. Adult animals have been kept submerged for periods ofup to 24 hr. With the exception of one animal (in poor general health), which died after

Gaseous exchange efficiency in Protopterus aethiopicus 5

only 7 hr., all the animals survived the submergence without harm. It was noticeable,however, that after submergence for over 15-20 hr. the animals' respiratory move-ments became very forceful and irregular, and they began to show signs of loss ofequilibrium. At the onset of the latter symptoms the animals were allowed access to

air.

Air/water

B

30

20

10

Q »

Confined in moist air Air/water

Opercular

Lung

r ve

ntila

tio

Ope

rcul

a

30

20

10

0

(

~Air/water

-

-

3 2

Confined

/

/l 1 l

under

1

4

water

6

1 _—•

A i r / "water

a o

1

X1

60

40

20 ]T_c

i0 **>

(4

60 .1

I!DO

- 40

- 20

4Hours

10 11

Fig. 2. The effect of confinement in air (upper graph) and water (lower graph) on the aquatic andaerial respiratory frequencies. Dotted line, average rate of lung ventilation (breaths per hour)determined for the whole experimental period. Solid line, closed circles represent averagebranchial (opercular) respiratory rate (beats/min.) during a period of 1-3 min. or recording.

Immediately following the first lung ventilation the rate of branchial irrigatorymovements fell rapidly, usually reaching the pre-submergence level in 30-60 min. Therate of lung ventilation following the submergence period was considerably greaterthan had been seen before confinement, and this rate decreased more slowly to theresting level at a rate dependent on the severity of the previous confinement.

All the experiments quoted above were carried out on adult (over 200 g.) animals.Table 1 shows the results of preliminary experiments on smaller animals. LarvalProtopterus (3-4 weeks old) with functional external gills were not affected by con-finement under well-aerated water even for periods in excess of 14 days. No branchialrespiratory movements were seen in these animals before or during the submergence.

6 B. R. M C M A H O N

In juvenile animals the branchial hyperirrigation response develops gradually, withincreasing size. As the animals' size increases, however, the length of time the animalcan survive without lung ventilation decreases.

Table i. Viable submergence time for different ages of Protopterus

Stage

Larval

Youngjuvenile

JuvenilesAdults

Weight(g.)

o-i

i-7

SO

500 +

Maximum viablesubmergence time

At least 15 days

At least 6 days

One to 4 daysUp to 1 day

Average branchialrespiratory rate

EnforcedNormal submergence

0 0

1-5 Up to 10

1 Up to 241 Up to 24

Development

Ext. gills functionalMouth open, lungs functional?Ext. gills very much reducedInt. gills functionalBoth functionalBoth functional

Inwater

L.V. L.V.

Inair 4.

L.V. L.V. L.V.

Fig. 3. Pressures recorded from the buccal cavity before and during exposure to air. Uppertrace: before exposure to air. Branchial respiratory movements seen. Lower trace: after 1 hr.exposure to air. Note the suppression of all branchial movement, even that which normallyconcludes the air-breathing cycle. L.V., Air-breathing cycle (lung ventilation).

In all experiments where the animals were confined in moist air the branchialrespiratory rate was always low even in the pre-confinement period. Branchialrespiratory movements were usually only recorded when the animals rose to the surfaceto breathe air, or when they were otherwise active. One branchial movement has beenshown to be an integral part of the air-breathing cycle (McMahon, 1969), but after 1 hr.exposure to air no branchial irrigatory movements are seen, even this obligatoryflushing stage of the air-breathing cycle having been suppressed (Fig. 3). The animalswere usually restless when first exposed to air and no measurements of the breathingrates were made in the first hour of each exposure. The animals' activity decreasedafter this time and recordings showed that the lung ventilation rate had been markedlyincreased (average of ten experiments, 6 x ) despite the abundance of air. This increased

Gaseous exchange efficiency in Protopterus aethiopicus 7

rate was maintained throughout the air exposure, which was limited to 5 hr. or less, asit is well known that Protopterus can survive long periods of air exposure as long as itis kept moist. On termination of the air exposure a dramatic but transitory increase inthe branchial respiratory rate was observed as soon as the animal was able to submergethe mouth (Fig. 2B).

Evidence from the measurement of pulmonary gas concentration

Samples of pulmonary gas were removed from the lungs at intervals throughout anumber of natural and artificially prolonged submergences. Immediately following alung ventilation the oxygen concentration in the lung gas was high, generally overl5% (> 110mm. Hg POt), and the carbon dioxide concentration was low, rarely

20 fA breaths ° ° » \ Voluntary — * O 1 Prolonged°CO J b • CO[ b

15

OU

.,10

2

° ° » \ Voluntary — * O 1 Prolonged| ° C O , J submergence — • • CO,[ submergence

I

0 25 50 75 100 125 1SQTime (min.)

Fig. 4. Oxygen and carbon dioxide concentration in samples of pulmonary gas withdrawn fromthe lung of Protopterus during prolonged submergences. Air breaths are indicated by arrows.The dotted arrow indicates the air breath following a long voluntary submergence. The opencircles and squares indicate the concentrations in samples taken during and just after this volun-tary submergence. The closed circles and squares indicate samples taken during a submergencewhich was artificially prolonged by preventing the animal from reaching the surface. The lines(dotted, CO] concentration; solid, O» concentration) are drawn through the points from theartificially prolonged submergence.

more than i-5-2-5% (11-22 mm. Hg J°co,)- During the length of an average sub-mergence (20-25 min., at rest in the home aquarium) the oxygen concentration fellrapidly (Fig. 4) to 4-5% (30 mm. HgP02). If the submergence period was prolonged bydenying the animal access to the surface the oxygen concentration continued todecrease, but much more slowly, reaching a level of O'3-O'5% (3 mm. Hg POt after150 min.

The carbon dioxide concentration rose rapidly in the first 5-10 min. after lungventilation, reaching a level of 4-5 % (25-30 mm. Hg PCot)- Little or no further

8 B. R. MCMAHON

increase occurred, however, and pulmonary carbon dioxide concentration exceeded5 % in only one animal after a 150 min. submergence (Fig. 4). The original level of bothgases was always restored at the next lung ventilation after a natural submergence, buta second ventilation was often needed to restore the levels fully when the submergencehad been prolonged for more than one hour.

Evidence from the measurement of gas tensions in inhalant and exhalantbranchial water

Samples were removed from the buccal and opercular cavities by means of implantedcannulae. The water in the buccal cavity (JP/,O,) varied little from that in theambient water, but changes in the concentration of both oxygen and carbon dioxide

80 r

60 -

I 40o"

20 -

8

-

•

a

•

•

a •

a

V

•

D

•

a

•

D

taa

I

1

•a

•

•

a

•

_

•

••

- 15

810 I

8

- 5

1 2 3

Log BRR

Fig. 5. Oxygen uptake and carbon dioxide production in water passing over the gills plotted as afunction of the branchial respiratory rate (BRR). PI,O, — PI,O, (tension of oxygen in inhalantwater — tension of oxygen in the exhalant water) = oxygen uptake. -Pjt.o, —-P/.o (tension ofCOj in the exhalant water — tension of CO, in the inhalant water) = carbon dioxide production.Solid circles = Po,- Open squares = PCo,-

occurred in passage over the gills and were detected in the exhalant branchial water(P£)o2). These changes are plotted as a function of respiratory rate in Fig. 5. Itcan be seen that both oxygen and carbon dioxide are exchanged at this site, and thatthe rate of this exchange varies with the rate of branchial respiration. Unfortunately,no estimate of ventilation volume could be made.

Evidence from the respirometry experiments

The actual consumption of oxygen and production of carbon dioxide were measuredin the specially designed respirometer illustrated in Fig. 1. Because of the differencein size of the experimental animals (150-600 g.), and perhaps because of differences in

Gaseous exchange efficiency in Protopterus aethiopicus 9

physiological state, some variation of the individual rates of gas exchange was seen.Mean figures for oxygen and carbon dioxide exchange have been calculated from thedata obtained from nine experiments on four different animals. These figures arepresented in histogram form in Fig. 6.

7#

u

ml.

180-

60-

40-

Air/water

CO2

No air Air/water

. 80-u

7 6 0 -oi

E 40-

20-

Air/water No air Air/water

Fig. 6. Average oxygen consumption and carbon dioxide production occurring via aerial andaquatic routes in normal animals, animals confined under water, and animals recovering fromconfinement under water. Exchange rates are expressed in cubic millimetres per kilogram bodyweight per hour (ml./kg.'Vhr."1). The figures are averaged from a number of experiments. Therange of variation is given in the text. The upper line indicates the total exchange under thestipulated conditions. The black area indicates the proportion of total exchange via the aquaticroute, and the clear area the proportion passing via the aerial route. • , Exchange at gills;• , exchange in lungs.

The first column shows the results obtained from animals free to breathe air orwater. The average figure for total oxygen consumption was 62-5 c.c. oxygen/kg./hr.(range of variation 27-8-86-5 ml./kg.~1/hr.~1). The average figure for carbon dioxideproduction was 47*4 c.c. carbon dioxide/kg./hr. (range of variation = 19-9—56-3 ml./kg.~1/hr.~1 CO2). The calculated total RQ for the averaged results is 0-755,a reasonablefigure for a carnivorous animal. The results show that the exchange ratio was verydifferent at the two respiratory rates. In every experiment the oxygen consumptionover the pulmonary surface was much greater than over the gills. Average aerialoxygen consumption was 91-7% of the total (range 86-5-94-0%). It is evident thatunder these conditions the lung is the principal site for oxygen exchange. Examinationof the figures, however, showed that only 32-5 % of the carbon dioxide is excreted viathe aerial route, the remainder passing via the aquatic route.

In five experiments the animals were denied access to the surface for periods of

io B. R. M C M A H O N

about 60 min. during the course of the experiment. Branchial respiratory rate wasincreased by up to four times and increased gaseous exchange was seen across the gillsurface (Fig. 6, second column). Carbon dioxide production via the gills was increasedso that up to 100% (average 85 %) of the pre-confinement production now passed bythis route. The aquatic oxygen consumption, however, though considerably increased,could provide only 17% (11 •8-30*0%) of the animals' total pre-confinement oxygenrequirements.

Rates of gas exchange were also measured for 1 hr. after the end of the confinement.A very marked increase in the total oxygen consumption was seen (49*8-72*6 % abovethe pre-confinement level.) The rate of lung ventilation also increased and all theadditional oxygen was consumed by this route. The increased lung ventilation alsoaffected the carbon dioxide production ratio, as on the average 57 % of the total carbondioxide produced was now eliminated via the lungs, while the amount passing via thegills was correspondingly reduced. Before evaluating the results of these experimentsit must be mentioned that the method of estimation of carbon dioxide concentrationin the water samples was accurate to 5 % only. This level of inaccuracy, while high,was not sufficient to influence the conclusions drawn from these results.

DISCUSSION

The experimental evidence presented here demonstrates clearly that gaseous ex-change at the gill surface accounts for very little of the total oxygen uptake in theadult Protopterus. This is in agreement with the work of Lenfant & Johansen (1968).The rate of branchial respiration is very low in animals at rest and the percentageutilization{P/Ol — PEfOl)/P/>Ol} xioo is very low when compared with the figurespublished for other fishes in Table 2. Percentage utilization seen in Neoceratodus

Table 2. Percentage utilization of oxygen at the gills of various fishes

DogfishTrigger fishCarpTroutEelNeoceratodusLepidosirenProtopterusProtopterus

BRR/min.

4240-60—-17-19—

3°var.o-51 0

Ventil.vol. /min.

248 ±42200-330300

13364

315—

——

Pi,o2

155151

Air sat.Air sat.131—140140

Percentageutilization

4858-8149up to 80%6836-3

0-404613

Source

Piiper & Schumann (1967)Hughes, G. M. (1967)Saunders, R. L. (1962)Van Dam (1938)Van Dam (1938)Lenfant et al. (1966)Johansen & Lenfant (1967)Present survey (1969)Present survey (1969)

compares with that seen in the other fishes but that of Protopterus is only 50 % efficientat very low irrigation rates. If the animal increases the rate of branchial irrigation, asseen in response to protracted submersion, the percentage utilization falls to a verylow level. Oxygen consumption thus falls by over 80% when adult Protopterus areconfined underwater for protracted periods. The oxygen consumption also falls inNeoceratodus similarly confined but by only 20-25% (Grigg, 1965 c). Direct evidenceas to the inefficiency of the gills in oxygen uptake is given by the respirometry experi-ments, where increases of branchial respiratory rate of up to four times in confined

Gaseous exchange efficiency in Protopterus aethiopicus 11

animals could provide only 10-30% of the animals' oxygen requirements. The animalswere forced into oxygen debt when prevented from breathing air, and this oxygen debtwas paid off by the marked hyperventilation seen once access to air was possible. Theincrease in aquatic oxygen uptake during long submergence is, however, of value inallowing even adult animals to remain submerged for relatively long periods.

Carbon dioxide excretion occurs over both lung and gill surfaces, with perhaps themajor part passing aquatically in the resting animal. The percentage of carbon dioxidepassing via either route can be increased by hyperventilation, but, whereas moderatelyincreased branchial respiration was able to remove all the carbon dioxide during aqua-tic confinement, a very marked increase in aerial respiration was needed to remove thecarbon dioxide accumulated during exposure to air.

If the data obtained from direct measurement of the respiratory media are plottedin the form of Oa/COa diagrams, as first used by Willmer (1934) and more recentlyby Rahn & Fenn (1955), confirmation of the results expressed above can be seen.Figures 7A, B show O2/CO2 plots for pulmonary air and expired water respectively.In Fig. 7B a regression line has been calculated from the plotted data to show therelationship between aquatic oxygen and carbon dioxide exchange. Theoretical gasexchange lines where R = unity are also drawn for aerial and aquatic routes. Theslope of the plotted regression line (R = 4*9) is considerably greater than R = 1 forwater, and this figure is in good agreement with the exchange ratio of 5-4 calculatedfrom the respirometry data. A high aquatic gas exchange ratio indicates that much morecarbon dioxide than oxygen is being exchanged across the gill surface. This imbalancecould be explained by a prior oxygenation of the blood reducing the amount of possibleoxygen uptake, or could be due to a thickening of the gill epithelium—such as is seenin Lepidosiren (Fullarton, 1931)—which would reduce the possible exchange for theless soluble oxygen while having much less effect on carbon dioxide. As very littlehas yet been published on the degree of separation occurring in the partially dividedheart of Protopterus, the extent of the former is difficult to estimate. The lengtheningof the diffusion path would be of adaptive benefit in an animal where the blood passingthrough the gills may have a higher POt than that of the ambient water. Under theseconditions a short diffusion path would result in the loss of oxygen to the ambientwater and a consequent loss of efficiency of the lung.

When the pulmonary gas data are plotted on an O2/CO2 diagram all the points areseen to lie beneath the R = 1 line for air, indicating that more oxygen uptake thancarbon dioxide elimination takes place in the lung. This conclusion is in agreementwith the exchange ratio deduced from respirometry data (R = 0-27). Above carbondioxide tensions of 20-25 mm. Hg the slope of the relationship approximates to zero,indicating that above this level no further carbon dioxide is eliminated into the lung,though oxygen is still being removed. The time course for gas exchange in the lung(Fig. 4) shows that this does, in fact, occur. This indicates that the level of carbondioxide in the lung is very low immediately following lung ventilation, but that thetension rises very quickly as carbon dioxide diffuses into the lung from the pulmonaryblood stream. Equilibration quickly occurs between the blood and gas and theyremain in equilibrium while any further carbon dioxide produced by the animal iseliminated through the gills. This explanation presupposes a very much higher tensionof carbon dioxide in the circulating blood than has been shown to occur in the blood

12 B. R. M C M A H O N

of the fishes studied to date (3*3 mm. Hg PCOi! in dogfish venous blood (Piiper &Schumann, 1967); 5-7 in trout ventral aortic blood (Stevens & Randall, 1967); 7-7 mm.Hg in the venous blood of Neoceratodus (Lenfant et al. 1966). A high level of circula-ting carbon dioxide might, however, be expected in the blood of an obligatory air-breathing form. In fact, Lenfant & Johansen (1968) show levels of up to 30 mm. HgP c 0 , in the dorsal arterial blood of this animal.

60

40

ou

20

Lung air

R=l(air)

R=l(water)

40 80 120

60

40

20

Expired water

-

R=l (water)— _

1 1 1

\R=l(air)Nv 6

\

1 1 1 1

20 40 60 80 100 120 140

Fig. 7.02/CO2 diagrams plotted for expired branchial water and pulmonary gas in Protopterus. Thelower figure shows the tensions in expired branchial water. The regression line was calculatedby the method of least squares. The upper graph contains evidence from three separate experi-ments, each plotted with different symbols. Theoretical R = i lines are shown for both air andwater for comparison. (R = gas exchange ratio).

The occurrence of a high level of carbon dioxide in the blood of Protopterus isof considerable interest. Rahn (1966) considers that the lungs of the emergent tetra-pods, though efficient in oxygen exchange, were much less efficient carbon dioxideexchange mechanisms than were the gills of the ancestral aquatic forms. This, in fact,is the case in Protopterus (B. R. McMahon, in preparation). Rahn postulates that acutaneous carbon dioxide exchange route was needed in the first terrestrial animalsto complement the lung exchange and thus prevent dangerously increased carbondioxide levels in the blood. He considers that the next step would have been theevolution of a tolerance of high carbon dioxide which would have rendered the lungtidal ventilation mechanism sufficient for all gas exchange, and would allow thecutaneous exchanger with its additional problems of water loss to be abandoned.

Gaseous exchange efficiency in Protopterus aethiopicus 13

The presence of high circulating carbon dioxide levels in the aquatic Protopterus,however, indicates that the early air-breathing fishes, including the Rhipidistia, mayhave already evolved a degree of tolerance of high carbon dioxide levels, partly due tothe development of an aerial respiratory mechanism but mostly in response to thepresence of high carbon dioxide levels frequently found in the environment. In thiscase the ancestral tetrapods may well have been pre-adapted to the terrestrial habitatin this respect and the evolution of an intermediate cutaneous carbon dioxide exchangermay not have been essential.

No measurement has been made of the respiratory exchange occurring across theskin of Protopterus. Though the importance of cutaneous carbon dioxide exchange hasbeen demonstrated in Lepidosiren (Cunningham, 1934), the skin of the adult Protop-terus is neither particularly thin nor particularly vascular and would not appear to bean efficient exchange surface. The proven efficiency of the fish gill in gaseous exchangeindicates that the skin of Protopterus is unlikely to be important in this role in the sub-merged animal, though it may be important during aestivation when the gills are col-lapsed in air (Lenfant & Johansen, 1968).

It has been demonstrated that adult Protopterus aethiopicus obtain 90% of theiroxygen consumption from the aerial exchange occurring in the lungs, even when theanimals are immersed in well-aerated water. If the animals are prevented from ventilat-ing the lungs for /ong periods, branchial hyperirrigation is seen. This response cannotprovide the whole of the animals' oxygen requirement, but the additional oxygenconsumption, though small, is of importance in prolonging the possible submergencetime. The results obtained by Jesse et al. (1968) for either P. aethiopicus or P. dolloi(not specified) would suggest that the gills were of greater importance than is indicatedhere. These workers used juvenile specimens, however, in which the degree ofdependence on aerial respiration is less well developed (B. R. McMahon, in prepa-ration).

Carbon dioxide excretion can occur via either aquatic or aerial routes, and althoughthe major part normally passes over the gills the fraction passing aerially can be in-creased by hyperventilation. As the animal can utilize aerial oxygen and is tolerantof high external carbon dioxide concentrations (B. R. McMahon in preparation), itis extremely well suited to its periodically hypoxic and hypercarbic environment.Protopterus is thus more similar to Lepidosiren than to Neoceratodus, both in habitat andin the degree of dependence on aerial respiration. If we imagine the rhipidistianfishes as having been similarly adapted to their rather similar environment, then theywere eminently pre-adapted to colonize the terrestrial habitat.

SUMMARY

1. The efficiency of gas exchange over the lung and gill surfaces of Protopterus hasbeen investigated.

2. Animals confined in water or in air showed an increased respiratory frequencyin the remaining medium, indicating that both routes were important in the totalgas exchange.

3. Direct measurement of the oxygen and carbon dioxide tensions of pulmonary airand inspired and expired branchial water showed gas exchange ratios (R) of 0-2 for

14 B. R. M C M A H O N

the lung and 5-0 for the gills approximately, demonstrating that more oxygen wasconsumed via the lungs and more carbon dioxide excreted via the gills.

4. Oxygen consumption and carbon dioxide production were measured directly ina respirometer in which respiratory air and water streams could be kept separateexcept during lung ventilation. At least 90% of the animals' oxygen consumptionoccurred in the lung, while 60% of the carbon dioxide excreted passed via theaquatic route.

5. The results are discussed with reference to the animals' adaptation to its environ-ment and with reference to the evolution of the terrestrial vertebrates.

I am greatly indebted to Professor G. M. Hughes, Department of Zoology,University of Bristol, in whose laboratory and under whose supervision this workwas carried out, and to the Science Research Council who provided financialsupport.

REFERENCES

BERG, T. & STEEN, J. B. (1965). Physiological mechanisms for aerial respiration of the Eel. Comp.Biochem. Physiol. 15, 469-84.

CARTER, G. S. & BEADLE, L. C. (1930). Notes on the habitat and development of Lepidosiren paradoxa.J. Linn. Soc. (Zool.) 37, 197-203.

CUNNINGHAM, J. T. (1934). Experiments on the interchange of oxygen and carbon dioxide between theskin of Lepidosiren and the surrounding water, and the probable emission of oxygen by the male Sym-branchus. Proc. zool. Soc. Lond. 102, 875-87.

DEAN, B. (1906). Notes on the living specimens of the Australian lungfish Ceratodus forsteri in theZoological Society's collection. Proc. zool. Soc. Lond. 74, 387-436.

DYE, J. F. (195 I) . Calculation of the effect of temperature on pH, free CO2, and the three forms ofalkalinity. J. Am. Wat. Wks Ass. 44, 356-72.

FULLARTON, M. H. (1931). Notes on the respiration of Lepidosiren. Proc. zool. Soc. Lond. 99, 1301-6.GRIGG, C. (1965 a). Studies of the Queensland lungfish Neoceratodus forsteri (K). I. Anatomy, histology

and functioning of the lung. Aust. J. Zool. 13, 243-53.GRIGG, C. (19656). Studies of the Queensland lungfish Neoceratodus forsteri (Krefft). II. Thermal

acclimation. Aust. J. Zool. 13, 407-11.GRIGG, C. (1965 c). Studies of the Queensland lungfish Neoceratodus forsteri (K). III. Aerial respiration

in relation to habits. Aust. J. Zool. 13, 413-21.HUGHES, G. M. (1967). Experiments on the respiration of the Trigger fish (Batistes capriscus). Experientia

23, 1077.JESSE, J., SHUB, C. & FISHMAN, A. P. (1968). Lung and gill ventilation of the African lungfish. Respir.

Physiol. 3, 267-287.JOHANSEN, K. & LENFANT, C. (1967). Respiratory function in the South American lungfish Lepidosiren

paradoxa (F). J. exp. Biol. 46, 205-18.JOHANSEN, K. & LENFANT, C. (1968). Respiration in the African lungfish Protopterus aethiopicus. II.

Control of breathing. J. exp. Biol. 49, 453-68.KERR, J, G. (1897). Habits of Lepidosiren in the dry season. Proc. zool. Soc. Lond. 1897.KERR, J. G. (1898). Habits and development of Lepidosiren. Proc. zool. Soc. Lond. 1898.LENFANT, C , JOHANSEN, K. & GRIGG, C. (1966). Respiratory properties of blood and the pattern of gas

exchange in the lungfish Neoceratodus forsteri (K). Respir. Physiol. 2, 1-21.LENFANT, C. & JOHANSEN, K. (1968). Respiration in the African lungfish. I. Respiratory properties of

blood and normal patterns of breathing and gas exchange. J. exp. Biol. 49, 437-52.LONGMAN, F. L. S. (1928). Notes on Epiceratodus. Mem. Qd Mus. 1928.MCMAHON, B. R (1969). A functional analysis of the aquatic and aerial respiratory physiology of an

African lungfish Protopterus aethiopicus with reference to the evolution of vertebrate lung ventilationmechanisms. J. exp. Biol. 51, 407-30.

PIIPER, J. & SCHUMANN, N. (1967). Efficiency of O, exchange in the gills of the Dogfish Scyliorhinusstellaris. Respir. Physiol. 2, 135-48.

RAHN, H. & FENN, W. O. (1955). A Graphical Analysis of Respiratory Gas Exchange. The O2/CO2Diagram. The American Physiological Society, Washington, D.C.

RAHN, H. (1966). Aquatic gas exchange theory. Respir. Physiol. 1, 1-12.

Gaseous exchange efficiency in Protopterus aethiopicus 15SAUNDERS, R. L. (1962). The irrigation of the gills in fishes. II. Efficiency of oxygen uptake in relation

to respiratory flow, activity and concentrations of oxygen and carbon dioxide. Can. J. Zool. 40,817-62.

SAWAYA, P. (1946). Sobre a biologia de alguns peixes de respiracao aerea. (L. paradoxa (Fitz) €Arapaimagigas (Cuvier)). Bolm Fac. Filos. CiSnc. Letr. Univ. S Paulo 11, 255-86 (quoted in Johansen& Lenfant, 1967).

SCHOLANDER, P. F. (1947). Analyser for accurate estimation of respiratory gases in one half cubiccentimetre samples. J. biol. Chem. 167, 1-17.

SPENCER, W. BALDWIN (1891) Notes on the habits of Neoceratodus forsteri. Proc. R. Soc. Viet. 4.STEVENS, E. DON & RANDALL, D. J. (1967). Changes in blood pressure, heart rate and breathing rate

during moderate swimming activity in rainbow trout. J. exp. Biol. 46, 307-16.VAN DAM, L. (1938). On the utilisation of oxygen and regulation of breathing in some aquatic animals.

Doctoral dissertation. University of Groningen.WILLMER, E. N. (1934). Some observations on the respiration of tropical fresh water fishes. J. exp. Biol.

11, 281-306.