Vestnik Zoologii, 53(2): 165–172, 2019 Morphology DOI: 10.2478/vzoo-2019-0017

UDC 597.551.2:612.335 HISTOLOGICAL AND ULTRASTRUCTURAL STUDIES ON THE INTESTINE OF GUNTEA LOACH, LEPIDOCEPHALICHTHYS GUNTEA (, )

S. K. Ghosh

Department of Zoology, Bejoy Narayan Mahavidyalaya, Itachuna, Hooghly-712 147, West Bengal, India E-mail: saroj.fi [email protected]

Histological and Ultrastructural Studies on the Intestine of Guntea loach, Lepidocephalichthys guntea (Cypriniformes Cobitidae). Ghosh, S. K. — Th e cellular organizations of intestine in Lepidocephalichthys guntea (Hamilton, 1822) have been described by light as well as scanning and transmission electron microscopy. Th e intestine is short and straight like, marked into anterior, middle and posterior region based on mucosal folds, number and size of columnar epithelial cells and mucous cells, thickness of submucosa and muscularis layer. Th e mucosa of anterior intestine forms high folds, which are lined with compactly arranged columnar epithelial cells and mucous cells. In the middle intestine, folds are pointless whereas the posterior intestine is without folds. Th e submucosa is formed of thin layer of connective tissue, contained collagen bundles and blood capillaries, comparatively well developed in the posterior intestine. By scanning electron microscopy, outlines of the luminal surface of anterior and middle intestine is embossed with oval or rounded columnar epithelial cells contained densely packed stubby microridges. Th e posterior intestine has closely set longitudinal folds characterized with minute blood capillaries and columnar epithelial cells having inconspicuous microridges. Ultrastructurally, the mucosal surface of the intestine consists of mucous cells with electron dense granules and columnar epithelial cells having numerous microvilli, mitochondria, endoplasmic reticulum, lysosomes and Golgi body. Cellular components of the anterior and middle intestine participate in the absorption whereas the presence of enormous blood vessels and capillary net work of posterior intestine probably responsible for air breathing. Key words: Lepidocephalichthys guntea, intestine, histoarchitecture, fi ne structure.

Introduction

Fishes exhibit enormous variations in their food habits and feeding specialization. Th e macroscopic and microscopic features of the alimentary canal along with associated structures also become modifi ed accordingly. Among certain fi sh species, diff erent parts of the digestive tract function as respiratory organ. Th e posterior intestine is used for gas exchange by some species belonging to families like Loricaridae, Cobitidae and Callichthyidae (Graham and Baird, 1982; McMahon and Burggren, 1987; Podkowa and Goniakowska- Witalińska, 2002). In some loaches, the digestive phase of the intestine exchanges with the respiratory phase 166 S. K. Ghosh and is specially adapted for aerial breathing. Th e vascular reservoir of the intestine extracts oxygen from the swallowed air and the resting gases are then exhaled through the vent (Yadav, 2006). Morphohistological and microanatomical characteristics of intestine of teleosts in relation to diet has been studied by many researchers (Buddington et al., 1997; Arellano et al., 2002; Banan Khojasteh et al., 2009; El-Bakary and El-Gammal, 2010; Mokhtar et al., 2015; Khadse and Gadhikar, 2017). Th e intestine primarily serves for digestion, its form is immensely mercurial corresponding with various feeding habits and functional necessitates. Investigations on the mucosal variation of the gastro-respiratory tract in air breathing fi shes have been limited to the reports by few workers (Moitra et al., 1989; Park et al., 2003; Ghosh et al., 2011). However, lacunae still exists on the respiratory effi cacy of the intestinal gas exchange systems among freshwater loaches. Lepidocephalichthys guntea (Hamilton, 1822) belongs to family cobitidae of the order cypriniformes. It feeds preferably on small crustaceans, insect larvae, worms, bottom detritus matter along with mud and sand particles; likes to burrow into the substrate to hide under freshwater (Dobriyal, 2013). Th is scavenger loach has a unique intestine that can perform as a respiratory organ. With a view to identify the diff erent cell types occurred on the mucosal surface of the intestine of L. guntea by histological and ultrastructural analysis; the present topic has been undertaken.

Material and methods

Tissue collection. Living mature specimens of L. guntea (total length ranging from 9 ± 1.74; n = 10) were collected from the local freshwater body of Purba Bardhaman (23.2333° N, 87.8667° E), West Bengal, India. Fishes were anaesthetized with tricaine methone-sulphonate (MS 222, Sigma Aldrich) solution (100 mg/L) and sacrifi ced following the guidelines of the Institutional Ethical Committee. Specimens were dissected through midventral incision in the abdominal cavity and the entire intestine was removed and immediately processed for respective studies. Light microscopical (LM) preparation. For histological study, small pieces of intestine (proximal, middle and distal part) were fi xed in aqueous Bouin’s fl uid for 16–18 hours. Aft er fi xation, the samples were washed repeatedly with 70 % ethanol, dehydrated with ascending series of ethanol and cleared in xylene. Th e tissues were infi ltrated with paraffi n wax of 56–58 °C under a thermostat vacuum paraffi n embedding bath for 1 hour. Transverse paraffi n sections were cut at 4 m thickness using a rotary microtome (Weswox MT-1090A). Aft er routine histological process, dewaxed sections were stained with Delafi eld’s Haematoxylin-Eosin (HE) (Suvarna et al., 2013) and Mallory’s triple (MT) stain (Mallory, 1936). Slides were mounted with DPX, viewed and photographed under Olympus-Tokyo PM-6 compound microscope. Scanning electron microscopical (SEM) preparation. To expose the luminal surface, the samples of intestine were incised longitudinally, spread out and pinned with luminal surface upper side on the cork sheets. Th e adhering mucus of the luminal surface was removed by repeated rinsing with 1 % Tween 40 solution. Aft er rinsing in 0.1 M cacodylate buff er, the tissues were infi ltrated with 2.5 % glutaraldehyde solution for 24 hours at 4 C and post fi xed with 1 % osmium tetroxide (OsO4) in 0.1 M cacodylate buff er (pH 7.4) for further 2 hours at room temperature. Fixed tissues were washed repeatedly in the same buff er and dehydrated through graded acetone followed by isoamyl acetate. Th e tissues were then dried by critical point method by liquid carbon dioxide, cemented to metal stubs, coated with gold palladium (approximately 20 nm thick) and examined under scanning electron microscope, Hitachi S-530. Transmission electron microscopical (TEM) preparation. Small fragments of intestine were fi xed in a mixture of 2.5 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 M sodium phosphate buff er (pH 7.3) for 4 hours at 4 °C. Aft er that, the tissues were washed in the same buff er and post fi xed in1 % osmium tetroxide (OsO4) in 0.1 M sodium phosphate buff er (pH 7.3) for further 2 hours at room temperature. Aft er proper fi xation, the samples were rinsed thoroughly with same buff er, dehydrated through ascending series of acetone, and embedded in araldite resin. Semithin sections were cut at 1 m thickness using ultramicrotome (Leica EM UC7), stained with 1 % Toluidine blue (TB) (Robinson, 1977) and observed under light microscope for gross observation of the region. Th en ultrathin sections of gray-silver colour interference (60–90 nm) were cut and mounted on copper grids. Sections were stained with uranyl acetate and lead citrate (Reynolds, 1963) and examined under a Tecnai G2 20S-Twin transmission electron microscope (FEI, Th e Netherlands) operating at 200 kV.

Results

In L. guntea, the intestine is short and almost straight like and the relative length of the gut is less than 1 (RLG < 1). Th e intestine is distinguished into anterior, middle and posterior regions according to diff erences in the structural organization of the mucosal folds. Th e wall of the intestine consists of four usual histological layers throughout the whole length viz., mucosa, submucosa, muscularis and serosa commencing from lumen to periphery (fi gs 1, a; 2, a–b). Th e mucosa of the intestine is thrown into many folds and Histological and Ultrastructural Studies on the Intestine of Guntea Loach… 167

Fig. 1. Photomicrographs of various regions of the intestine of Lepidocephalichthys guntea by scanning electron microscopy (SEM) as well as transmission electron microscopy (TEM) and histological sections stained with Delafi eld’s Haematoxylin-Eosin (HE) and Mallory’s triple (MT) stain: a — anterior intestine consists of mucosa (M) with numerous fi nger like projections (V) toward the lumen (L), submucosa (solid arrows), muscularis (ML) and serosa (arrow heads) (MT) ×100; b — middle intestine exhibits blunt projections lined with tightly packed columnar epithelial cells (CEC) and mucous cells (broken arrows). Th e submucosa (SM) is distinguished from mucosa (M) by basement membrane (BM) and made up of connective tissue, collagen fi bres and blood vessels (solid arrows) forming lamina propria (LP). Note the presence of apical top plate (arrow heads) over the mucosal border (HE) ×400; c — higher magnifi cation of middle intestine showing mucosa (M) packed with CEC having conspicuous nuclei (N) and top plate (arrow heads). Broken arrow marks secreted mucin from mucous cell (HE) ×1000; d — luminal surface of the middle intestine provided with prominent microridges (arrow heads) represent the apical surface of CEC. Note the presence of mucous cells (solid arrows) in between CEC and mucin mass (broken arrows) over CEC (SEM) ×3500; e — folds (V) of middle intestine provided with columnar epithelial cells having numerous microvilli (MV) apically and basally located nuclei (N). Note the presence of scattered mucous cells (broken arrows) in between columnar epithelial cells. Solid arrows indicate blood vessels with erythrocytes (TEM) ×570; f — mucosa of middle intestine showing CEC with microvilli (MV) and nucleus (N) connected with neighbouring cells by tight junctions (broken arrows). Cytoplasm contains tuft of mitochondria (solid arrows), lysosome (arrow head) and lipid droplets (LD). Note the presence of MC and BV (TEM) ×2550. 168 S. K. Ghosh form many fi nger-like projections. Th e number and form of folds shows variation in the diff erent regions of the intestine. In the anterior intestine (fi g. 1, a), projections are long in comparison to middle (fi g. 1, b) and posterior (fi g. 2, a) regions, incline to shorter and capacious. Th e mucosa in all the three regions is made up of simple columnar epithelial cells having basally or centrally placed conspicuous oval nuclei. Th e columnar epithelial cells are border towards the luminal surface. Th e mucous secreting cells either oval or rounded in shape are scattered in between the columnar epithelial cells along the outer boundary of the mucosa of anterior and middle intestine (fi gs 1, b–c). Th e columnar epithelial cells are sheathed with completely with thin top plate except the regions where the mucous cells are located. Th e submucosa is distinguished from mucosa by basement membrane and invades into the mucosal folds forming lamina propria (fi gs 1, b–c). Th e submucosa is vascular and contains connective tissue fi bres with scarce collagen bundles, comparatively well developed in posterior intestine. Th e muscularis consists of an inner circular layer and outer longitudinal layer of smooth muscle fi bres (fi gs 2, a–b). It is very thin in the anterior intestine but becomes thicker posteriorly. Th e inner circular layer is thicker than outer longitudinal one. Th in serosa forms the outermost boundary of the intestine and consists of simple fl attened epithelium, provided with blood vessels (fi gs 1, a; 2, a). Under scanning electron microscope, the luminal surface of the middle intestine is provided with various folds, lined with regularly packed oval or rounded columnar epithelial cell embossed with stubby microvilli (fi g. 1, d). In some regions the packing of columnar epithelial cells are disjoined by the opening of mucous cells. Mucin mass are also found to be adhered on the microvilli of the epithelial cells. Under transmission electron microscope, middle intestinal mucosa contains tall columnar epithelial cells marked with well microvilli towards the lumen forming brush border (fi gs 1, e–f). Th e columnar epithelial cells are allied to form tight junctions with neighbouring cells. Each cell is characterized with basally placed oval or spherical nucleus with small lumps of heterochromatin dispersed all around its interior. A large number of oval shaped mitochondria are distributed throughout the cytoplasm. Flask or oval shaped mucous cells bearing clear cytoplasm and low electron dense granules are scattered in the mucosal surface in between columnar epithelial cells. Some lipid droplets and electron dense bodies having variable sizes are encountered in the apical cytoplasm of the columnar epithelial cell (fi g. 1, f). Few blood capillaries with erythrocyte and lysosomes are also evident in certain regions. Th e wall of the posterior intestine is delicate and translucent as its lumen is stretched due to holding of gulped air. Histologically, the posterior intestine is similar in nature to the other parts of intestine but show some diff erences in the shape and folding patterns, number of mucous cells, thickness of submucosa and muscularis layer. Th e folds diminish signifi cantly in size as well as in numbers and in some regions become more or less fl at. Th e columnar epithelial cells become reduced in magnitude due to extensive development of blood capillaries in the mucosal surface (fi gs 2, a–b). Th e submucosa is comparatively well developed, uniformly thick and highly vascular. Certain granular cells are present in the intestinal submucosa. Th e muscularis is very thin and followed by serosa. Th e serosa is extremely fi ne and constituted of simple squamous epithelium, penetrated by blood vessels. Scanning electron microscopy investigation shows that the mucosa of the posterior intestine is marked with closely set long wavy irregular folds (fi g. 2, c). Th e columnar epithelial cells having inconspicuous microridges are less in number due to penetration of blood capillaries. In certain areas of mucosal folding minute blood capillaries are observed, encircled by epithelial cells. Ultrastructural studies of the posterior intestine show that columnar epithelial cells are embossed with numerous microvilli made up of fi ne fi laments. Polymorphic nuclei having one or more nucleoli covered with nuclear envelope are located at the base of columnar epithelial cells (fi gs 2, d–e). A moderate number of rough and smooth endoplasmic Histological and Ultrastructural Studies on the Intestine of Guntea Loach… 169 reticulum is found adjacent to the nucleus. Lysosome with large electron dense granules and numerous polymorphic mitochondria and Golgi apparatus are present in the upper cytoplasm of the cell. Few mucous cells fi lled with mucus granules, vesicles and electron dense bodies inhabiting the entire cytoplasm of columnar epithelial cells. Blood vessels

Fig. 2. Photomicrographs of posterior intestine of L. guntea by scanning electron microscopy (SEM) as well as transmission electron microscopy (TEM) and transverse sections stained with Delafi eld’s Haematoxylin- Eosin (HE) and Toluidine blue (TB) stain: a — histological section of posterior intestine shows mucosa (M) with columnar epithelial cells (CEC), submucosa (SM), muscularis (ML) and serosa (S). Note predominantly presence of blood vessels (solid arrows) in M and SM (HE) ×400; b — semithin sections of intestine illustrates fl at mucosa (M) with CEC and blood vessels (solid arrows). Note the presence of inner circular muscle layer (CL), outer longitudinal muscle layer (LL) and highly vascular submucosa (SM). VL mark vesicles (TB) ×1000; c — long irregular mucosal folds (MF) exhibits minute blood capillaries (arrows) and columnar epithelial cells (CEC) (SEM) ×200; d — mucosa is lined with columnar epithelial cells having numerous microvilli (MV), basally located nucleus (N) encircled by rough endoplasmic reticulum (arrow heads), polymorphic mitochondria (MT), lysosome (solid arrow) and Golgi apparatus (G). Note the presence of blood vessel (broken arrow) packed with erythrocyte (E) (TEM) ×5000; e — intestinal epithelium shows mucous cells (MC) and CEC contains nucleus (N), mitochondria (arrow heads), rough endoplasmic reticulum (broken arrows) and vesicle (VL). Note the presence of blood capillaries (solid arrows) with erythrocytes (E) (TEM) ×2500; f — submucosa holds blood vessels (BV) with erythrocytes (solid arrows). Arrow heads mark the nuclei of erythrocytes (TEM) ×1550. 170 S. K. Ghosh with erythrocytes are predominantly located in the mucosa and submucosa of posterior intestine (fi gs 2, d–f).

Discussion

Th e consensus among the researches is that the short gut length is characteristic feature of the carnivorous teleosts. However, scavenger loach (Lepidocephalichthys guntea) is a typical freshwater omnivorous fi sh possessing enormously short alimentary canal (Mishra et al., 1991). According to them the morpho-anatomical peculiarities of the alimentary canal can be correlated with the specifi c nature of the food, which does not need prolonged digestion. It has been observed in the present study that the folds of the anterior intestine are comparatively long and provided with columnar epithelial cells which are covered by means of a thin top plate. Under scanning electron microscope, it has also been observed that the mucosal folds in the anterior, middle and posterior intestine modify accordingly to retain food for absorption and eff ective functioning for respiration in L. guntea. In the present study, variations in the pattern of microvilli of the columnar epithelial cells in diff erent regions of the intestine are observed in relation to their functional signifi cance. In the proximal intestine, the microvilli are broad and stubby, may hold profuse amount of mucin secreted from nearing mucous cells for easy passing of semidigested food components. Mandal and Chakrabarti (1996) also reported similar observations in the intestine of Notopterus notopterus and Oreochromis mossambicus. Moreover, microridges on the columnar epithelial cells are suggestive of their active participation in absorption (Chakrabarti et al., 1992; Mokhtar et al., 2015). Th e presence of densely packed columnar epithelial cells and top plate indicates absorptive nature of anterior and middle intestine of L. guntea. At the ultrastructural level, the epithelium of proximal intestine consists of columnar epithelial cells, characterized by junctional complexes, mitochondria, lysome and regular microvilli that increase surface area. Th e cytoplasm of columnar epithelial cells contains vesicles, which may concern pinocytotic activity to some nutrients. Th e columnar epithelial cells perform both absorptive and secretory in function (Cyrino et al., 2008). Th e brush border conceives the momentous digestive/absorptive interface, a functional microenvironment where enzymes involved in further food breakdown are located and where absorption and transport will eventuates (Kuzmina and Gelman, 1997; Mokhtar et al., 2015). Electron dense bodies are the place for aggregation of surfactant, which minimizes the surface tension of inner lining of the respiratory organs and provide guard for epithelial cells (Podkowa and Goniakowska-Witalińska, 2002). Mucous cells having mucinogen granules concentrated at the proximal region have been harmonized with assorted digestive function. Th e occurrence of connective tissue fi bers in the submucosa probably increase in eff ectiveness to distention for communicating the food particles apart from the absorptive role of the tract. In the present study, lamina propria is feeble developed in the posterior region and the submucosa is provided with numerous patches of blood capillaries indicating its possible role in respiration. Th e muscularis of the anterior and middle intestine shows greater development due to need for greater effi ciency in contraction of the intestinal wall for easy transmission of the undigested food materials in the short intestine. On contrary, the wall of posterior part is thin and transparent due to its lumen is enlarged by bubbles of air. Similar fi nding was also reported in the posterior intestine of Corydoras aeneus (Podkowa and Goniakowska-Witalińska, 2002). Th erefore, the structural organization can be mediated as conceivable adaptability to omnivorous feeding habit. Drastic reduction in the thickness of mucosal epithelium and the development of huge capillary bed in the mucosal epithelium and submucosa are the peculiarities of the posterior intestine in L. guntea. Th e mucosal respiratory epithelium contains columnar epithelial cells intercalated with large blood vessels. By scanning electron microscopy, Histological and Ultrastructural Studies on the Intestine of Guntea Loach… 171 the peculiar distribution of wavy mucosal folds along with minute blood capillaries in the posterior intestine indicates that this region is probably related to gas exchange. Th e gaseous exchange probably takes place between the air contained in the intestine and the blood circulating in the respiratory epithelium of the intestinal mucosa. Park et al. (2003) also noticed short mucosal folds, thinner wall and extensive vascular capillary network in the mucosa of the intestine and the rectum of mud loach, Misgurnus anguillicaudatus. Th e mucin secreted by mucous cells shields the delicate posterior epithelium, forms slim lining that expedites gas exchange and also tolerates lubrication of fecal matter to exit (Moitra et al., 1989). Large number of mitochondria in the basal cytoplasm of columnar epithelial cells is probably due to an increased demand of energy for gas exchange. In L. guntea, columnar epithelial cells in the proximal region of the intestine are commonly involved in absorption while the highly vascularized distal region is modifi ed and serves for respiratory function.

Th e author is thankful to the authority of Sophisticated Analytic Instrumentation Facility (SAIF), All India Institute of Medical Sciences (AIIMS), New Delhi for providing electron microscope facility.

References

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Received 14 May 2018 Accepted 5 March 2019