JOURNAL OF MORPHOLOGY 257:147–163 (2003)

Morphology of the Air-Breathing Stomach of the Catfish plecostomus

Dagmara Podkowa and Lucyna Goniakowska-Witalin´ ska*

Department of Comparative Anatomy, Institute of Zoology, Jagiellonian University, 30-060 Krako´w, Poland

ABSTRACT Histological and ultrastructural investiga- round, with a diameter ranging from 88–177 nm, while tions of the stomach of the catfish Hypostomus plecosto- elongated ones, 159–389 nm long, are rare. EC of III mus show that its structure is different from that typical are numerous and also closed. The whole cytoplasm is of the stomachs of other teleostean fishes: the wall is thin filled with large DCV: round, with a diameter from 123– and transparent, while the mucosal layer is smooth and 283 nm, and oval, 230–371 nm long, both with a core of devoid of folds. The epithelium lining the whole internal irregular shape and a wide, irregular halo. EC are in- surface of the stomach consists of several types of cells, the volved in the regulation of digestion and probably local gas most prominent being flattened respiratory epithelial exchange. In conclusion, the thin-walled stomach of Hy- cells. There are also two types of gastric gland cells, three postomus plecostomus, with its rich network of capillaries, types of endocrine cells (EC), and basal cells. The epithe- has a morphology suggesting it is an efficient organ for air lial layer is underlain by capillaries of a diameter ranging breathing. J. Morphol. 257:147–163, 2003. from 6.1–13.1 ␮m. Capillaries are more numerous in the © 2003 Wiley-Liss, Inc. anterior part of the stomach, where the mean number of capillary sections per 100 ␮m of epithelium length is 4, KEY WORDS: air breathing; stomach; ultrastructure; en- compared with 3 in the posterior part. The cytoplasm of docrine cells; the epithelial cells, apart from its typical organelles, con- tains electron-dense and lamellar bodies at different stages of maturation, which form the sites of accumulation of surfactant. Small, electron-dense vesicles containing In water, the gills of fishes are the main gas ex- acidic mucopolysaccharides are found in the apical parts change organ. The amount of oxygen dissolved in of some respiratory epithelial cells. Numerous gastric water fluctuates continuously. The oxygen concen- glands (2 glands per 100 ␮m of epithelium length), com- tration in water varies, especially in tropical areas, posed of two types of pyramidal cells, extend from the where the elevated water temperature reduces oxy- surface epithelium into the subjacent lamina propria. The gen solubility. Shallow, stagnant waters, swamps, gland outlets, as well as the apical cytoplasm of the cells and waters which dry up seasonally are habitats are Alcian blue-positive, indicating the presence of acidic mucopolysaccharides. Zymogen granules have not been characterized by low oxygen levels. In tropical wa- found, but the apical parts of cells contain vesicles of ters there are no vertical currents, which are vital variable electron density. The cytoplasm of the gastric for the transport of dissolved oxygen to lower gland cells also contains numerous electron-dense and depths. In hypoxic (oxygen deficient) environments, lamellar bodies. Gastric gland cells with electron-dense gills are not sufficient for gaseous exchange and cytoplasm and tubulovesicular system are probably in- several fish additionally take up oxygen di- volved in the production of hydrochloric acid. Fixation rectly from the atmosphere through well- with tannic acid as well as with ruthenium red revealed a vascularized accessory respiratory organs (Graham, thin layer of phospholipids and glycosaminoglycans cover- 1997). ing the entire inner surface of the stomach. In regions of the epithelium where the capillaries are covered by the Among 430 families of teleostean fishes, accessory thin cytoplasmic sheets of the respiratory epithelial cells, respiratory organs have evolved in 42 families and a thin air–blood barrier (0.25–2.02 ␮m) is formed, thus in more than 350 species (Graham, 1997). There is a enabling gaseous exchange. Relatively numerous pores great diversity in the structure of these organs: closed by diaphragms are seen in the endothelium lining the apical and lateral parts of the capillaries. Between gastric gland cells, solitary, noninnervated endocrine cells (EC) of three types were found. EC are characterized by Contract grant sponsor: the State Committee for Scientific Re- lighter cytoplasm than the surrounding cells and they search (KBN); Contract grant number: DS/14/IZ/2001. contain dense core vesicles (DCV) with a halo between the electron-dense core and the limiting membrane. EC of *Correspondence to: Lucyna Goniakowska-Witalin´ ska, Department of Comparative Anatomy, Institute of Zoology, Jagiellonian Univer- type I are the most abundant. They are of an open type, sity, R. Ingardena 6, 30-060 Krako´w, Poland. reaching the stomach lumen. The round DCV of this type, E-mail: [email protected] with a diameter from 92–194 nm, have a centrally located core surrounded by a narrow halo. EC of type II are rarely observed and are of a closed type. They possess two kinds of DCV with a very narrow halo. The majority of them are DOI: 10.1002/jmor.10102

© 2003 WILEY-LISS, INC. 148 D. PODKOWA AND L. GONIAKOWSKA-WITALIN´ SKA finger-like protrusions of the mucosal layer in The catfish Hypostomus plecostomus, from the branchial chambers; paired air sacs extending from , is a bottom-dweller which branchial chambers; the labyrinth — folded mucosa reaches more than 30 cm in length. The native range of suprabranchial chambers (Munshi and Hughes, of the Hypostomus extends from the Rio de la 1992); well-vascularized epithelium lining the buc- Plata system throughout Panama and Costa Rica. copharynx (Singh et al., 1991); as well as various This catfish swims to the surface of the water and types of airbladders (Liem, 1989; Podkowa and gulps air, which is then stored in the stomach (Cart- Goniakowska-Witalin´ ska, 1998). Some parts of the er, 1935; Armbruster, 1998). Behavioral and physi- digestive tract can also be used for gas exchange. In ological studies (Gee, 1976) showed that H. plecos- Dalia pectoralis (Umbriidae) (Crawford, 1974), this tomus initiates air-breathing at low water oxygen function is served by the posterior part of the esoph- tensions. The detailed physiological examination agus. Among catfishes, there are two families of carried out by Graham and Baird (1982) confirmed bottom-dwellers inhabiting South American fresh- that the catfish uses gills for gaseous exchange, but waters that have digestive tracts modified for air is a facultative air breather. breathing. Many genera from the family Loricari- In the light of the foregoing physiological re- idae, including Ancistrus, Chaetostoma, Hyposto- search, it seemed interesting to examine the histol- mus, Liposarcus, Loricaria, Loricariichthys, Rhinel- ogy and ultrastructure of the stomach of the catfish epis, Rhineloricaria, Pogonopoma, Pogonopomoides, Hypostomus plecostomus to check whether its struc- Pterygoplichthys, and Sturisoma, have evolved mod- ture is modified for air breathing. ifications of the stomach which enable its usage as an accessory respiratory organ (Carter and Beadle, MATERIALS AND METHODS 1931; Gradwell, 1971; Gee, 1976; Silva et al., 1997; Armbruster, 1998; Satora, 1998; Takasusuki et al., Five young specimens of the catfish Hypostomus plecostomus, 1998; Oliveira et al., 2001). Pygidium (Armbruster, 1.7–2.7 g, were used for the observations. They were obtained from a local pet shop. Prior to the experiment the fishes were kept 1998) and Eremophilus mutisii (Cala, 1987) of the in an aquarium at 23–25°C for 3 weeks and fed with Tetramin, a family also use the digestive tract tropical fish food. The specimens were previously anesthetized in in respiration. 0.1% MS 222 (3-amino-benzoin acid ethyl ester), killed by decap- The posterior intestine is used for gas exchange by itation, and their stomachs were removed by dissection. some catfishes belonging to the Callichthyidae, e.g., Hoplosternum thoracatum (Huebner and Chee, Light Microscopy (LM) 1978), Corydoras aeneus (Kramer and McClure, 1980; Podkowa and Goniakowska-Witalin´ ska, Stomachs from two specimens were immersed in Bouin’s fixa- tive for 24 h, dehydrated in ethanol, and embedded in paraffin. 2002), and Brochis splendens (Gee and Graham, Sections 6 ␮m thick were stained routinely with hematoxylin/ 1978). Some species of Cobitidae also use the poste- eosin and with Passini stain (Goniakowska-Witalin´ ska and Ku- rior intestine for air breathing, including Misgurnus biczek, 1998). Histochemical studies were performed by the peri- fossilis (Jasin´ ski, 1973), Lepidocephalus guntea (Ya- odic acid Schiff (PAS) reaction for neutral mucopolysaccharides and by Alcian blue staining at pH 2.5 for acidic mucopolysaccha- dav and Singh, 1980), and M. anguilicaudatus (Mc- rides (Kiernan, 1990). Mahon and Burggren, 1987). Although several physiological and behavioral studies concerning respiration in air-breathing spe- Transmission Electron Microscopy (TEM) cies have been made, only a few reports have dealt The stomachs were fixed with bubbles of air in their lumen. with the histology and ultrastructure of accessory After1hoffixation the stomachs were cut into pieces and fixed respiratory organs in the digestive tract. Histologi- for a further hour. One stomach was fixed in an isoosmotic solu- cal studies of accessory respiratory organs have been tion containing 2.5% glutaraldehyde in 0.1 M cacodylate buffer published on the esophagus of Dalia pectoralis with 1.5% picric acid, pH 7.4, and 20°C. Another stomach was fixed in glutaraldehyde with tannic acid (1 mg/1mloffixative (Crawford, 1974), the stomach of Liposarcus anisitsi solution) replacing picric acid in order to provide contrast for (Oliveira et al., 2001), and the intestine of Lepido- phospholipids (Goniakowska-Witalin´ ska, 1984). A third stomach cephalus guntea (Yadav and Singh, 1980; Moitra et was fixed in glutaraldehyde containing ruthenium red (1 mg / 1 al., 1989), Misgurnus anguillicaudatus (McMahon ml of fixative solution) to visualize glycosaminoglycans (Luft, 1971). After fixation the material was washed and postfixed in 2% and Burggren, 1987), and M. mizolepis (Park and osmium tetroxide with the same buffer for 2 h, quickly dehy- Kim, 2001). Other studies have dealt only with the drated in acetone, and embedded in Epon 812. Semithin sections ultrastructure of the air–blood barrier in the stom- were stained with methylene blue and Azure II. Ultrathin sec- ach of Ancistrus multispinnis (Satora, 1998) and in tions, mounted on copper grids and stained with uranyl acetate the posterior intestine of M. fossilis (Jasin´ ski, 1973). and lead citrate, were examined with a JEM 100 SX at 80 kV. Detailed histology and ultrastructure have been de- scribed only for the stomach of Loricariichthys Morphometric Measurements platymetopon (Silva et al., 1997) and in the posterior The following measurements were made on semithin sections, intestine of Hoplosternum thoracatum (Huebner using an eye-piece micrometer: the thickness of the wall of the and Chee, 1978), and Corydoras aeneus (Podkowa stomach and its components, the diameter of the gastric glands, and Goniakowska-Witalin´ ska, 2002). the length of erythrocytes, and the diameter of capillaries. The AIR-BREATHING STOMACH OF 149 TABLE 1. Range and arithmetic mean of thickness of two to three layers of smooth muscle cells sur- of the stomach wall and its components (␮m) rounding the thin lamina propria. Range Mean N* The respiratory epithelium consists of several types of cells: 1) respiratory epithelial cells (the most Stomach wall 49.3–89.4 74.6 50 Tunica muscularis 22.1–32.6 29.3 50 prominent); 2) gastric gland cells of pyramidal Mucosa 12.2–34.2 22.4 50 shape; 3) single endocrine cells (EC); and 4) basal Bodies of epithelial cells 2.7–8.4 5.4 30 cells. ␮ *N ϭ number of measurements. The bodies of epithelial cells, 2.7–8.4 m high (Table 1), with large centrally located nuclei fre- quently extend inward between neighboring capil- number of capillary sections and gastric glands per 100 ␮mof laries, while thin surface sheets cover underlying epithelium length was also counted. capillaries (Figs. 1c, 4a). The cell bodies of some Measurements of the diameter of the following organelles were epithelial cells are strongly flattened and lie in di- made with TEM micrographs: electron-dense and lamellar bod- rect contact with capillaries (Figs. 1c, 2a), a relation- ies, dense core vesicles in endocrine cells, pores in endothelial cells, vesicles in epithelial and gastric gland cells, as well as the ship more frequently seen in the posterior part of the height of microvilli. stomach. Scarce, short microvilli from 0.12–0.38 ␮m The thickness of the air–blood barrier and its components was long are occasionally observed on their free surface calculated from TEM micrographs using the method of Weibel (Fig. 2a,b). The epithelial cells are joined by tight (1979) and several measurements were taken from each micro- graph to give a mean value of the thickness. junctions, zonulae adherentes, desmosomes, and also deep interdigitations of cell membranes (Fig. 2a). The central part possesses numerous mitochon- RESULTS dria, profiles of rough endoplasmic reticulum, well- Dissection of Hypostomus revealed that the short developed Golgi complexes, numerous free ribo- esophagus opens into a greatly expanded S-shaped somes, and polysomes (Fig. 2a,b). The apical parts of stomach enlarged by bubbles of air, which occupies some cells contain numerous, small electron-dense the right side of the body cavity. Its wall was thin vesicles of diameter 0.13–0.43 ␮m (Figs. 2b, 3a) and and transparent, like that of the intestine, which also filaments arranged parallel to the cell surface. formed several loops ventral to the stomach. Catfish Fixation with ruthenium red revealed an electron- of total body length varying from 50–60 mm had a dense coat on the surface of the epithelial cells, stomach about 10 ϫ 5 mm in diameter and an in- indicating the presence of glycosaminoglycans (Fig. testine about 350 mm long, which exceeded several 3a). The supranuclear cytoplasm contains round or times the length of the body. oval dense bodies of diameter 0.28–1.38 ␮m and The mean thickness of the stomach wall is 74.6 lamellar bodies of diameter 0.58–1.28 ␮m (Table 2) ␮m (Table 1). It is composed of four layers: 1) an in various stages of transformation (Figs. 2b, 3b,c). external serosal layer of simple squamous epithe- Small vesicles of moderate electron density were lium underlain by loose connective tissue containing observed within the Golgi apparatus. In the vicinity blood vessels; 2) a smooth muscular layer of mean of large dense bodies, small ones were observed. ␮ thickness 29.3 m not organized into distinct layers, Some dense bodies had a corrugated shape, which but consisting of thick bundles of varying orienta- indicates their growth by a process of aggregation of tion separated by connective tissue; 3) a submucosa small ones. Among electron-dense bodies there were of connective tissue; and 4) a mucosal layer consist- some with amorphous, homogenous, electron-dense ing of a lamina propria containing blood vessels and material, as well as more electron-lucent ones con- scarce collagen fibers as visualized by Passini stain, taining small vesicles or granules. Dense bodies as well as fibroblasts, lymphocytes, and granulo- with membranes, which gradually emerge from pe- cytes, and an internal single layer of epithelium ripheral parts of the electron-dense matrix, were with a mean thickness of 22.4 ␮m (Table 1), smooth, devoid of folds, and strongly flattened apically (Fig. also observed (Fig. 3b,c). The system of forming 1a). Gastric glands, lined by pyramidal cells, are membranes results in parallel or, more frequently, located in the mucosal epithelial layer (Fig. 1b). in a concentric configuration of membranes sur- The whole internal surface of the stomach, as well rounding an electron-dense core. Some bodies pos- as the surface of the epithelial and gastric gland sess two, or rarely more, electron-dense cores with cells, showed a weak, positive Alcian blue stain, separated membrane systems. Bodies with more indicating the presence of acidic mucopolysacchar- than two cores were rarely visible. Mature lamellar ides (Fig. 1b). The PAS reaction was negative, indi- bodies are secreted into the stomach lumen. How- cating that neutral mucopolysaccharides are not ever, the actual process of secretion has not been synthesized in the stomach. observed (Fig. 3b). Lamellar structures have not Observations by TEM showed that the submuco- been observed in the stomach lumen. They are prob- sal layer contains a few collagen bundles, fibro- ably washed away from the smooth surface during blasts, lymphocytes, and granulocytes. The mucosal fixation procedures. Fixation with the addition of layer includes a thin muscularis mucosae consisting tannic acid revealed an electron-dense coat on the Fig. 1. a: Cross section through the stomach of Hypostomus plecostomus. The wall of the stomach consists of an external serosal layer with large blood vessels, muscular layer (M) of smooth muscle cells arranged in bundles, and an internal mucosal layer devoid of folds and lined by flattened epithelial cells. Capillaries of lamina propria are adjacent to the epithelium (arrows). Gastric gland cells (Gg) of pyramidal shape are visible within the mucosa. L, stomach lumen. Light microscope (LM). Semithin epon section, ϫ950. b: Cross section of stomach stained with Alcian blue. Shallow epithelial pits are lined by gastric gland cells. Apical parts of epithelial cells, gastric gland cells as well as the whole internal surface of the stomach show a positive reaction to Alcian blue (arrow), which indicates the presence of acidic mucopolysaccharides. L, stomach lumen. LM. Alcian blue stain, ϫ80. c: Cross section through the stomach wall. Epithelial cells lining the internal surface are flattened. Capillaries (C) are adjacent to the epithelial layer. Two capillaries in lamina propria are filled by lymphocytes. In the capillary lying on the right side of the figure the endothelial cell body (En) is situated basally. There is a granulocyte (asterisk) underlying the epithelial layer. Mm, Muscularis mucosae; L, stomach lumen. TEM, ϫ4,500. AIR-BREATHING STOMACH OF CATFISH 151

Fig. 2. a: Flattened epithelial cell with centrally located oval nucleus (N) lies above a capillary with an erythrocyte (E). On the free surface of epithelial cells there are short microvilli. The cell is joined with neighboring ones by tight junctions and desmosomes (arrowheads), as well as by numerous interdigitations of cell membranes (arrows). Supranuclear cytoplasm contains numerous mitochondria, Golgi complexes (G), single cisternae of rough endoplasmic reticulum, and free ribosomes. L, stomach lumen. TEM, ϫ14,000. b: Cuboidal respiratory epithelial cell with basally located nucleus (N) situated adjacent to a capillary (C). The apical cytoplasm of this cell contains numerous, small electron-dense vesicles (arrows). In the supranuclear cytoplasm there are mitochon- dria, Golgi complexes (G), single cisternae of rough endoplasmic reticulum, electron-dense bodies, lamellar body (white arrowhead), and free ribosomes. L, stomach lumen. TEM, ϫ15,000. 152 D. PODKOWA AND L. GONIAKOWSKA-WITALIN´ SKA

Fig. 3. a: Fixation with ruthenium red revealed the presence of glycosaminoglycans on the surface of respiratory epithelial cells. Numerous vesicles of moderate and high electron density are situated in the apical cytoplasm. L, stomach lumen. TEM. Ruthenium red staining, ϫ28,000. b: Fragments of two respiratory epithelial cells joined by desmosomes and interdigitations of cell membranes. Apical cytoplasm of one cell contains lamellar body just before secretion to the stomach lumen (asterisk). The second cell contains electron-dense bodies with distinct corrugated shape and a lamellar body with electron-dense eccentric core. Golgi complexes (G) and mitochondria are present in the vicinity of these bodies. L, stomach lumen. TEM, ϫ24,000. c: Fragment of thin epithelium lying above a capillary (C). Cytoplasm contains lamellar bodies in different stages of transformation. One body with an electron-dense core and a system of lamellae, another with two cores of moderate electron density, and a third with concentric lamellae are seen. The cytoplasm contains also mitochondria, ribosomes, and a network of filaments (arrowhead). L, stomach lumen. TEM, ϫ31,000. surface of the epithelial cells (Fig. 4a). This coat is The respiratory epithelium is underlain by a cap- morphological evidence of the presence of phospho- illary network (Fig. 4a). Capillary sections have an lipids. oval shape, due to the distension of the stomach by AIR-BREATHING STOMACH OF CATFISH 153 TABLE 2. Range and arithmetic mean of the diameter gulped air. Their long axis ranges from 6.1–13.1 ␮m, of electron-dense and lamellar bodies in respiratory while the length of the oval nucleated erythrocytes epithelium and gastric gland cells (␮m) is similar and ranges from 7.5–13.0 ␮m. Capillary Range Mean N sections are more numerous in the anterior part of ␮ Electron-dense bodies 0.28–1.38 0.68 100 the stomach (4 capillaries per 100 m of epithelium Lamellar bodies 0.58–1.28 0.89 85 length), while in the posterior stomach the corre- sponding number is 3. In areas where capillaries are covered by thin cytoplasmic sheets of epithelium (Figs. 4a,b, 5a), the

Fig. 4. a: Fixation with tannic acid revealed an electron-dense coat of phospholipids on the surface of epithelial cells. The cell body with large nucleus (N) is situated between capillaries (C) covered by thin epithelial sheets. Apical part of the cell contains large electron-dense bodies and smaller lamellar ones. The endothelial cells form pyramidal projections protruding into the capillary lumen (arrowheads). These projections contain pinocytotic vesicles and ribosomes. The body of an endothelial cell is situated laterally in the capillary on the left and contains a lamellar body (arrow). E, Erythrocyte; L, stomach lumen. TEM, ϫ14,000. b: The air–blood barrier is composed of three layers: external, thin cytoplasmic sheets of respiratory epithelial cells, narrow interstitial space with basal lamina, and thin parts of endothelial cells. An erythrocyte (E) with large nucleus (N) is visible in the capillary lumen as well as lamellar structures (arrow). The body of an endothelial cell (En) is situated laterally in the capillary. L, stomach lumen. TEM, ϫ15,000. 154 D. PODKOWA AND L. GONIAKOWSKA-WITALIN´ SKA

Figure 5 AIR-BREATHING STOMACH OF CATFISH 155 TABLE 3. Range and arithmetic mean of the thickness ated principally at the base of capillaries (Fig. 5a), of whole air–blood barrier and its components (␮m) although some are located in their lateral parts (Fig. Range Mean SD* N 4b). Their cytoplasm contains numerous pinocytotic vesicles, single mitochondria, rough endoplasmic re- Barrier 0.25–2.02 0.86 0.414 90 ticulum, Golgi apparatus, free ribosomes, and also Epithelium 0.10–1.76 0.71 0.366 80 Interstitial space 0.023–0.059 0.038 0.010 30 scarce electron-dense and lamellar bodies. The lat- Endothelium 0.024–0.207 0.095 0.041 70 ter are rarely seen also in the capillary lumen (Fig. ϭ 5a). *SD standard deviation. Gastric glands are located at regular intervals in the epithelial layer (two glands per 100 ␮m of epi- thelium length). The mean diameter of these glands ␮ air–blood barrier is 0.25–2.02 m thick (Table 3). It is 32.5 ␮m. A cross section of a single gland has is composed of three layers: cytoplasmic sheets of about 12 cells, with cytoplasm of differing electron epithelium, interstitial space, and endothelium. The density (Fig. 5b). The cells are joined by tight junc- cytoplasmic sheets of epithelial cells are the thickest tions, desmosomes, and interdigitations of mem- ␮ component of the barrier (0.10–1.76 m) (Table 3) branes; their nuclei are located basally. On their free and they contain organelles such as mitochondria, surface the gastric gland cells have numerous mi- profiles of rough endoplasmic reticulum, single crovilli varying in length from 0.30–1.38 ␮m, which dense and lamellar bodies, and free ribosomes. The are devoid of a core of actin filaments. The apical interstitial space, restricted to the regions between cytoplasm contains numerous round and oval vesi- the basal laminae of the epithelium and the endo- cles of differing electron density with a diameter thelium, is the thinnest part of the barrier (0.023– ranging from 0.13–0.38 ␮m. Some gland cells are in ␮ 0.059 m). The capillary endothelium is relatively contact with the stomach lumen. The central part of ␮ thin in the barrier regions (0.024–0.207 m) and these cells is occupied by numerous mitochondria, contains mainly numerous pinocytotic vesicles and profiles of rough endoplasmic reticulum, well- single organelles such as mitochondria, free ribo- developed Golgi complexes, free ribosomes, and nu- somes, and scarce rough endoplasmic reticulum. merous electron-dense and lamellar bodies in differ- The surface of endothelial cells is smooth, but cyto- ent stages of maturation. Narrow cells with plasmic projections of a pyramidal shape are seen electron-dense cytoplasm are seen rarely (Fig. 5b). protruding into the capillary lumen (Fig. 4a). Pino- These cells contain tightly packed organelles, nu- cytotic vesicles are present in the cytoplasm of the merous mitochondria, and a poorly developed tubu- endothelial cells and some of them communicate lovesicular system considered to be responsible for with the capillary lumen and also with the intersti- the synthesis and secretion of hydrochloric acid. tial space (Fig. 5a). Single coated vesicles, larger This system is not observed in the electron-lucent than pinocytotic ones, are also present. Relatively cells. In the outlets of the gastric glands, amorphous numerous pores of a diameter from 69–95 nm and and lamellar structures are seen. Fixation with ru- closed by diaphragms are seen in the endothelium thenium red as well as with tannic acid revealed an lining the apical and lateral parts of capillaries (Fig. electron-dense coat on the surface of the gastric 5a). The endothelial cells are joined by tight junc- gland cells, indicating the presence of both glycos- tions and scarce desmosomes. Their bodies are situ- aminoglycans and phospholipids. Solitary, noninnervated endocrine cells (EC), sep- arated by gland cells characterized by cytoplasm lighter than that of the surrounding cells, are lo- Fig. 5. a: The air–blood barrier. The cytoplasm of an epithe- cated exclusively within the gastric glands. The EC lial cell contains numerous mitochondria, single cisternae of are joined with neighboring cells by zonulae adher- rough endoplasmic reticulum, Golgi apparatus (G) and free ribo- somes. Interstitial space (asterisk) is restricted to the basal lam- entes and also scarce interdigitations of cell mem- ina of epithelial and endothelial cells. The part of endothelial cell branes. The basally located nucleus possesses in- participating in the formation of the air–blood barrier is thin and vaginations of the nuclear envelope. The cytoplasm contains numerous pinocytotic vesicles. There are vesicles com- contains very characteristic secretory vesicles, the municating with the capillary lumen as well as with the intersti- tial space (arrowheads). Pores closed by diaphragms are visible in so-called dense core vesicles (DCV), with a halo be- the endothelial cells (arrows). The endothelial cell body (En) lies tween the electron-dense core and the limiting mem- in the basal part of a capillary and contains pinocytotic vesicles brane. Ultrastructural observations enabled identi- and small, electron-dense bodies. There is a fragment of lamellar fication of three types of endocrine cells with DCV of ϫ body in the capillary lumen. L, stomach lumen. TEM, 28,000. b: various shapes and diameters. Shallow pit in the epithelial layer is lined by gastric gland cells of pyramidal shape with basally located nuclei (N). The cells have EC of type I are the most numerous. They have a numerous microvilli. Apical cytoplasm contains small vesicles of pyramidal shape and oval, basally located nuclei. variable electron density. Deeper are numerous mitochondria, Some of them, referred to as an open type, reach the electron-dense bodies, and lamellar bodies. Two cells with very stomach lumen by a narrow apical part with long electron-dense cytoplasm and numerous mitochondria are seen. One of these cells has single tubules in its apical cytoplasm (white microvilli extending into the lumen (Fig. 6a). In arrow). O, Outlet of the gastric gland; Ct, connective tissue of their apical cytoplasm centrioles are occasionally lamina propria. TEM, ϫ4,500. seen. DCV have a diameter ranging from 92–194 nm 156 D. PODKOWA AND L. GONIAKOWSKA-WITALIN´ SKA

Fig. 6. a: Electron-lucent endocrine cell (EC) of type I situated between two darker gastric gland cells (Gg). Note numerous electron-dense bodies in the gastric gland cells. The EC has microvilli projecting into the stomach lumen. The centriole in the apical part of the cell (arrowhead), elongated mitochondria, single dense core vesicles, and numerous polysomes are also visible. TEM, ϫ28,000. b: Basal fragment of EC of type I with light cytoplasm and basally located nucelus (N), situated between gastric gland cells (Gg). Cytoplasm contains characteristic DCV, some of which are in the vicinity of the cell membrane (arrow). DCV have a centrally located core and a prominent halo; some have an eccentric core and a widened halo, while others appear to lack a core. Cytoplasm contains mitochondria, well-developed Golgi apparatus (G), polysomes, and a network of filaments. TEM, ϫ31,000. AIR-BREATHING STOMACH OF CATFISH 157

Fig. 7. EC of type II with centrally located nucleus (N) in the vicinity of a gastric gland cell (Gg). Ct, Connective tissue of lamina propria. TEM, ϫ16,000. Inset: Higher magnification of a part of the figure (left) showing a round DCV with electron-dense core and narrow halo, elongated DCV with rod-shaped electron dense cores. TEM, ϫ42,000. and are dispersed throughout the cytoplasm. They the elongated ones are 155–389 nm long and 88–106 are round or oval with a central core of high electron nm wide. There is perhaps only one type of DCV but density surrounded by an electron-lucent halo. Some sectioned in different planes. In these cells mito- DCV have an eccentric core and a broader halo. chondria, scarce rough endoplasmic reticulum, poly- Others in the vicinity of the cell membrane are de- somes, microtubules, and microfilaments are void of the core, suggesting that they may have present. released secretory products into the intercellular Endocrine cells of type III are also of the closed space. Cytoplasm of the EC of type I contains elon- type. They are round, or more frequently oval with a gated mitochondria, rough endoplasmic reticulum, large nucleus (Fig. 8a). Large DCV throughout the Golgi apparatus, polysomes, single microtubules, whole cytoplasm are observed as either round with a and a network of microfilaments (Fig. 6b). diameter from123–283 nm, or elongated (230–371 ϫ The type II EC is of the closed type and does not 106–283 nm). The core of the vesicles is irregular in contact the stomach lumen. These cells are very shape and is surrounded by a wide halo (Fig. 8b). rarely observed. They have an oval shape (Fig. 7) The mean diameter of round vesicles as well as the and two kinds of DCV grouped predominantly in the mean width of oval ones is the same, which means apical part of the cytoplasm. DCV have a core of very that they are different sections of one type of vesicle. high electron density and a very narrow halo. The The cytoplasm of type III cells contains only scarce round ones have a diameter from 88–177 nm and organelles, including isolated mitochondria, free ri- 158 D. PODKOWA AND L. GONIAKOWSKA-WITALIN´ SKA

Figure 8 AIR-BREATHING STOMACH OF CATFISH 159 bosomes, and single cisternae of rough endoplasmic mus, correlated with the high distension capacity of reticulum, as well as solitary lamellar bodies (Fig. these organs to fill with air. The muscular layer of 8a). respiratory stomachs is also relatively thin. The The epithelial layer also contains round and oval muscularis of the stomach of Liposarcus is not ar- basal cells. They lie deep in the epithelium on a ranged into distinct layers but in bundles of smooth basal lamina and have light cytoplasm, centrally muscle surrounded by connective tissue, as in the located round or oval nuclei, and scarce organelles. stomach of Hypostomus. The greatest differences between the typical stom- achs of teleosts and those adapted to respiration are DISCUSSION observed in the mucosal layer. In typical stomachs it The typical stomach of teleostean fishes is the site forms high folds, reaching 577 ␮m, as in Misgurnus of food digestion and its thick wall is composed of mizolepis (Park and Kim, 2001), and is covered by several layers: an external serosa, a thick muscular simple columnar epithelium. On the other hand, in layer of smooth muscle cells, a submucosa of connec- respiratory stomachs the mucosa is lined by flat- tive tissue, and an internal mucosa. A capillary net- tened epithelium. In the stomach of Hypostomus the work is present in the lamina propria of the mucosal mucosa is smooth and its mean thickness is only layer under the epithelium. This stomach morphol- 22.4 ␮m. The epithelium of the respiratory stomachs ogy has been described for several teleostean fishes, of Ancistrus, Liposarcus, and Hypostomus, as well as including Gasterosteus aculeatus (Hale, 1965), the respiratory sacs of Loricarichthys, is closely un- Monopterus albus (Liem, 1967), Esox lucius (Bucke, derlain by a network of capillaries, a relationship 1971), Perca fluviatilis (Noaillac-Depeyre and Gas, never observed in typical stomachs of teleosts. 1978), Ictalurus punctatus (Sis et al., 1979), An- The network of capillaries in the stomach of Hy- guilla anguilla (Clarke and Witcomb, 1980), Girella postomus is relatively rich, as observed on semithin tricuspidata (Anderson, 1986), Sparus auratus (El- sections. The mean number of capillaries is 4 per bal and Agulleiro, 1986a), Anarhichas lupus (Hel- 100 ␮m of epithelium length. There are more capil- berg and Bjerkas, 2000), and Misgurnus mizolepis laries in the anterior part of a Hypostomus stomach (Park and Kim, 2001). (4/100 ␮m) than in the posterior part (3/100 ␮m). In However, several species of teleosts from the fam- the stomach of Ancistrus there are seven capillaries ily Loricariidae and Trichomycteridae use the stom- per 100 ␮m of epithelium length (Satora, 1998) ach as an accessory respiratory organ. There are (measurements done on semithin sections from the only a few reports describing the structure of this author’s collection). part of the digestive tract, modified for gas ex- The apical cytoplasm of epithelial cells in diges- change. Silva et al. (1997) observed the histology tive stomachs of teleosts is filled by numerous vesi- and ultrastructure of the stomach of Loricariichthys cles containing mucopolysaccharides: acidic in An- platymetopon, consisting of two blind respiratory guilla anguilla and Sparus auratus (Clarke and sacs communicating by a common duct with the Witcomb, 1980; Elbal and Agulleiro, 1986a), and esophagus, which passes into the intestine. In An- neutral in Esox lucius, Ictalurus punctatus, Tilapia cistrus multispinnis the middle part of the stomach nilotica, A. japonica, and Anarhichas lupus (Bucke, (corpus) functions as a respiratory organ and the 1971; Sis et al., 1979; Osman and Caceci, 1991; Kuo histology of the whole stomach and the ultrastruc- et al., 1994; Helberg and Bjerkas, 2000). Mucus is ture of the air–blood barrier have been described secreted into the stomach’s lumen and forms a coat (Satora, 1998). Oliveira et al. (2001) examined the protecting the epithelium from mechanical damage histology of the respiratory stomach of Liposarcus and enzyme activity and also acts as a lubricant for anisitsi. The respiratory stomachs of the above- the stomach contents. In the stomach of Hypostomus mentioned species are enlarged by bubbles of gulped a few epithelial cells contain scarce electron-dense air and have thin, transparent walls. A similar vesicles. However, apical parts of the epithelial cells structure is also characteristic of the stomach of the as well as their surface show a weakly positive re- catfish Hypostomus plecostomus. Collagen fibers are action to Alcian blue, suggesting that these vesicles scarce in the thin walls of the respiratory sacs of contain low amounts of acidic mucopolysaccharides. Loricarichthys and also in the stomach of Hyposto- The PAS reaction was negative for these vesicles, which means that neutral mucopolysaccharides are not synthesized in the stomach of this species. Tubular glands in the connective tissue of the mucosa, consisting of pyramidal or trapezoidal cells Fig. 8. a: EC of type III with centrally located large nucleus (N) lying between gastric gland cells (Gg). The whole cytoplasm of with basally located nuclei, are characteristic of the the EC is filled by large DCV. Cytoplasm also contains rough stomachs of teleostean fish. Such glands were ob- endoplasmic reticulum, polysomes, and a lamellar body (arrow). served in the stomach of Perca fluviatilis (Noaillac- Ct, connective tissue of lamina propria. TEM, ϫ16,000. b: Frag- Depeyre and Gas, 1978), Sparus auratus (Elbal and ment of an EC of type III filled by large DCV which are round or oval and have an electron-dense core of irregular shape and a Agulleiro, 1986a), Anguilla japonica (Kuo et al., wide irregular halo. Their cytoplasm contains numerous poly- 1994), and Piaractus mesopotamicus (Aires et al., somes. N, nucleus. TEM, ϫ35,000. 1999). In respiratory stomachs, glands are a compo- 160 D. PODKOWA AND L. GONIAKOWSKA-WITALIN´ SKA nent of the mucosal epithelial layer, as in the respi- Characteristic large electron-dense and lamellar ratory sacs of Loricariichthys (Silva et al., 1997), and bodies were observed in the epithelial and gland the corpus of the Ancistrus stomach (Satora, 1998). cells of the respiratory sacs of Loricariichthys (Silva In the stomach of Hypostomus, glands form shallow et al., 1997), in the epithelial cells of the stomach pits within the epithelial layer. corpus of Ancistrus (Satora, 1998), in the posterior, Teleostean fishes, as a rule, possess cells of one respiratory part of the intestine of Misgurnus fossi- type in the gastric glands, producing hydrochloric lis (Jasin´ ski, 1973), as well as in the epithelial and acid and pepsinogen. Pepsinogen is stored as zymo- gland cells of the stomach of Hypostomus. The size of gen granules of various diameter, from 0.8 ␮min lamellar bodies measured in figures published in the Girella tricuspidata (Anderson, 1986), to 2.5 ␮min article by Silva et al. (1997) ranged from 0.23–0.73 Anarhichas lupus (Helberg and Bjerkas, 2000), ␮m, while in Hypostomus their diameter ranges while hydrochloric acid is produced by the tubulo- from 0.58–1.28 ␮m. Bodies of similar size occur in vesicular system observed in the apical cytoplasm of the epithelial cells of the respiratory part of the gland cells of Perca fluviatilis, Girella tricuspidata, intestine of Corydoras aeneus (Podkowa and and Piaractus mesopotamicus (Noaillac-Depeyre Goniakowska-Witalin´ ska, 2002). Lamellar bodies and Gas, 1978; Anderson, 1986; Aires et al., 1999). were also observed in the epithelium lining the gas Zymogen granules are absent from the gastric gland bladders of teleosts (Prem et al., 2000), as well as in cells of Hypostomus. The gastric glands of Sparus the respiratory epithelium of the airbladder of the auratus (Elbal and Agulleiro, 1986a) consist of two catfish Pangasius hypophthalmus (Podkowa and cell types: electron-lucent cells producing pepsino- Goniakowska-Witalin´ ska, 1998). Single lamellar gen and electron-dense ones with more numerous bodies are visible in the endothelial cells and in the mitochondria and a well-developed tubulovesicular capillary lumen in the respiratory sacs of Loricar- network in the apical cytoplasm, producing hydro- ichthys, the stomach of Hypostomus, and in amphibian chloric acid. In the stomach of Hypostomus electron- lungs (Goniakowska-Witalin´ ska, 2000). Electron- dense cells containing numerous mitochondria, sin- dense and lamellar bodies are most characteristic gle tubules, and vesicles in the apical cytoplasm of the epithelium of vertebrate lungs: lungfishes were also observed. The apical cytoplasm of both cell (Klika and Lelek, 1967; Hughes, 1973; Zaccone et types of Hypostomus contains small vesicles of vary- al., 1995; Power et al., 1999), Polypterus (Zaccone et ing electron density and diameters from 0.13–0.38 al., 1989), amphibians (Goniakowska-Witalin´ ska, ␮m. These are Alcian blue-positive at pH 2.5, indi- 1984, 1986, 1995, 2000), reptiles (Pastor, 1995), cating that the vesicles probably contain acidic mu- birds (Lopez, 1995), and mammals (Weibel, 1973; copolysaccharides, similar to the gastric gland cells Robertson et al., 1984). The range of lamellar bodies of the respiratory sacs of Loricariichthys. Silva et al. (0.41–2 ␮m) in the lungs of amphibians is greater (1997) concluded that respiratory sacs are used only than that in fish because amphibians have larger for air breathing and have no relation to digestive cells (Goniakowska-Witalin´ ska, 1995). processes. These authors never observed food parti- Lamellar bodies are the site of surfactant accumu- cles in the lumen of respiratory sacs. Such particles lation: surface-active material, which reduces sur- were also rarely observed in the Hypostomus stom- face tension at the inner lining of lungs and airblad- ach. The whole digestive tract of Hypostomus is thin- ders. Surfactant is also involved in antimicrobial walled. Gastric glands are numerous, but zymogen defense mechanisms and facilitates gas exchange as granules were not observed in these cells and the well as the removal of extraneous substances reach- tubulo-vesicular system present in the scarce dark ing the respiratory organs via inspired air cells is poorly developed. Algae and partly decom- (Jarstrand, 1984; Daniels et al., 1995; Rubio et al., posed organic material are predominant compo- 1996). Recent investigations have shown that sur- nents of the Hypostomus diet. Digestion in the stom- factant can be found in other organs, including the ach is probably not intensive and occurs mainly in oral cavity, stomach, and the intestine of the diges- the long intestine. tive tract of mammals (Rubio et al., 1996; Bourbon The ultrastructure of the epithelial cells of the and Chailley-Heu, 2001). Being a component of the respiratory sacs of Loricariichthys and the epithelial mucus, surfactant protects the epithelium from di- cells of the stomach of Hypostomus are similar. The gestive enzymes and helps in transport of food par- epithelial cells of both species are flattened and have ticles. Biochemical and immunohistochemical anal- basally located nuclei. Their free surface is equipped ysis of surfactant from the gas bladders of teleosts with short microvilli. These cells are joined by tight (Daniels and Skinner, 1994; Prem et al., 2000), junctions and desmosomes. Additionally, in the cells lungs of lungfishes (Smits et al., 1994; Orgeig and of Hypostomus distinct interdigitations of cell mem- Daniels, 1995; Power et al., 1999), as well as from branes are visible. The supranuclear cytoplasm of the digestive tracts of mammals (Bourbon and the epithelial cells of both species contains rough Chailley-Heu, 2001) have shown that the surfactant endoplasmic reticulum and well-developed Golgi ap- is composed of phospholipids and scarce amounts of paratus, while in Hypostomus stomach the cells also proteins. contain numerous mitochondria and a network of Numerous capillary vessels underlie the epithe- filaments. lium lining the respiratory sacs of Loricariichthys, AIR-BREATHING STOMACH OF CATFISH 161 the respiratory part of the stomach of Ancistrus, the calva (Youson et al., 2001), gar Lepisosteus osseus stomach of Liposarcus anisitsi, and Hypostomus. (Groff and Youson, 1997), and several species of The majority of capillaries are located under flat- teleosts (Tagliafierro, 1999). tened parts of the epithelium, enabling the forma- Among teleost fishes with respiratory parts in the tion of a thin air–blood barrier. digestive tract EC have been observed only in the Ultrastructural observations carried out on the respiratory intestine of Corydoras aeneus. Two types lungs of vertebrates have shown that the air–blood of closed EC, dispersed in the epithelium with DCV barrier is composed of three layers: 1) cytoplasmic of diameter 69–230 nm, were found (Podkowa and sheets of epithelial cells; 2) an interstitial space Goniakowska-Witalin´ ska, 2002). In the respiratory composed of the basal lamina of epithelial and en- sacs of Loricariichthys, EC cells were observed dothelial cells together with interlaminal connective mainly within the gastric glands (Silva et al., 1997). tissue; and 3) the plasmalemma of endothelial cells. Three types of EC in the stomach of Hypostomus In some species of fishes the interstitial space is with DCV of diameter 88–389 nm are located only restricted to the basal lamina of epithelial and en- within the gastric glands and one type of EC is open. dothelial cells, as in the airbladder of Pangasius EC of type I in the stomach of Hypostomus possess hypophthalmus, in which the mean thickness of the DCV with a mean diameter of 132 nm, which resem- barrier is 0.7 ␮m (Podkowa and Goniakowska- ble DCV in EC of type I in the respiratory intestine Witalin´ ska, 1998). The thickness of the air–blood of Corydoras aeneus and are similar to DCV found in barrier in the respiratory parts of the digestive EC producing catecholamines in other fish species: tracts of fishes is similar, e.g., 0.18 ␮m in the poste- Perca fluviatilis and Ameiurus nebulosus (Noaillac- rior intestine of Misgurnus fossilis (Jasin´ ski, 1973), Depeyre and Gas, 1982), and Sparus auratus (Elbal ␮ 1–2 m in the intestine of Hoplosternum thoracatum and Agulleiro, 1986b). EC of type II in the stomach ␮ (Huebner and Chee, 1978), 0.24–3.00 minthe of Hypostomus have one type of DCV. Depending on intestine of Corydoras aeneus (Podkowa and the plane of section, these may appear round with ␮ Goniakowska-Witalin´ ska, 2002), and 0.6 minthe diameter 148 nm or may appear elongated. These stomach of Ancistrus (Satora, 1998). In the stomach DCV are similar to the DCV found in EC of type II in of Hypostomus the thickness ranged from 0.25–2.02 the stomach of Pimelodus (Gremski and Ferri, 1982) ␮ m. producing gastrin, EC of types I and VIII in the In all investigated vertebrate species the thick- stomach and intestine of Sparus (Elbal and Agul- ness of the barrier in the lungs is similar and does ␮ leiro, 1986b) with mean diameters of 60 nm and 150 not reach more than 3 m, which enables gaseous nm, respectively. exchange (Goniakowska-Witalin´ ska, 1995; Klika EC of type III in the stomach of Hypostomus DCV and Lelek 1967; Lopez, 1995; Pastor, 1995; Weibel, are oval, with a large core of irregular shape and a 1973). diameter of 193 nm. They are similar to DCV in cells In the part of endothelial cells forming the air– producing serotonin, e.g., EC of type I in the stom- blood barrier in the respiratory sacs of Loricariich- ach of Pimelodus (Gremski and Ferri, 1982). Single, thys, in the stomach of Hypostomus, as well as in the lamellar bodies were found in EC of type III in the respiratory intestines of Misgurnus fossilis (Jasin´- stomach of Hypostomus. Such bodies have also been ski, 1973) and Corydoras aeneus (Podkowa and observed in the EC of the lungs of Bombina orien- Goniakowska-Witalin´ ska, 2002), single pores are talis (Goniakowska-Witalin´ ska, 1997). They have seen. They are characteristic of capillaries of the not been observed in EC of the digestive tract of digestive tract, which are involved in the transport of digested food to the blood (Jasin´ ski, 1973). other fishes. The endocrine cells (EC) of the digestive tracts of In the stomach of Hypostomus, as in the digestive fishes are noninnervated, solitary cells, or are in tracts of other fishes, EC probably play a role in small groups dispersed in the epithelial layer of the regulation of digestion, but since the stomach of this alimentary canal. 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