Cell Tissue Res (1987) 250:191 196
andT'ts e
The morphology of the lung
of the African lungfish, Protopterus aethiopicus
A scanning electron-microscopic study
J.N. Maina
Department of Veterinary Anatomy, University of Nairobi, Nairobi, Kenya
Summary. The lung of the African lungfish (Protopterus
aethiopicus) is paired, long and cylindrical. It is situated
on the dorsal aspect of the coelomic cavity ventral to the
ribs. Much of the gas exchange tissue is found in the proxi-
mal aspect of the lung with the caudal part largely taken
up by a centrally situated air-duct with a few large peripher-
ally located alveoli. Interalveolar septa, arranged at differ-
ing hierarchical levels from the air-duct, subdivide the lung
into alveoli, the gas exchange compartments. The alveolar
surface is covered by some cells characterized by microvilli
on their free surface, while others are devoid of such struc-
tures. The general organization of the lung of Protopterus
aethiopicus is similar to that of the other genera of Dipnoi,
Neoceratodus and Lepidosiren, with the interalveolar septa
increasing the surface area for gas exchange through pulmo-
nary compartmentation. The abundant septal smooth mus-
cle fibres and elastic tissue may contribute to the physiome-
chanical compliance of the lung. The undifferentiated alveo-
lar pneumocytes and the double capillary system, observed
in Protopterus, in general appear to characterize the very
primitive lungs of the lower air-breathing vertebrates.
Key words: Lung Scanning electron microscopy Pneu-
mocyte Fish - Dipnoi African lungfish (Protopterus
aethiopicus)
The lungfishes (Order: Dipnoi) are unique among fishes
in both their global ecological distribution and their aerial
mode of breathing. They are restricted to three land masses
with the genus Lepidosiren in South America, Protopterus
in Africa and Neoceratodus in Australia. To varying extents,
the lungfishes utilize, for gas exchange, a lung formed as
an endodermal diverticulum from the primitive pharynx.
According to the prevailing theories on the organic evolu-
tion of the lower and higher vertebrates, the antecedents
of the extant tetrapods are presumed to have been aquatic.
The lung or its many possible progenitors like the pharyn-
geal air-pouches, the air-bladder and the swimbladder are
apparently phylogenetically older than the gills (Torrey and
Feduccia 1979). Nevertheless, the eventual transition from
water to air-breathing was a primary requirement for the
evolution and adaptive radiation exhibited by the present
day vertebrates and called for diverse pulmonary organiza-
Send offprint requests to." J.N. Maina, Department of Veterinary
Anatomy, University of Nairobi, P.O. Box 30197, Nairobi, Kenya
Cell
Resealw
9 Springer-Verlag 1987
tional modifications to meet the concomitant increases in
oxygen demands.
The study of the respiratory system of the lungfish is
not only of special interest in our understanding the func-
tional and structural design of the lungs in the early air-
breathers but also illustrates how the mode of respiration
has over time influenced the general development and orga-
nization of the closely related organ systems, mainly the
cardiovascular system. The remarkable interest in these as-
pects is supported by the numerous studies directly or close-
ly associated with the lung in these rare species. Some of
the notable functional and ecological accounts are those
by Smith (1930, 1931), Carter (1957), Grigg (1965a), Len-
fant et al. (1966), Johansen and Lenfant (1967), Johansen
and Reite (1967), Johansen et al. (1967), Jesse et al. (1967),
Johansen (1968, 1970), Bishop and Foxon (1968), Johansen
and Hanson (1968), Lenfant and Johansen (1968), McMa-
hon (1969, 1970), Lomholt et al. (1975), Johansen et al.
(1976), Hughes (1976) and DeLany and Fishman (1977).
The notable anatomical studies are those by Grigg (1965b),
Klika and Lelek (1967), Hughes (1973) and Hughes and
Weibel (1976, 1978). In our earlier study (Maina and Ma-
loiy 1985), we attempted morphometrically to characterize
the lung of the African lungfish (Protopterus aethiopicus)
to gain insight into the basis for its function. In the present
study, the lung of this species has been examined using
the scanning electron microscope to facilitate the study of
the lung surfaces which are essential to gas exchange. Scan-
ning electron microscopy has only infrequently been used
on respiratory organs of fish (Olson and Fromm 1973; Raj-
banshi 1977; Hughes and Munshi 1978; Lewis 1979;
Hughes 1979; Hughes and Mondolfino 1983 ; Hughes and
Umezawa 1983).
Materials and methods
Fifteen specimens of the African lungfish (Protopterus aeth-
iopicus Peters) were caught from creeks leading into Lake
Victoria, a fresh water lake that forms a common border
between Kenya, Uganda and Tanzania. The fish were trans-
ported to Nairobi in aerated plastic water bags, surviving
the journey remarkably well. In the laboratory they were
killed by intraperitoneal injection with sodium pentobarbi-
tone (May and Baker, Dagenham, England). After exposing
the buccal cavity by dissecting away the lower jaws, the
lung was cannulated. With the fish in a supine position,
the lungs were fixed by instillation of 2.3% glutaraldehyde
192

Fig. 1. A scanning electron micrograph of a transverse section of the lung of Protopterus aethiopicus showing the eccentrically located
main air conducting duct (d) with the alveoli (a) in the gas exchange tissue. The arrows indicate the pleural covering overlaid by
a peritoneal sheath, x 26

buffered with 0.2 M potassium phosphate (pH 7.8, total os-
molarity 330 mOsm). After the fixative stopped flowing,
the lungs were ligated very close to the buccal cavity, care-
fully dissected out from the ribs, and immersed in fixative
for about 24 h. Transverse and longitudinal slices of the
lung were cut and processed for scanning electron microsco-
py by dehydration in five changes of absolute ethanol, criti-
cal-point drying in liquid carbon dioxide, sputtering with
gold-palladium complex, and viewed with a Philips PSEM
275 scanning electron microscope.
Results
The paired lungs of Protopterus aethiopicus are long cylin-
drical structures that run on the dorsal aspect of the visceral
cavity from the pharynx and terminate bluntly close to the
anus. They are symmetrical and very intimately attached
to the ribs as shown by the notable costal impressions on
their dorsal surface, after their removal from the body. The
trachea was insignificant in size, the left and right lungs
intercommunicating close to the pharynx through a very
small glottis. An eccentrically located air duct runs the
length of the tubular lung with the gas-exchange tissue
largely confined to the lateral aspect (Fig. 1). In each lung,
the pulmonary artery was located laterally and the pulmo-
nary vein medially. The pulmonary veins joined together
about a third of the way along the length of the lungs
towards the pharyngeal end. The exchange tissue was large-
ly confined to the cranial one-third of the lung where it
constituted about 60% of the cross-section fo the lung while
the distal two-third had fewer and larger alveoli, confined
peripherally to the large now centrally located air duct.
The alveoli were separated by well developed septa ar-
ranged, from the luminal aspect of the air duct outwards,
in three hierarchical levels. The primary septa were more
prominent and lined the air duct, with the secondary and
tertiary alveoli located deeper towards the pleural surface
(Figs. 2, 3). The alveoli decreased in size both from the
adluminal surface to pleural surface and from the cranial
to the caudal aspect of the lung. The diameter of the alveoli
ranged from 0.32 mm to 1.76 mm. The septa were largely
made up of smooth muscle, elastic tissue, collagen and
ground substance (Maina and Maloiy 1985). The blood
capillaries protruded from the adjacent surfaces of the septa
into the alveoli (Fig. 4). The alveolar surface was covered
by similar but distinguishable cells. Those situated in alveo-
lar depressions (Fig. 5) had microvilli on their immediate
free surface and on their cytoplasmic extensions, whereas
those that bulged into the alveoli had a smoother surface
with few stubby microvilli.
Discussion
The three genera of lungfish, Neoceratodus, Lepidosiren and
Protopterus, exhibit bimodal gas exchange characteristics to
varying extents. Neoceratodus forsteri is an obligate water-
Fig. 2. The gas exchange surface of the lung showing the primary septa (p) that line the air duct with the secondary septa (s) and
tertiary septa (t) situated towards the pleural surface. The septa increase the surface area available for gas exchange in the lung.
The area indicated with a star is enlarged in Fig. 4. x 46
Fig. 3. A close up of the interalveolar septa showing the alveolar cells situated in depressions (arrows). These cells appear to decrease
in number towards the pleural surface. The primary septa (p), secondary septa (s) and tertiary septa (t) are clearly visible, x 120
193
breather (Lenfant et al. 1966) while Lepidosiren paradoxa
(Johansen and Lenfant 1967) and Protopterus aethiopicus
(Lenfant and Johansen 1968) are obligate air-breathers.
This respiratory feature may account for the fact that Neo-
ceratodus has a single lung (Grigg 1965b) while both Lepi-
dosiren (Hughes and Weibel 1978) and Protopterus (Klika
and Lelek 1967; Maina and Maloiy 1985), have paired
lungs. In addition, the gills of Neoceratodus are better devel-
oped than those of the other two species of lungfish (Grigg
1965b). In general, the lungs of the Dipnoi are similar in
their shape and topographical position. These lungs are
long and situated ventral to the spinal column and the ribs
to which they are firmly attached (Grigg 1965b; Hughes
and Weibel 1976; Maina and Maloiy 1985). This close ver-
tebrocostopulmonary relationship may enhance the filling
and the emptying of the air in such elongated lungs as
any changes in the position of the animal will greatly influ-
ence the shape and the size of the lung. The inspiratory
phase in the Dipnoi is effected by buccal pumping (Bishop
and Foxon 1968), but the expiratory one is largely due
to the elastic recoil of the contractile elements (Grigg
1965b; Bishop and Foxon 1968; McMahon 1969) like the
smooth muscle and elastic tissue found in abundance in
the lung.
Structurally, the organization of the lung of Protopterus
aethiopicus is similar to that observed in Neoceratodus
(Grigg 1965b; Hughes 1973), Lepidosiren paradoxa
(Hughes 1973; Hughes and Weibel 1976, 1978), Protopterus
annectens (Klika and Lelek 1967) and Protopterus dolloi
(De Groodt et al. 1960). In these lungs, pulmonary trabecu-
lation and septation tremendously increases the overall sur-
face area available for gas exchange over that which could
be possible in a saccular lung of comparable size like that
encountered in Polypterus senegalensis by Klika and Lelek
(1967). The septa also impart rigidity to the lungs of Dipnoi
but unavoidably reduce the volume of the exchange tissue
in the lung (Hughes 1978). The concentration of the ex-
change tissue in the cranial parts of the lung promotes a
more efficient ventilation during both inspiratory and expir-
atory phases, and inevitably enhances the quantity of gas
exchanged (Maina and Maloiy 1985). The blood capillaries
in the thick interalveolar septa protrude only towards one
side into the alveolus, a morphological feature that has
been described as a double capillary system by Perry (1983).
This arrangement drastically reduces the effective gas ex-
change capillary surface and appears to be a transient de-
sign feature of the lungs of the higher vertebrates during
the initial stages of their development (Burri 1974; Pinker-
ton et al. 1982). The ectotherms, which for similar body
weight have a lower resting oxygen requirement (Hutchson
et al. 1968), appear to manage with such poorly designed
lungs.
In Protopterus aethiopicus the alveolar cells exhibit
structural features that characterize the mammalian type-2
pneumocytes (the granular or cuboidal pneumocytes), i.e.
microvilli as well as those of the type-1 cell (the smooth
7~
4~

or squamous pneumocyte) i.e. long cytoplasmic extensions.
The lack of clear morphological differentiation of the alveo-
lar pneumocytes observed in Protopterus aethiopicus ap-
pears to characterize the lung of Dipnoi (Grigg 1965b;
Klika and Lelek 1967; Hughes 1973; Hughes and Weibel
1978) and also the amphibian lung (Czopek 1965; Bils and
Hughes 1978). In the reptilian lung, although the type-1
and the type-2 alveolar pneumocytes can be distinguished,
these cells are not as well differentiated as in the m a m m a l i a n
and avian lungs (Perry 1983). The distinct differentiation
of the alveolar pneumocytes in the lungs of the higher verte-
brates (mammals and birds) decreases the overall alveolar
pneumocyte density (Maina 1986). This would firstly give
rise to a thin blood-gas barrier, thus increasing the diffusing
capacity of the lung, and secondly reduce the oxygen con-
sumption by the p u l m o n a r y tissue. These fine structural
refinements are presumably not necessary in the lungs of
most lower vertebrates because ectotherms have lower oxy-
gen demands than endotherms.
Acknowledgements. This work was carried out at the Institute of
Anatomy, University of Bern. I am most grateful to Prof. E.R.
Weibel, the Director of the Institute, for the use of the facility,
and Dr. P. Gehr and Mr. K. Babl for their immense help. The
work was funded in part by the Leverhulme Trust Fund of London
to whom I am particularly grateful.
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Accepted October 30, 1986