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- MATERIALS AND METHODS
- LITERATURE CITED
We describe the structural modifications that occur in the alimentary canal of the African lungfish Protopterus annectens during aestivation and after arousal. With fasting, all gut segments undergo structural modifications. The epithelium covering the intestinal vestibule undergoes bursts of activation at 4 months of aestivation, adopting a more quiescent appearance at 6 months. The ridge area of the spiral intestine shows, at 4 months of aestivation, epithelial disintegration, cell desquamation, cell death, and loss of the freshwater phenotype. Surprisingly, the epithelium adopts a stratified appearance at 6 months of aestivation. Except for epithelial disintegration, the smooth portion of the spiral intestine follows a similar pattern of modifications than the ridge area. The entire epithelium of spiral intestine appears to be renewed during aestivation. The presence of intraepithelial mast cells suggests that inflammation is part of the cellular response to aestivation. After arousal, cell phenotypes are restored in about 6 days, but full structural recovery is not attained during the experimental period (15 days post-aestivation). Several aspects of the cellular response to fasting are shared by a wide range of animal groups. This commonality agrees with the presence of a character that allows to adjust the structural and functional properties of the gut to food availability and food quality, and to the characteristics of the fasting episodes. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.
African lungfish (Dipnoi) have the ability to aestivate through the dry seasons that constitute part of tropical life (Fishman et al., 1986; Graham, 1997). Aestivation is a state of dormancy that presents, among other characteristics, exclusive air-breathing, slowdown of the general metabolism, and suppression of kidney and digestive functions (Burggren and Johansen, 1986; Fishman et al., 1986). The adaptive changes in function that occur throughout the aestivation cycles have been mainly studied from the physiological and biochemical points of view (Chew et al., 2003, 2004; Ip et al., 2005; Wood et al., 2005; Amelio et al., 2008; Perry et al., 2008). However, the function of any organ relies on its structure. Surprisingly, the study of structural modifications that accompany functional adaptation to aestivation has received little attention. These modifications do occur. They range from discrete cellular changes to gross histological modifications. Although the decrease in heart function occurs without any significant modification of the heart structure (Icardo et al., 2008), the aestivating kidney shows glomerular collapse and considerable thickening of the filtration barrier (Ojeda et al., 2008). These changes are accompanied by modifications in the activity of nitric oxide (Amelio et al., 2008). Structural changes in the lungs and gills have also been reported during aestivation (Sturla et al., 2002). However, to our knowledge, the study of structural modifications that may occur in the gut of the lungfish during aestivation has not received any attention. On the other hand, several species of the Callichthydae and Cobitididae fish families use a section of the intestine as an air-breathing organ (McMahon and Burggren, 1987), and they are known to be able to survive in mud for hours (see, Graham, 1997). However, this is not equivalent to aestivation and, to our knowledge, modifications in gut structure have not been reported.
The alimentary canal of the African lungfish Protopterus annectens is comprised of a short oesophagus, an intestinal vestibule (a true stomach is not present in lungfish; see Kardong, 2006), a spiral intestine, and a cloaca (Parker, 1892; Purkerson et al., 1975; Rafn and Wingstrand, 1981; Icardo et al., 2010, 2011). The intestinal vestibule, interposed between the oesophagus and the spiral intestine, is a thin-walled sac that appears to serve as a simple passage for food and/or as a food reservoir (Icardo et al., 2010, 2011). The spiral intestine appears to be the single part of the entire gut involved in food processing. It consists of a first chamber with mucosal ridges followed by a longer portion that shows a smooth surface. The two portions are lined with a columnar, pseudostratified epithelium that contains up to six cell types: enterocytes, goblet cells, ciliated cells, dark pigment cells, wandering leukocytes, and endothelial cells pertaining to intraepithelial vessels (Icardo et al., 2011). The functional meaning of some of these cells is unclear as yet. Other cell types could not be identified. The correct structural organization of this epithelium is required for the functional processes associated with digestion. Its structure would likely be modified in the absence of food intake.
In fact, the intestine is a very flexible organ capable to regulate the digestive function to adjust to diet changes and to dietary restrictions (Starck and Rahmaan, 2003; Secor, 2005; Andriamihaja et al., 2010). This is accomplished by structural modifications that range from an increase/decrease of the intestinal mass to more specific cellular and subcellular changes (Waheed and Gupta, 1997; Dunel-Erb et al., 2001; Starck and Beese, 2001, 2002; Hume et al., 2002; Secor, 2008; German et al., 2010). Loss of intestinal mass, downregulation of intestinal performance, and several structural modifications, such as the reduction in microvillous height, are common during fasting episodes. Modifications are more severe in species that, like snakes and aestivating anurans, undergo long periods of fasting in a repetitive manner (see Cramp et al., 2005; Secor, 2005). The gut of the African lungfish P. annectens does not show gross anatomy modifications during aestivation (Icardo et al., 2010). However, we hypothesized that the structure of the epithelium would have to be modified to adjust to the lack of food and water ingest. The study of these modifications is reported here.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
The epithelium of the lungfish gut undergoes profound structural modifications in response to fasting. Several aspects of this response are shared by a wide range of animal groups, from insects to mammals. This commonality agrees with the presence of a character that has been acquired early in evolution. It allows to adjust the structural and functional properties of the gut to food availability and food quality and to the characteristics of the fasting episodes (see Secor, 2005). In birds and mammals, fasting is accompanied by a decrease in intestinal mass and in the surface area of villi (Dunel-Erb et al., 2001; Hume et al., 2002; Starck and Rahmaan, 2003; Karasov et al., 2004). Fasted catfish also undergo significant reduction in intestine mass (German et al., 2010). In species that undergo long episodes of fasting in a repetitive manner, such as snakes and aestivating anurans, these modifications are more severe, reaching up to a loss of 60% of the wet intestine weight (Starck and Beese, 2001, 2002; Secor, 2005; Cramp et al., 2005; Secor, 2008). In P. annectens, there is no apparent specific mass reduction. Furthermore, shortening is not possible since the alimentary canal is a straight organ attached at both ends. Except for some irregularities in the ridges, architectural changes in the gut were not apparent (also, see Icardo et al., 2010). In contrast, most fasting species undergo a reduction in villous surface, number of villi, or changes in villi length (Waheed and Gupta, 1997; Dunel-Erb et al., 2001; Starck and Beese, 2001, 2002; Cramp and Franklin, 2005; Secor, 2008).
In the lungfish, the different segments of the alimentary tract become affected differently. In addition, temporal differences were detected. The transitional epithelium of the intestinal vestibule shows, at 4 months of aestivation, bursts of secretory activity in discrete areas of the epithelium. We could not distinguish any particular localization, nor could we find any indication of structural differences in freshwater conditions (Icardo et al., 2011) that may be responsible for this unequal response. More curiously, the epithelium showed a uniform, close-to-freshwater structure after 6 months of aestivation. A plausible explanation is that this segment needs some time to adjust to aestivation, responding with bursts of activity until reaching a more quiescent state. This is not unique. Lungfish need a period of about two months to adjust respiratory frequency and heart rate to aestivation conditions (Delaney et al., 1974). Furthermore, green-striped burrowing frogs (Cyclorana alboguttata) undergo significant reductions in the mass of the small intestine, together with discrete cellular changes, between 3 and 9 months of aestivation (Cramp et al., 2005). In aestivating lungfish, the final structure of the intestinal vestibule is close to that observed in freshwater conditions reinforcing the suggestion (Icardo et al., 2010, 2011) that this segment is not involved in food processing. The same could be applied to the cloaca even when this segment does not show bursts of activity. Further studies are needed to confirm differences in function along the lungfish gut. However, the structural data indicate that only the segments that appear to be specifically involved in digestion undergo drastic structural modifications. This is restricted to the spiral intestine.
In the spiral intestine, structural differences are observed between 4 and 6 months of aestivation. As occurs with the intestinal vestibule, the period between 4 and 6 months appears to be necessary to reach some kind of structural equilibrium. Furthermore, differences between the ridge area and the smooth portion of the spiral intestine were detected. At 4 months, the pseudostratified columnar epithelium that covers the ridges is almost disintegrated, cell desquamation appears to be massive, and the epithelium and the lamina propria show numerous dead cells. Disintegration of the epithelium and cell death occurs in fasted and food-restricted birds (Karasov et al., 2004) and in fasting rats (Kakimoto et al., 2008). Cell death also occurs in more distant animals like the cockroach Periplaneta Americana (Park et al., 2009). Although cellular changes appear to be less intense in food-restricted than in fasted mammals, lack or decrease of supranuclear vesicles in enterocytes and intense cell desquamation appear to be a common feature in most animal groups subjected to fasting (Dunel-Erb et al., 2001; Starck and Beese, 2001; Karasov et al., 2004). This is accompanied by a decrease in cytoplasmic staining (German et al., 2010). It should be mentioned that the epithelium of the smooth portion of the spiral intestine does not appear disintegrated. Small differences in response could be related to functional differences. Segments with more important digestive functions (see Icardo et al., 2011) may respond more intensely to aestivation. Of note, the inner portion of the spiral intestine, which appears to be less important in digestion (Icardo et al., 2011), undergoes less intense modifications.
Surprisingly, the epithelium of the entire spiral intestine thickens and adopts a stratified appearance at 6 months of aestivation. In addition, many cells are organized into columns and appear to be migrating across the epithelium. In fact, the entire epithelium appears to be renewed by cells that lack secretory vacuoles, are PAS-negative, and they do not show any of the phenotypic characteristics found in freshwater conditions. The origin of these cells is uncertain. In birds and mammals, continuous cell proliferation allows for fast and reversible changes of the epithelium (Starck and Rahmaan, 2003). In the lungfish, the lack of a distinct germinal zone in freshwater conditions (Icardo et al., 2011) and the absence of mitoses during aestivation indicate an extraepithelial origin. The fact that many cells appear halfway between the lamina propria (or the vascular plexus) and the epithelium, and the similarity of the nuclear phenotypes between cells in the epithelium and in the lamina propria, suggest a vascular origin. Although we know nothing about the presence of stem cells in the lungfish, they are not a late acquisition in evolution. For instance, stem cells give rise to various adult cell types in flatworms, cnidarians, and sponges (Mochizuki et al., 2000; Saló and Baguñá, 2002; Juliano and Wessel, 2010). In mammals, haematopoietic stem cells are able to migrate to sites of injury to regenerate damaged tissues (Kucia et al., 2004; Stroo et al., 2009), and epithelial stem cells appear to be responsible for tooth replacement in zebrafish (Huysseune and Thesleff, 2004).
It is worth to mention that cell death, cell desquamation, and transient stratification of the columnar epithelium, also occur in the gut epithelium of tadpoles at climax of metamorphosis (Bonneville, 1963; Schreiber et al., 2005). However, metamorphosis is driven by high levels of thyroid hormone. We do not know how hormone secretion is modified during aestivation. In the lungfish, the presence of intraepithelial mast cells, many of them with empty granules, indicates a possible role of inflammation as part of the cellular response to aestivation. Mast cells are not detected in freshwater conditions (see Icardo et al., 2011).
It should be underscored that aestivation cannot be looked upon as a simple downregulation of metabolic functions. Many cellular activities are upregulated (Icardo et al., 2008; Ojeda et al., 2008), and energy consumption is required to maintain the aestivation state. During fasting, animals rely upon stored energy to meet metabolic demands (Secor, 2008). A fundamental question is how this energy is generated. In the heart, myocardial cells appear to rely upon stored glycogen (Icardo et al., 2008). In the gut, in addition to preventing desiccation, the enormous amount of mucus and cell debris shed into the lumen may be retrieved for energy consumption. Cell debris can be utilized as a source of energy (Park et al., 2009). The liberation of enzymes that accompanies plasma membrane rupture degrades the mucous material and many components such as amino acids may be freed into the lumen (Hume et al., 2002) and may traverse the epithelium by nonspecific ways (Secor, 2008). Hibernating squirrels (Carey, 1990; Carey and Sills, 1992) and aestivating anurans (Secor, 2005) increase glucose uptake between 50% and 90% despite a reduction in the surface of the villi and a substantial decrease in intestinal mass. Similarly, uptake of several amino acids increases in fasting rats (Waheed and Gupta, 1997). In lungfish, the epithelium during aestivation may simply be more permeable, allowing nutrient passage by a mere gradient concentration. The presence of large intracellular spaces would facilitate absorption. Thus, we hypothesize that the material shed into the lumen is reutilized as a source of energy for general body maintenance. Instead of reabsorbing the intestinal tissue, as premigratory birds (Piersma and Gill, 1998) and aestivating frogs (Cramp et al., 2005) appear to do, continuous cell transit and desquamation would add new cellular material that could serve as fuel for consumption. Identification of brush border membrane enzyme activities, and study of the expression of key transporters, are needed to confirm the present hypothesis. However, the facts that epithelial vessels are dilated, and that the lymphatic micropumps are very apparent, suggest that some kind of transport activity is present throughout the epithelium. In the absence of transport activity, vessels and lacteals appear collapsed (Dunel-Erb et al., 2001; Karasov et al., 2004).
Dipnoi constitute the single fish group where the presence of lymphatic vessels and lymphatic micropumps has been documented (Vogel and Mattheus, 1998). An extensive lymphatic system also occurs in the wall of the alimentary canal (Icardo et al., 2011). It was previously suggested that the intraepithelial vessels could be part of the lymphatic system of the gut (Icardo et al., 2011). The fact that they contain red blood cells during aestivation and after arousal casts doubts on that assertion since erythrocytes are not normally found within lymphatics. Further studies are needed to elucidate this matter.
After arousal, the epithelium of the lungfish intestine recovers many of its features when the lungfish is in water. This occurs in about 6 days. The recovery time is longer than that observed in birds and mammals after experimental fasting (Dunel-Erb et al., 2001; Karasov et al., 2004), but similar to that observed after mammalian hibernation (Carey, 1990; Hume et al., 2002). In fact, recovery appears to be slower after long fasting periods (Hume et al., 2002). Of note, the epithelium of the spiral intestine appears stratified at 6 months of aestivation. Epithelia of this kind can easily be transformed into columnar epithelia (Starck and Beese, 2001) without recurring to high mitotic activity that requires elevated energy costs. This is a strategy that appeared unique to the Burmese python (Python molurus bivittatus) (Starck and Beese, 2001), but seems to be shared by lungfish. The difference is that, in the lungfish, epithelial cells have to develop full phenotype characteristics. This makes the structural recovery more remarkable and explains the growing microvilli and cilia, and the progressive acquisition of the PAS-positive staining. Nonetheless, the lungfish gut does not fully attain freshwater features during the arousal period studied here. It should be mentioned that, although the animals may swallow water, they were not supplied with food. In fact, lungfish refuse to eat until at least the beginning of the second week after arousal (unpublished observations). This may indicate that the animals will only feed when some restructuring of the epithelium has taken place, that initial recovery is independent of food intake, and that it relies on internal mechanisms rather than on the availability of nutrients. However, feeding may be necessary for full structural recovery (Cramp and Franklin, 2003). Complete functional recovery may take up to three months (Cramp and Franklin, 2003).