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 (Burggren and Johansen,1986; Fishman et al.,1986). Food intake is also suppressed.
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.
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 Na+, 0.32 mM K+, 0.72 mM Ca++, 0.06 mM Mg++, 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.
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.
For conventional light microscopy, selected gut fragments were dehydrated in graded ethanol, embedded in Paraplast (Sherwood, St. Louis, MN), and serially sectioned at 8 μm. The sections were stained with hematoxylin and eosin for general observations, or Sirius red for the detection of collagen (see: Sheehan and Hrapchak,1980).
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).
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, 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.
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 demarcation lines separated the two components. However, SEM revealed the presence of several internal protrusions, (Fig. 3) which appeared to constitute a discrete boundary between the two segments. The stomach was a thin-walled, 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 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 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).
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 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.
The authors thank B. Gallardo for technical assistance.