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The Anatomy of the Gastrointestinal Tract of the African Lungfish Protopterus annectens.

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THE ANATOMICAL RECORD 293:1146–1154 (2010)
The Anatomy of the Gastrointestinal
Tract of the African Lungfish,
Protopterus annectens
JOSÉ M. ICARDO,1* WAI P. WONG,2 ELVIRA COLVEE,1 AI M. LOONG,2
2
AND YUEN K. IP
1
Department of Anatomy and Cell Biology, University of Cantabria, Santander, Spain
2
Department of Biological Sciences, National University of Singapore, Singapore,
Republic of Singapore
ABSTRACT
The gastrointestinal tract of the African lungfish Protopterus
annectens is a composite, which includes the gut, the spleen, and the
pancreas. The gut is formed by a short oesophagus, a longitudinal stomach, a pyloric valve, a spiraling intestine, and a cloaca. Coiling of the
intestine begins dorsally below the pylorus, winding down to form six
complete turns before ending into the cloaca. A reticular tissue of undisclosed nature accompanies the winding of the intestinal mucosa. The
spleen is located along the right side of the stomach, overlapping the
cranial end of the pancreas. The pancreas occupies the shallow area,
which indicates on the gut dorsal side the beginning of the intestine
coiling. In addition, up to 25 lymphatic-like nodes accompany the inner
border of the spiral valve. The mesenteric artery forms a long axis for
the intestine. All the components of the gastrointestinal tract are
attached to each other by connective sheaths, and are wrapped by connective tissue, and by the serosa externally. We believe that several previous observations have been misinterpreted and that the anatomy of
the lungfish gut is more similar among all the three lungfish genera
than previously thought. Curiously, the gross anatomical organization is
not modified during aestivation. We hypothesize that the absence of
function is accompanied by structural modifications of the epithelium,
and are currently investigating this possibility. Anat Rec, 293:1146–
C 2010 Wiley-Liss, Inc.
1154, 2010. V
Key words: Protopterus annectens; gut; aestivation; spleen;
pancreas
Lungfish (Dipnoi) are air-breathing fish that thrive
in freshwater in Africa (Protopterus), South America
(Lepidosiren), and Australia (Neoceratodus). The African
genus, Protopterus, constitutes the main line in dipnoan
evolution and appears to have remained unchanged
since the Devonian period (Graham, 1997). An important biological characteristic of the lungfish is its ability
to undergo aestivation through the dry seasons that
characterize tropical life. During aestivation, there is a
depression of the general metabolism, a depression of
heart function, a suppression of urine production, and
an increase in the accumulation of metabolites (BurggC 2010 WILEY-LISS, INC.
V
ren and Johansen, 1986; Fishman et al., 1986). Food
intake is also suppressed.
Grant sponsor: Ministerio de Educación y Ciencia, Spain;
Contract grant number: CGL2008-04559/BOS.
*Correspondence to: José M. Icardo, Departamento de Anatomı́a y Biologı́a Celular, Facultad de Medicina, c/ Cardenal Herrera Oria, s/n, 39011-Santander, Spain. Fax: 34942201903.
E-mail: icardojm@unican.es
Received 3 November 2009; Accepted 11 February 2010
DOI 10.1002/ar.21154
Published online 13 April 2010 in Wiley InterScience (www.
interscience.wiley.com).
THE GUT OF Protopterus annectens
The extraordinary capability of the lungfish to adapt
to extreme climate changes has been mainly studied
from the physiological and biochemical viewpoints
(Chew et al., 2003, 2004; Ip et al., 2005; Wood et al.,
2005; Perry et al., 2008). However, less attention has
been focused on the morphology of the various organs,
which have to decrease (or to suppress) function during
aestivation and, subsequently, have to attain full functional recovery after the aestivating period. In recent
studies we have made a morphological analysis of two
key organs in the African lungfish, the heart and the
kidney, and have shown both the normal structure
(Icardo et al., 2005a, b; Ojeda et al., 2006) and the structural modifications which occur during aestivation
(Icardo et al., 2008; Ojeda et al., 2008). Functional
aspects of the kidney related to nitric oxide activity
(Amelio et al., 2008), and the morphology of the lung
(Sturla et al., 2002), have also been studied. Much less
attention has been given to other organs, like the intestine. Indeed, several anatomical features of the lungfish
gut remain obscure as yet.
The gastrointestinal tract of the lungfish has been
described as a longitudinally organized organ, which
includes a very short oesophagus, a stomach, a pyloric
region, a spiraling intestine, and a cloaca (Parker,
1892; Purkerson et al., 1975; Rafn and Wingstrand,
1981). The segment located between the oesophagus
and the pyloric aperture is considered to be the stomach of these fish. However, it lacks many of the structural features typical of the stomach of the tetrapods.
The organization of the spiraling intestine appears to
be species specific. For instance, the spiral valve
appears to start behind the glottis in Neoceratodus,
and is, therefore, much more complex in the Australian
lungfish than in the other two lungfish genera (Rafn
and Wingstrand, 1981). The relationships of the intestine with two other organs, the spleen and the pancreas, are also unclear. While most studies have
reported the presence of a single spleen (Parker, 1892;
Coujard and Coujard-Champy, 1947), an anterior and a
posterior spleen have been described in Neoceratodus
(Rafn and Wingstrand, 1981). The pancreas appears to
be a single organ, but discrete masses of pancreatic tissue may be embedded to varying degrees in the intestinal wall (Parker, 1892; Rafn and Wingstrand, 1981).
Other uncertainties about the real composition of the
gut arise from the fact that several studies have used
animals living in freshwater, whereas others have used
fish taken in aestivation. The appearance of the different components of the gut could vary with the physiologic state, explaining different or contradictory
observations. There is also the possibility that the species under study were identified erroneously (see Rafn
and Wingstrand, 1981). The present work tries to clarify all the uncertainties related to the anatomical organization of the gastrointestinal tract of the African
lungfish Protopterus annectens. The study has been
performed in animals living in freshwater and after 4
and 6 months of aestivation. We believe that several
previous morphological observations have been misinterpreted and that the anatomy of the intestine
presents greater similarity among all the three lungfish
genera than previously thought. This study constitutes
the basis for further analysis of the intestinal structure, and of the structural changes that may occur during lungfish aestivation.
1147
MATERIALS AND METHODS
Maintenance of Specimens
The study was performed on 12 specimens of the lungfish Protopterus annectens weighing between 100 and
150 g. The specimens were collected from Central Africa
and imported through a local fish farm in Singapore.
Species identification was performed according to Poll
(1961). Sex identification within this weight range is not
possible due to the lack of distinct external features. The
animals were acclimatized to laboratory conditions for at
least 1 month. They were maintained in plastic aquaria
filled with dechlorinated tap water (pH 7.1–7.2), containing 0.71 mM Naþ, 0.32 mM Kþ, 0.72 mM Caþþ, 0.06
mM Mgþþ, 2.2 mM Cl, and 0.2 mM HCO3, at 25 C
(see Ojeda et al., 2008). The water also contained small
amounts of phosphates (0.10 mM) and sulphates (0.04
mM). Water was changed daily. During the adaptation
period, the fish were fed midge larvae (Bio-Pure Blood
Worm, Hikari Sales USA, CA). Food was withdrawn 96
hr before the experiments.
Aestivation
Seven fish were induced to aestivate at 25–30 C individually in plastic tanks containing a small volume (15
mL) of water, as previously described (Chew et al., 2004;
Ip et al., 2005). The water dried up in 6 days. During
this time the animals formed a mucus cocoon which
enveloped the entire body. The time required for cocoon
formation was counted as part of the aestivation process.
The animals were sacrificed after 4 and 6 months of
aestivation.
All the fish were killed by a blow to the head and the
ventral body wall was opened. The entire gastrointestinal tract of six fish (three maintained in fresh water and
three after 6 months of aestivation) was excised and
fixed in 3% glutaraldehyde in phosphate buffered saline.
In addition, the gastrointestinal tract of another six animals (two in freshwater, two after 4 months of aestivation, and two after 6 months of aestivation) was divided
into segments and fixed in methanol:acetone:water,
(40:40:20).
Dissection and Preparation of the Specimens
The entire gastrointestinal tract was dissected free
from the pharynx to the cloaca. To this end, a ventral
and a dorsal mesentery (see Rafn and Wingstrand, 1981)
had to be cut. Further dissections allowed to expose the
spleen and the pancreas, and the inner aspect of the gut
wall. Digital photographs were taken with an Olympus
digital 800 camera (Olympus Imaging, Japan). Alternatively, cross sections of the gastrointestinal tract were
performed at several levels and processed for light microscopy or for scanning electron microscopy. Longitudinal sections were performed at the pyloric level and
processed similarly.
Light Microscopy
For conventional light microscopy, selected gut fragments were dehydrated in graded ethanol, embedded in
Paraplast (Sherwood, St. Louis, MN), and serially sectioned at 8 lm. The sections were stained with hematoxylin and eosin for general observations, or Sirius red for
1148
ICARDO ET AL.
Fig. 1–2. Fig. 1. Gastrointestinal tract of P. annectens. Freshwater.
The same specimen has been dissected progressively. Cl, cloaca; Pa,
pancreas; Si, spiraling intestine; Sp, spleen; St, stomach. (a) Frontal
view: The line of attachment of the ventral mesogastrium is in front.
The entire tract is covered by the serosa. The spleen runs longitudinally along the right side of the stomach. (b) Right view: The serosa
has been dissected. The pancreas is now visible. It runs caudally and
dorsally. The spleen and the pancreas overlap for a short segment.
(c) The spleen and the pancreas have been dissected. Arrow indicates
the shallow area occupied by the pancreas, and the oblique pyloric
aperture. Two-headed arrow indicates the area of spleen-pancreas
overlapping. (d) The spiral intestine has been opened through the
external wall. It is formed by six cones piled one on top of the next.
The coiling starts at the cranial pyloric border. The first chamber
shows oblique mucosal ridges (thick arrows). Tenuous ridges can also
be observed (arrowhead) in the most external parts of the second and
third coils. The rest of the intestine shows a smooth surface. A longitudinal mucosal fold (double arrow) enters the cloaca. Thin arrow
indicates left pyloric leaflet. Magnification bar: 1 cm. Fig. 2. Gastrointestinal tract of P. annectens. Six months of aestivation. The same
specimen has been dissected progressively. It is a little bit larger than
that shown in Fig. 1. Cl, cloaca; Pa, pancreas; Si, spiraling intestine;
Sp, spleen; St, stomach. (a) Dorsal view: The spleen and the pancreas
can be recognized under the serosa. (b) Right view: The serosa has
been dissected. Both the spleen and the pancreas appear very dark.
The cranial part of the pancreas (arrow) holds the spleen. (c) The spiral
intestine has been opened through the external wall. The inner intestine surface is very dark. Note the presence of the six coils. The mucosal ridges are more tenuous and disorganized. The lumen of the
final part of the gut is filled with mucus. Thick arrow indicates the
oblique pyloric aperture and the beginning of coiling. Thin arrow indicates the mesenteric artery. Magnification bar: 1 cm.
the detection of collagen (see: Sheehan and Hrapchak,
1980).
whereas Fig. 2 represents that of a 6-month aestivating
animal. A comparison between Figs. 1 and 2 showed that
the gross anatomical organization was similar in the two
cases. In addition, no differences were observed between
4 and 6 months of aestivation (not shown). However, two
minor differences were noted between freshwater and
aestivating animals. First, the guts from aestivating fish
showed a darker coloration, which was very conspicuous
at the inner surface of the gut (Figs. 1d, 2c), but was also
evident in the spleen and pancreas (Figs. 1a,b, 2a,b). Second, the guts from aestivating fish were slightly thinner.
This appeared to be mostly because of collapse of the gut
lumen (see below). In addition, the lumen of the caudalmost part of the gut was filled with a mucous substance
in aestivating fish (Fig. 2c). Variable amounts of mucus
were also observed in other parts of the gut. This was
much less apparent in freshwater fish. Despite these differences, it was clear that aestivation did not modify the
structural organization of the gut. Thus, the following
description could be applied to either case. Specific features will be highlighted when considered necessary.
Scanning Electron Microscopy (SEM)
For SEM, selected gut fragments were dehydrated in
graded acetone, dried by the critical point method
(Anderson, 1951), coated with gold, and observed with a
Quanta Inspect microscope (FEI Company).
RESULTS
The gastrointestinal tract of P. annectens showed a longitudinal organization, being enveloped by the peritoneal
serosa (Fig. 1a). Dissection of the serosa allowed to identify the main components of the gastrointestinal tract:
the gut, the spleen, and the pancreas (Fig. 1b). On further
dissection the spleen and the pancreas were removed
(Fig. 1c) and the gut was opened to reveal its internal organization (Fig. 1d). Figure 1 represents the gastrointestinal tract of a specimen maintained in freshwater,
THE GUT OF Protopterus annectens
1149
Fig. 3–6. Fig. 3. P. annectens. Freshwater. SEM. The cranial part of
the gut has been opened. The pharynx (Ph), the oesophagus (Oe), and
the stomach (St) are exposed. The oesophagus is a very short segment
which appears separated from the stomach by a series of discrete protrusions (arrowheads). Longitudinal ridges are restricted to the lateral
parts of the stomach. Magnification bar: 500 lm. Fig. 4. P. annectens.
Freshwater. SEM. Same specimen as in Fig. 3. Longitudinal ridges are
much more prominent in the middle and caudal parts of the stomach.
Magnification bar: 400 lm. Fig. 5. P. annectens. SEM. Cross sections
through the middle part of the stomach (St) from freshwater (a) and
aestivating (b) fish. The stomach is open in a and collapsed in b. In
both cases, the spleen (Sp) appears to the right of the stomach. The
serosa (arrowheads) envelops both organs. A system of connective
sheaths (arrows) attaches the two organs to each other and to
the serosa, leaving empty spaces which appear more collapsed in b.
Inset: Four-month aestivation, thick section, and Hematoxylin-eosin.
The collapsed stomach shows a dentate lumen. Compare to b. Magnification bars: a, b 500 lm; inset:100 lm. Fig 6. P. annectens. SEM. (a)
Freshwater. Cross section through the cranial part of the spiraling
intestine (Si). This portion is formed by a large chamber which shows
regular mucosa ridges (arrowheads). On the dorsal side, the intestinal
wall has started to coil to form the first cone (arrow). The mesenteric
artery (A) runs along the inner border of the coil. The location of the
vena porta (v) is indicated. The pancreas (Pa) appears dorsally. Asterisks indicate the reticular tissue underlying the mucosa. Inset shows
the head of a nematode buried between two ridges. (b) Six-month aestivation. The mucosal ridges are irregular and appear covered by mucus. Magnification bars: a, 500 lm; b, 200 lm; inset, 30 lm.
Below the pharynx, the gut was initially formed by a
very short oesophagus which opened into a longitudinal
(cranio-caudal) stomach (Figs. 1, 2). No clear demarca-
tion lines separated the two components. However, SEM
revealed the presence of several internal protrusions,
(Fig. 3) which appeared to constitute a discrete boundary
1150
ICARDO ET AL.
Fig. 7–8. Fig. 7. P. annectens. Thick-sections stained with Sirius
red. Pa, pancreas; Si, spiraling intestine; Sp, spleen. (a) Freshwater.
The first intestinal chamber shows regular ridges. The cranial part of
the first coil is indicated by arrow. The pancreas occupies the shallow
area corresponding to the first coil. The caudal end of the spleen overlaps the pancreas. Arrowhead indicates a vessel common to the two
organs. The entire system is enveloped by a layer of connective tissue
and by the serosa. (b) Six-month aestivation. This section has been
made at a caudal level than that shown in a. The first intestinal chamber shows irregular ridges. This is the single morphological difference
between the two situations. Arrow indicates the first coil. Within the
coil, several lymphatic-like nodes (L) appear. The pancreas occupies a
dorsal position. Asterisks indicate reticular tissue. In a and b, black
dots throughout the tissue correspond to dark pigment cells. Magnification bars: a, b, 800 lm. Fig. 8. P. annectens. SEM cross sections of
the spiraling intestine, from the midgut to the cloaca. Sections from
freshwater and aestivating animals alternate to illustrate the absence
of gross morphological changes during aestivation. Black arrows in
(a–c) indicate the inner border of the spiral valve. Black and white
arrows in (a-c) indicate the mesenteric artery. (a) Freshwater. Midgut.
Two entire intestine turns are visible. The innermost part of the lumen
is occupied by mucus. The intestinal epithelium (white arrows) is separated from the subjacent reticular tissue (asterisks) by connective
sheaths (arrowheads). (b) Six-month aestivation. Hindgut. At a more
caudal level, one and a half epithelial turns are visible. (c) Freshwater.
Most caudal level of spiral intestine. White arrows indicate the mucosa. One single intestine turn appears. The connective sheaths and
the large spaces between them are very conspicuous. L, lymphoidlike node. Asterisks indicate in b and c the reticular tissue. (d) Sixmonth aestivation. The cloaca (Cl) has been sectioned. The epithelium
is attached to the thick outer wall by connective sheaths (arrowheads).
The reticular tissue is much less evident. Inset: Four-month aestivation. Sirius red. Midgut. The intestine coils and the associated reticular
tissue are the predominant structures. Magnification bars: a–d, 500
lm. Inset: 1 mm.
THE GUT OF Protopterus annectens
between the two segments. The stomach was a thinwalled, flattened sac. It appeared narrower in its cranialmost part, but lacked regional specializations (Figs.
1c, 2c). Its internal surface showed several longitudinal
folds, (Fig. 4) which gave the stomach an irregular
appearance in cross sections (Fig. 5, and inset). This was
more evident during aestivation, when the stomach
lumen was collapsed (Fig. 5). The caudal end of the
stomach opened through an oblique pyloric aperture into
the intestine (Figs. 1d, 2c). The pyloric aperture was
1151
guarded by a pyloric valve (Fig. 1d) consisting of two
thick lateral leaflets with a dentate free border.
The intestine presented a spiraling organization (Figs.
1d, 2c). Below the pylorus, the intestine consisted initially of a large chamber (Figs. 1d, 2c, 6). Spiraling of
the intestine started from the dorsal wall of this chamber, at the cranial level of the pyloric aperture (Figs. 1d,
2c, 6a, 7). It continued downward forming six coils
attached to the outer wall. Each coil was shaped like a
cone with the cones piled one on top of the next. The beginning of the coiling appeared at the cranial intestine
level as a dorsal protrusion into the first large chamber
(Figs. 6a, 7). The protruding surface of the first coil is
shown in en face views in Figs. 1d and 2c. The beginning
of the spiral valve was marked externally by the presence of a furrow (Fig. 1c). This shallow area was occupied by the pancreas (Figs. 1c, 6a, 7).
The structure of the intestine at the level of the first
coil (Figs. 6a, 7) was complex because of the presence
both of the first large chamber and of the associated
organs (see below). Below this level, the structure of the
spiraling intestine was dominated by the presence of the
successive coils (Fig. 8). The highest number of coils in
any cross section was of two and it was observed at the
midgut level (Fig. 8a). The coiling decreased progressively (Fig. 8b), until a single coil was observed at the
lowest level (Fig. 8c). The last coil was continuous with
a dorsal, double fold that entered the cloaca (Fig. 1d).
The cloaca was a short chamber with a thick wall and
an irregular contour in cross sections (Fig. 8d).
The inner surface of the intestine presented regional
specializations. The mucosa of the first large chamber
displayed oblique ridges, which were regularly arranged
in freshwater fish (Figs. 1d, 6a, 7a), but appeared more
irregular in aestivating animals (Figs. 2c, 6b, 7b). In
freshwater fish, the deep area between the ridges frequently contained nematodes (inset Fig. 6a). Parasites
were never observed in aestivating animals. Mucosal
ridges were also present in the most external areas of
the second and third coils. They ended abruptly following a vertical line, with no internal or external demarcation. The rest of the intestinal mucosa was smooth (Figs.
1d, 2c). Under SEM, the cloacal mucosa displayed
Fig. 9–10. Fig. 9. P. annectens. Details of the reticular tissue.
(a) Six-month aestivation. Hindgut. SEM. The reticular tissue is packed
with cells. Connective tissue sheaths attach the several components
of the reticular tissue (asterisks) to each other and to the epithelium
(E). The spaces between sheaths appear empty. Inset: Freshwater. The
cells populating the reticular tissue appear full of granules. (b) Fourmonth aestivation. Thick-section stained with Sirius red. The reticular
tissue constitutes the main separation between the epithelial (E) coils.
The collagen component appears in red. The cellular component (pale
yellow) is unstained. The connective sheaths (and the spaces) are not
evident in histological sections. Arrowhead indicates a small artery.
(c) Freshwater. Thick-section stained with Hematoxylin-eosin. The
reticular tissue under the epithelium is full of cells. Several sheaths
(arrowheads) appear collapsed in lower part of the photograph. Black
dots throughout the tissue in b and c correspond to dark pigment cells.
Magnification bars: a, 500 lm; b, 150 lm; c, 100 lm; and inset, 5 lm.
Fig. 10. P. annectens. Freshwater. SEM. Longitudinal section through
the spiraling intestine. Two lymphatic (L)-like nodes appear attached to
each other and to the epithelium (E) through connective sheaths. The
spaces between sheaths are empty. Inset: One node has been dissected after sectioning. Magnification bars: 300 lm; inset, 300 lm.
1152
ICARDO ET AL.
discrete parallel furrows in its cranialmost part (not
shown).
The wall of the spiraling intestine was always associated with a well-vascularized reticular tissue (Figs. 6–8,
inset Fig. 8). This tissue followed the basal aspect of the
mucosa (Fig. 9), contained rounded cells packed with
granules (inset Fig. 9), and constituted the main separation between adjacent coils. Despite this close relationship, the mucosa and the reticular tissue were
independent structures. They were linked by sheaths of
connective tissue (Figs. 8, 9). These sheaths also
enclosed large spaces which always appeared empty
under the microscope.
In addition to the reticular tissue, two solid organs,
the spleen and the pancreas, were closely associated
with the lungfish intestine. The two organs could be
observed under the gut wrappings, tracing a slightly spiral course along the right-dorsal wall of the gut (Figs.
1b, 2b). The spleen appeared as a brownish, compact,
elongated organ located along the right wall of the stomach (Figs. 1, 2). It ended caudally at the midlevel of the
pyloric aperture. The pancreas appeared as a dark organ
located in the shallow area that marked the beginning of
the spiral valve. The pancreas was also an elongated
organ, but rather irregular in shape due to the presence
of small marginal lobes (Fig. 1c). The two organs maintained a close relationship. Usually, the caudal end of
the spleen overlapped the cranial end of the pancreas
(Figs. 1b, 7a). In other cases, the cranial end of the pancreas was cup-shaped and received the caudal end of the
spleen (Fig. 2b). The two organs were attached to the
surrounding structures (and to each other) by sheaths of
connective tissue similar to those described above, by
small amounts of adipose tissue, and by blood vessels
(Fig. 7a). Cutting the blood vessels made dissection of
the two organs easy. As an exception, the connective tissue located in the cranial part of the pyloric aperture,
where the spleen and the pancreas were always in direct
contact, was denser and more difficult to dissect. This
area was traversed by the mesenteric artery (Fig. 2c)
before entering the intestine. The mesenteric artery ran
longitudinally along the inner border of the spiraling
intestine, and could easily be observed in cross sections
(Fig. 8).
In addition to the spleen and pancreas, cross (Figs. 7b,
8c) and longitudinal (Fig. 10) sections revealed the presence of lymphatic-like tissue. Up to 25 rounded or oval
lymphatic-like nodules (Fig. 10) were observed associated with the inner portion of the spiraling intestine,
along its entire length. The nodules varied in size, but
their major axis was always less than 3 mm. They were
larger and more closely apposed at the level of the first
spiral turn, and smaller and more dispersed at the level
of the last spiral turns. Like the spleen and the pancreas, the nodes were attached to the surrounding structures by loose connective tissue and connective sheaths
(Fig. 10), and could readily be dissected (inset Fig. 10).
DISCUSSION
The first point that should be emphasized is that the
gastrointestinal tract of P. annectens is a composite
formed by different organs and structures, which are
packed together by connective tissue of variable density.
The serosa, instead of forming folds and mesenteries,
constitutes the external, unifying wrapping. The second
important observation is that the anatomical organization of the gut is not modified during aestivation. A general collapse of the gut lumen and darker coloration are
minor differences which appear after aestivation. The
collapse of the gut lumen could be considered normal in
the absence of any food intake for long periods. The
cause of the darker coloration is unclear. The entire gastrointestinal tract is populated by dark pigment cells,
and the possibility that these cells may increase in number during aestivation should be investigated further.
The gastrointestinal tract of P. annectens shows a longitudinal, straight organization. This is a primitive characteristic observed in lampreys (Kardong, 2006). It is
also a common feature in lungfish (Parker, 1892; Rafn
and Wingstrand, 1981). In these cases, the gut is short
and there are minor variations in the regional specialization of the different chambers. In agreement with this
general statement, the boundary between the stomach
and the oesophagus is quite tenuous, and the stomach is
a straight chamber that shows no gross regional specializations. There is some controversy in the literature on
whether this straight chamber should be considered a
stomach. It not only lacks regional (cardiac and pyloric)
specializations. Gastric pits and gastric glands, which
also define the stomach in other vertebrates, are absent
(Parker, 1892; Rafn and Wingstrand, 1981). Certainly, it
is not a ‘‘true’’ stomach and the terminology, when compared to that applied to more advanced vertebrates, may
not be very precise (see Chatchavalvanich et al., 2006).
However, both the shape and the function of the stomach
vary along the evolutionary scale (Kardong, 2006). For
instance, the stomach may simply serve to store food, or
to slow food transit. The stomach of P. annectens appears
to be well suited for the latter. The stomach distends
easily even when fixed, and the presence of longitudinal
ridges is most likely the result of the contraction of the
wall musculature (see Holmgren and Nilsson, 1999). We
believe that, in the absence of a more precise nomenclature (or of a better structural definition), the term stomach should be maintained. The caudal end of this
chamber ends into an oblique pyloric aperture which is
guarded by a twofold pyloric valve. Despite early reports
in Protopterus (discussed in Rafn and Wingstrand,
1981), the pyloric valve in P. annectens appears to be
very similar to that of Neoceratodus.
Below the pylorus, the gastrointestinal tract is formed
by a spiral intestine that constitutes more than half of
the total length of the tract. The presence of a spiral
intestine is a primitive feature described in sturgeons
and in some elasmobranches (Chatchavalvanich et al.,
2006; Kardong, 2006). It increases the time of food
transit and facilitates digestion and absorption (Holmgren and Nilsson, 1999). In P. annectens, it starts from
the cranial part of the pyloric boundary, winds down in
six coils, and ends in the cloaca (also, see Parker, 1892).
In the Australian lungfish N. forsteri, the spiraling
intestine has been reported to start behind the glottis, in
what has been presented as a major difference with the
other lungfish (Rafn and Wingstrand, 1981). However,
this appears to be a misinterpretation. Rafn and Wingstrand considered that the longitudinal fold of the stomach, where the spleen lies, constituted the beginning of
the spiral valve. However, the stomach is clearly separated from the intestine by the pyloric valve, and it does
1153
THE GUT OF Protopterus annectens
not form a spiral. Close examination of Fig. 7 of that article (Rafn and Wingstrand, 1981) reveals a configuration similar to that in P. annectens. Thus, the general
organization of the spiraling intestine appears to be similar in all the lungfish species. Only the number (and
the length) of the coils appears to be greater in N.
forsteri, as the highest number of coils observed in a single cross section of the Australian lungfish intestine was
six (Rafn and Wingstrand, 1981), whereas a maximum
of only of two were seen in P. annectens. The higher
number of coils indicates a delayed digestion time.
The mucosa of the spiral intestine has been described
in several lungfish species as containing villous projections (Coujard and Coujard-Champy, 1947; Purkerson
et al., 1975; Rafn and Wingstrand, 1981). However, this
appears to be a misinterpretation of the images obtained
with the conventional microscope, together with a direct
extrapolation from the mammalian intestine. The intestinal mucosa of P. annectens shows oblique ordered
ridges (also, see Parker, 1892) that when sectioned,
resemble the mammalian villi. The ridges are restricted
to the first intestinal chamber (the one surrounding the
first coil), and to the most external part of the second
and third coils. Furthermore, the ridges end abruptly
without any external or internal demarcation. The first
intestinal chamber was previously named bursa entiana
(Parker, 1892; Rafn and Wingstrand, 1981). We believe
that the use of this term should be discontinued. The
bursa entiana is a muscular, thick-walled, chamber-like
enlargement of the pyloric part of the stomach of some
elasmobranches (Holmgren and Nilsson, 1999), which
continues into the intestine. In the lungfish, the first intestinal chamber is not a separate entity or a connecting
segment and, from an anatomical point of view, pertains
to the intestine. In addition, it is where the bile duct
(the ductus choledochus communis) enters the gut (Rafn
and Wingstrand, 1981).
The presence of a reticular tissue associated with the
basal surface of the mucosa is a curious feature. It was
described as a layer of adenoid (Parker, 1892) or lymphoid (Rafn and Wingstrand, 1981) tissue forming part of
the intestine mucosa and submucosa. However, the presence of cells packed with granules of secretory appearance raises doubt about the validity of this assertion,
and the exact nature of this tissue needs to be determined. From an anatomical point of view, the tissue
cannot be considered an integral part of the mucosa.
Although it follows all the intestinal coiling, it is a separate entity, being attached to the mucosa by a system of
connective tissue sheaths. This system has not previously been reported, probably due to the use of histological sections. When conventional histology is used, most
of the sheaths appear collapsed. Similarly, the large
spaces between sheaths have gone unreported. The connective sheaths appear to form an attachment system to
hold all the components of the gastrointestinal tract together (including the spleen, the pancreas and the lymphatic-like nodes). The spaces between sheaths are
unlikely an artefact. They may constitute a reserve for
gut deformation. In addition, they could also be a liquid
reservoir.
In P. annectens, the spleen is shaped like a rod and
extends along the right side of the stomach. This is a
common feature in lungfish (Parker, 1892; Rafn and
Wingstrand, 1981). The rod-shaped spleen described
here corresponds to the anterior (foregut) spleen
reported in the Australian lungfish (Rafn and Wingstrand, 1981). The caudal part of the spleen overlaps the
cranial part of the pancreas, which appears embedded in
the deep furrow that marks the beginning of the intestinal coiling. Despite this close relationship, which is
maintained in all the lungfish (Parker, 1892; Rafn and
Wingstrand, 1981), the two organs are independent and
can easily be dissected. It is also shown here that the
pancreatic limits are quite distinct and that the entire
organ is situated under the connective sheet that envelops the gastrointestinal tract. Scattered pancreatic
masses, in direct contact with the intestinal mucosa,
have been described both in Protopterus and Neoceratodus (Parker, 1892; Rafn and Wingstrand, 1981). We
could not confirm those findings. It is possible that some
of the reticular tissue associated with the intestinal wall
was misinterpreted as pancreatic tissue. Another difference with previous reports in the lungfish is the absence
of a posterior spleen associated with the inner border of
the spiral intestine. The posterior spleen was described
as a large rod that formed a kind of long intestinal axis
(Rafn and Wingstrand, 1981). P. annectens (also, see
Coujard and Coujard-Champy, 1947) shows a large number of lymphatic-like nodules (or lymphoid tissue,
Parker, 1892), located in the same position as the posterior spleen described in N. forsteri. Although this may
be a major difference between lungfish species, histological cross sections might give the false impression that
the so-called posterior spleen is a continuous organ
instead of a succession of small nodes. Our results indicate that in P. annectens, the mesenteric (coeliaco-mesenteric) artery, and not a putative posterior spleen,
constitutes the main longitudinal axis of the spiraling
intestine. Indeed, the mesenteric artery and the intestine coiling develop together in Neoceratodus (Saito,
1985), and the artery constitutes the axis for gut rotation in the tetrapods (Langman, 2006). In this context,
the presence of lymphatic nodes may be equivalent to
the lymphatic chains which follow the mesenteric vessels
in mammals.
In conclusion, our results indicate that previous observations concerning the anatomical organization of the
gastrointestinal tract of the lungfish may have been misinterpreted. The gut gross anatomy appears to respond
to a general pattern and may be quite similar in all the
lungfish species. The collapse of the gut lumen, which
occurs during aestivation may simply reflect the absence
of function and, thus, it may be a mechanism to protect
the epithelium from desiccation. We hypothesize that
the absence of function is accompanied by modifications
of the epithelium at the structural level, and are currently investigating this possibility.
ACKNOWLEDGMENTS
The authors
assistance.
thank
B.
Gallardo
for
technical
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