Respiration in the African Lungfish Protopterus Aethiopicus: I. Respiratory Properties of Blood and Normal Patterns of Breathing and Gas Exchange^*

Members of the three existing genera of lungfishes represent different stages in the transition from aquatic to aerial breathing. Neoceratodus, the Australian lungfish, is primarily aquatic and utilizes gill breathing, while its lung offers an accessory means for O₂ absorption (Lenfant, Johansen & Grigg, 1966; Johansen, Lenfant & Grigg, 1967). Conversely, the South American lungfish, Lepidosiren paradoxa, is a typical air breather and the much reduced gills are of little or no importance for O₂ absorption but may have some functional significance for elimination of CO₂ (Johansen & Lenfant, 1967). The African lungfish, Protopterus, resemble L. paradoxa in being primarily an air breather. The purpose of the present paper is to evaluate the status of P. aethiopicus in the transition from aquatic to aerial breathing by analysis of the respiratory properties of blood and of the normal pattern of breathing and gas exchange.

Ten large specimens of Protopterus were transported by aircraft from Lake Victoria, Uganda, East Africa to Seattle, Washington. The fishes arrived in good condition and were kept in aquaria with water temperatures of about 20°C. for several days before the experiments were started. In addition, experiments were performed in the Department of Physiology at the Makerere University College in Kampala, Uganda, on material freshly caught from Lake Victoria. ^‡ This work not only permitted verification of the data obtained in the U.S.A., but made it possible to study additional problems precluded in the Seattle experiments because of the limited number of fish. All the fishes were fasting during the periods of study and observation.

Blood gases and pH were measured by means of a Beckman 160 gas analyser using an oxygen macro-electrode and a Severinghaus-type CO₂ electrode mounted in special micro-cuvettes. Blood pH was measured on a Beckman micro-assembly. Gas contents were measured using gas chromatography according to the method described by Lenfant & Aucutt (1967). The in vitro work on the respiratory properties of blood was doné according to descriptions by Lenfant & Johansen (1965).

The methods used to measure gas exchange involved a closed temperature-controlled chamber. This chamber, partially filled with water, was used to measure gas exchange in both the water and air phase. Fish placed in the chamber continued their normal aerial and branchial breathing pattern. When without water, the chamber was used to measure gas exchange during air exposure.

In preparation for an experiment the fish was anaesthetized by immersion in MS 222 (Tricaine methane sulphonate, Sandoz). An incision was made along the ventral midline slightly posterior to the heart and by careful dissection the large vena cava, the common pulmonary vein, the left pulmonary artery and the coeliac artery were all dissected free. All four vessels were cannulated in the direction of the heart using polyethylene catheters filled with heparinized saline. The catheters entered the main vessels through small side branches or alternatively through holes made in the vessel walls. All cannulations were non-obstructive, allowing continued free passage of blood in the vessels to be recorded or sampled from. Afferent branchial blood samples and blood pressures were obtained by cannulating the 1st or 2nd afferent branchial artery (aortic arches 3 or 4). It is recalled that in Protopterus these are direct thoroughfare arteries having no connexion with respiratory exchange circulation. Following cannulation all catheters were anchored in place and the incision was carefully closed. The indwelling catheters were kept in place for variable periods of time extending to more than 10 days. Most fishes were studied over periods lasting several days.

A catheter was also inserted behind an operculum close to the posterior gill arches to permit sampling of expired water, and another catheter was inserted via the mouth through the pneumatic sphincter in the pharyngeal floor into the anterior portion of the lungs to allow sampling of lung gas. Both of these catheters were carefully anchored by means of sutures, taking care not to cause undue mechanical interference with normal branchial breathing movements and the passage of air into and out of the lung.

Following the surgical procedures the fish were permitted to recover from anaesthesia in shallow, well-oxygenated water. This recovery was usually quick and was often aided by artificial inflations via the lung catheter. The fish were allowed to rest in water for several hours before any sampling or recording was done.

Blood for in vitro work was obtained early after recovery or from special fish used as blood donors only.

The following mean values were determined for three fish used as blood donors: Haematocrit, 25% Hb concentration, 6·2 g%; mean corpuscular haemoglobin concentration, 24·8%. The mean O₂ capacity was 6·8 vol%. Figure 1 shows a set of oxyhaemoglobin dissociation curves. The curves are typically sigmoid and attest to a relatively high affinity of haemoglobin for O₂. Figure 2 A compares the oxyhaemoglobin dissociation in Protopterus with earlier published curves for Neoceratodus and Lepidosiren. At similar the O₂ affinity is seen to be slightly higher for Protopterus and Lepidosiren than for Neoceratodus. Figure 2B allows comparison with earlier published curves for Protopterus. The curve reported by Fish (1956) shows a higher affinity at a similar ⁠. Data from Swan & Hall (1966) disclose a much lower affinity than we determined. Our curve at 40 mm. Hg is extrapolated, since the highest we used was 27 mm. Hg. Figure 3 compares the Bohr effect of Protopterus blood with our earlier published data from Neoceratodus and Lepidosiren and with data from Swan & Hall (1966) on Protopterus, The present results show a much higher Bohr effect than reported by these authors. Figure 4 shows the influence of temperature on the oxyhaemoglobin affinity in Protopterus and Neoceratodus in comparison with human blood. Protopterus has a much larger temperature shift than Neoceratodus. The annual range of water temperature in Lake Victoria where our animals were obtained is very small (25–28°C).

In Fig. 5 the CO₂ dissociation curve of Protopterus is compared with CO₂ dissociation curves for Neoceratodus and Lepidosiren. The CO₂ combining power of Protopterus blood is considerably higher than for the other lungfishes. Protopterus, moreover, showed no Haldane effect (Fig. 5), whereas the other species, especially Neoceratodus, did. In Fig. 6 the buffering capacities of the blood in the three species are compared. The buffering capacity of Protopterus blood exceeds that of the other lungfishes.

When Protopterus were observed in aquaria it became apparent that the animals normally utilized both branchial and pulmonary breathing. Both means of breathing, however, were quite irregular, although steady rhythms sometimes prevailed for long periods when the fish were kept in well-aerated water in which they were free to swim and surface for air. The activities of the two modes of breathing were clearly interrelated. In particular, the branchial breathing rate increased prior to each air-breath (Johansen & Lenfant, 1968).

Table 1 lists branchial breathing rates recorded for 5 min. intervals several times in each of the three specimens. The of external water, exhaled water and the extraction of O₂ from the branchial water current are also tabulated. Ideally the percentage extraction of O₂ from the water should have been correlated with actual ventilation of water, but such ventilation measurements were not technically feasible. Four measurements on two different specimens showed CO₂ tensions in exhaled water ranging from 5·4 to 10·6 mm. Hg, when the fish was in aerated water.

Figure 7 shows the branchial breathing rate for one lungfish plotted against time during continuous recording for 25 min. A great variability is evident. Exhaled water samples taken at 5 min. intervals for calculation of percentage extraction of O₂ from the water also showed a large variability.

Table 2 shows average intervals between air breaths recorded several times for each individual specimen listed. The table includes fish in which a fairly regular air-breathing rhythm prevailed. Air exposure of the fish by slowly draining the tank water elicited a drastic increase in the rate of air breathing. This increase could clearly be dissociated from excitement or arousal as the fish rarely responded by violent behaviour to air exposure. When air-exposed the fish usually started slow searching movements and after variable times attempted to dig with their noses, seemingly seeking to bury in the substratum. Fish air-exposed on a muddy surface where burrowing was possible did actually bury themselves, slowly but methodically. Not only did air exposure elicit an increased rate of air-breathing, it also changed the pattern of breathing into groups of several breaths at shortened intervals.

The relative contribution of gills and lungs in O₂ absorption and CO₂ elimination are indicated in Table 3. Gas exchange ratios have been calculated for aquatic versus aerial gas exchange as well as for over-all gas exchange when the fish is in water. The The lower horizontal column lists O₂ absorption, CO₂ production and over-all gasexchange ratios during air exposure. The data disclose that aquatic absorption of O₂ constitutes only between 10 and 12% of the entire O₂ uptake. Conversely, aquatic gas exchange is on the average 2·5 times more effective than the pulmonary gas exchange in eliminating CO₂ when the fish is in water. The over-all gas exchange ratio is quite variable. All data tabulated on gas exchange represent average values from readings every hour for 6–10 hr. The reliability of the absolute values suffers from the inevitable exchange between the gas phase and the water phase in the metabolism chambers. However, this exchange would tend to minimize the role of aquatic exchange for CO₂ elimination and the aerial exchange for O₂ absorption. It is notable that air exposure did not promptly reduce the over-all oxygen uptake. The apparent reduction in the gas-exchange ratio represented the only real change during the relatively short periods of observation.

Figure 8 illustrates the time course of changes in the partial pressures of pulmonary gases in the interval between air breaths. The time course of the gas exchange ratio is also plotted. This specimen and other fish used for these measurements were free to swim in large tanks and had not been subjected to any surgical treatment. The expired gas was collected by trapping the released gas into water-filled funnels suspended above the heads of the fishes.

Figure 9 shows the relationship between oxygen uptake and the gas composition in the ambient environment. In this typical experiment the fish is seen to maintain his down to a of approximately 85 mm. Hg with a corresponding increase in the metabolism chamber of about 35 mm. Hg. A further reduction in was accompanied by an abrupt fall in ⁠. The experimental arrangement did not make it possible to decide whether the or the or both are instrumental in bringing about this change. The experiments underlying Fig. 9 lasted for variable periods up to 8 hr. All fish survived the experiments. Consistently, prolonged air exposure was accompanied by a progressive vasodilation of the skin.

Table 4 shows the blood gas tensions in undisturbed fish resting in well aerated water as well as during air exposure. Figure 10A, B emphasizes the difference in blood gas tensions borne out in the table and shows the time course of blood gas changes occurring between consecutive breaths. Note that blood from the dorsal aorta shows a consistently higher oxygen tension than pulmonary arterial blood. Expressed in percentage oxygen saturation (Fig. 10B), the pulmonary arterial blood is seen to be about 65–70% saturated against more than 75% average saturation in systemic arterial blood from the dorsal aorta. Finally, pulmonary venous blood is about 85% saturated with oxygen. To the right in Fig. 10A are plotted the blood oxygen tensions following air exposure. The arrows indicate the number of air breaths and draw attention to a conspicuous stimulation of air breathing associated with air exposure. All blood gas values are increased and the gradient between systemic arterial blood and pulmonary arterial blood has become larger.

The lungfishes occupy habitats similar to those of many other air-breathing fishes, yet the O₂ capacity of their blood seems to be generally much lower. Thus, the blood of Protopterus compares well with values reported for the South American Lepidosiren and the Australian Neoceratodus (Johansen & Lenfant, 1967; Lenfant et al. 1966). In contrast, three South American air-breathing fishes—the electric eel, Electrophorus electricus (Johansen, Lenfant, Schmidt-Nielsen & Petersen, 1968); the hassa, Hoplo-sternum littorale (Willmer, 1934) and Symbranchus marmoratus (Johansen, 1966)— showed average O₂ capacities of 12·3, 18·1 and 14·7 vol.% respectively. Since tropical air-breathing fishes largely occupy similar habitats, the marked difference in O₂ capacity may be related to the efficiency in O₂ transport. Many air-breathing fishes have a permanently low arterial O₂ saturation because the blood draining the aerial gas-exchange organ is mixed with the general systemic venous blood before it perfuses the arterial circulation. In the lungfishes such extensive shunting of oxygenated blood to the venous circulation is prevented by a separate pulmonary vascular circuit and a partial division of the heart and its outflow channels (Johansen, Lenfant & Hanson, 1968).

The oxyhaemoglobin dissociation curves (Figs. 1, 2) conform in shape to those reported for lungfishes earlier but the actual affinity for O₂ shows definite variations and was higher in our experiments. Fish (1956), working on haemoglobin solutions, which in general have higher affinity than whole blood, reported a P₅₀ value of about 11 mm. Hg at 6 mm. Hg for P. aethiopicus. Swan & Hall (1966), working with whole blood from Protopterus, found an O₂ affinity much lower than our experiments revealed (Fig. 2B).

It has been suggested that the O₂ affinity of haemoglobin generally decreases with increasing dependence on aerial gas exchange (McCutcheon & Hall, 1937). This tendency should reflect an adjustment to the higher O₂ availability in air than water. More recent data from transitional forms among amphibians substantiate this hypothesis (Lenfant & Johansen, 1967). In regard to fishes, most workers endorse the early suggestion by Krogh & Leitch (1919) that higher O₂ affinity of haemoglobin correlates with the ability of a species to survive in an O₂ poor medium. Among air-breathing fishes, those showing accessory air breathing with a dominance of aquatic gas exchange may show adaptation to conditions in the water whereas obligatory air-breathing fishes, like Lepidosiren and Protopterus, should show adjustments towards atmospheric conditions.

Willmer (1934) offered convincing data demonstrating that blood from freshwater fish inhabiting well-aerated water of low CO₂ content displays a marked sensitivity to CO₂ changes (Bohr and Root’s shifts), whereas blood from fish in stagnant swamps and muddy creeks with a high CO₂ content was practically insensitive to CO₂. Later workers, notably Carter (1951, 1962) and Fish (1956), used such data to generalize that a transition from aquatic to aerial gas exchange is accompanied by a reduction in the CO₂ sensitivity of the blood.

Carter’s generalization on the evolution of the Bohr shift receives support from a comparison of blood from Neoceratodus (Lenfant et al. 1966) and Lepidosiren (Johansen & Lenfant, 1967). Lepidosiren, which is decidedly more of an air breather, shows a much smaller Bohr shift than Neoceratodus. An extension of this comparison to Protopterus, based on the present results or those of Swan & Hall (1966), does not support such a trend. A comparison of Bohr shifts in selected amphibians showing increasing dependence on air breathing similarly does not provide support for Carter’s hypothesis (Lenfant & Johansen, 1967). The latter comparison, however, is complicated by neoteny of the species of urodeles studied. Further work is obviously desirable to resolve the role of adaptive changes in the CO₂ sensitivity of the blood in the evolution of air breathing.

The effect of a temperature change on the O₂ affinity of haemoglobin is different in blood from the Australian and African lungfishes. Neoceratodus, which occupies waters that may show large annual and diurnal changes in temperature (up to 20° C.), has blood that is notably insensitive to temperature changes. Conversely, Protopterus, with very slight temperature variations in its habitat, possesses blood which is much more sensitive to temperature changes.

The CO₂ combining power and buffering capacity of blood from the lungfishes and other forms in the transition to air breathing reveals some consistent trends. Figure 5 shows an increasing CO₂ combining power from Neoceratodus through Lepidosiren to Protopterus. This tendency substantiates predictions made by Lenfant et al. (1966) that a greater dependence on air breathing is correlated with an increasing CO₂ combining power.

The role of the gills and skin in O₂ uptake in Protopterus was found to be about 10% of the total uptake when the fish rests in aerated water (Table 3). In Lepidosiren the importance of the gills for O₂ absorption is even less and reported to be only 2% of the total O₂ uptake (Sawaya, 1946). The relatively high extraction of O₂ from the water found presently (Table 1) was of little importance to oxygen uptake because of extremely low values of ventilation. On the other hand, aquatic exchange by gills and skin was 2·5 times as efficient as the pulmonary exchange for CO₂ elimination. The role of the skin could be important in this comparison since Cunningham (1934) reports that the skin in Lepidosiren has a gas-exchange ratio of more than 10 when the fish is in water. The progressive vasodilation of the skin in Protopterus during air exposure attests to an active role in gas exchange. The present results for Protopterus are in accord with the tendency for animals having bimodal, aquatic and aerial, gas exchange to show a low gas-exchange ratio for the air-breathing organ, and a gas-exchange ratio greater than unity for the water-breathing organ (Lenfant & Johansen, 1967).

Figure 9 demonstrates that Protopterus while in air retains respiratory independence down to environmental O₂ tensions of about 85 mm. Hg correlated with an external of 30–35mm-Hg. Most aquatic vertebrates show critical O₂ tensions as low as 30 mm. Hg. Hall (1929) compared factors limiting oxygen uptake in active and sluggish species of fishes. His data suggest that active fishes have a greater tolerance to variations in external oxygen tension. Similarly, fishes with a higher haemoglobin content were more capable of maintaining a steady oxygen uptake in spite of reduced external oxygen availability. If Protopterus was primarily a water breather, Hall’s generalizations would be applicable to explain the narrow range of O₂ independence, but being primarily an air breather, Protopterus should be more tolerant to external changes than water breathers. The profound metabolic adjustments occurring during fasting and aestivation (Smith, 1935) may be related to the unusual metabolic responses of Protopterus to changes in ambient gas composition.

The nomogram (Fig. 11) provides a basis for discussing differences in gas exchange between Neoceratodus, in which the lung functions as an auxiliary gas exchanger and Protopterus which depends predominantly on pulmonary breathing. When resting in well-aerated water Neoceratodus employs air breathing very infrequently. During such conditions the lung is of no consequence to gas exchange, and similar blood gas values prevail in systemic arterial, pulmonary arterial and pulmonary venous blood. Efficient branchial gas exchange maintains the arterial blood more than 90% oxygen saturated and arterial values are extremely low, a character shared with typical aquatic breathers. Protopterus, on the other hand, practises active, frequent air breathing and the lungs play a major role in gas exchange. The importance of the lungs is apparent in the differences in oxygen saturation as well as values of pulmonary arterial and pulmonary venous blood. The intermediate saturation value of systemic arterial blood sampled from the dorsal aorta testifies that pulmonary venous blood is channeled selectively into the cardiac outflow channels giving rise to the systemic circulation. The emphasis on air breathing in Protopterus is also borne out by the much higher blood values, being on the average more than 20 mm. Hg above the values in Neoceratodus.

The limitations of pulmonary air breathing in the bimodal gas exchange of lungfishes were tested by exposing the fish to the air and thus interrupting aquatic gas exchange altogether. Unlike Neoceratodus, Protopterus never reacted violently or became restless and excited when removed from water. Air-exposure accelerated air breathing in both species. In Neoceratodus this became manifest in a conspicuous increase in oxygen tension of pulmonary venous blood. However, systemic arterial oxygen tension dropped sharply and CO₂ tensions increased markedly in all blood samples. In Protopterus air exposure increased the oxygen saturation in both pulmonary venous and systemic arterial blood, while pulmonary arterial blood maintained the same saturation level. The increase in blood CO₂ tensions was much less marked than in Neoceratodus. Figure 12 A and B show the time course of blood gas tensions following air exposure. The inability of the lung of Neoceratodus to keep up the oxygen saturation of arterial blood seems attributable to a low pulmonary blood flow rather than to the efficiency of gas exchange in the lung. In contrast, the pulmonary blood flow in Protopterus is high enough to maintain the arterial oxygen tension when gill breathing is suspended (Fig. 12 A). In Neoceratodus the arterial CO₂ tensions were increased more than 4 times during 30 min, exposure to air. Protopterus blood showed a corresponding increase of about 30%. In Protopterus, however, CO₂ elimination represents the most severe limitation of pulmonary breathing. The ability of Protopterus to survive several months of terrestrial existence while aestivating during periodic droughts is most probably related to the moist conditions in the cocoon allowing some CO₂ to escape through the skin. Also the markedly reduced over-all metabolism during aestivation will reduce the requirements for gas exchange.