Comparative Studies on Histology and Histochemistry of Pancreas Between Labeo Calbasu (Hamilton, 1822) and Mystus Gulio (Hamilton, 1822)

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Iran. J. Ichthyol. (December 2016), 3(4): 251–265 Received: September 03, 2015 © 2016 Iranian Society of Ichthyology Accepted: November 18, 2016 P-ISSN: 2383-1561; E-ISSN: 2383-0964 doi: 10.7508/iji.2016.02.015 http://www.ijichthyol.org

Comparative studies on histology and histochemistry of pancreas between Labeo calbasu (Hamilton, 1822) and Mystus gulio (Hamilton, 1822)

Saroj Kumar GHOSH1*, Padmanabha CHAKRABARTI2

1Department of Zoology, Bejoy Narayan Mahavidyalaya, Itachuna, Hooghly-712 147, West Bengal, India. 2Fisheries Laboratory, Department of Zoology, The University of Burdwan, Burdwan- 713 104, West Bengal, India. * Email: [email protected]

Abstract: The cellular architecture and histochemical nature of the enzyme secretory acinar cells of pancreas have been investigated in two species of fish, Labeo calbasu and Mystus gulio having different feeding habits. Histological analysis illustrates that the exocrine pancreatic tissues are scattered within the hepatic parenchyma as hepatopancreas and spleen as spleenpancreas in L. calbasu. The structural analysis also shows that exocrine as well as endocrine pancreatic tissues are also diffused in the adipose tissues in between the intestinal coiling of L. calbasu. In M. gulio the discrete pancreatic tissue is attached to the outer wall of the stomach and formed cluster of acini interspersed with small areas of islet of Langerhans and blood vessels. The exocrine pancreatic acinar cells are provided with conspicuous nuclei and densely packed acidophilic zymogen granules. The endocrine component of pancreas, islet of Langerhans is encircled by a thin connective tissue and comprised of α, β and δ cells. Different cell types in the islets are differentiated based mainly upon their shape, position, staining intensity and density of the cytoplasmic secretory granules. α cells contain sparse cytoplasm and densely stained nuclei, are generally located to the periphery of the islet. Aldehyde fuchsin positive β cells bear spherical nucleus and granular cytoplasm, are in the center of islet of Langerhans contacted with blood vessels. δ cells are few in number with light stained nuclei disperse in the pancreatic islet. Histochemical detection displays that the zymogen granules of exocrine acinar cells showing variegated intensities of tryptophan reaction, the precursor of numerous pancreatic enzymes which probably related to the food and feeding habits of the fishes concerned. The zymogen granules are larger in size and heavily deposited in the acinar cells of hepatopancreas of L. calbasu in comparison to M. gulio may be correlated with the herbivorous feeding habit in respect to secretion of digestive enzymes. Keywords: Histoarchitecture, Tryptophan, Pancreas, Structural analysis, Cyprinidae, Bagridae. Citation: Ghosh, S.K. & Chakrabarti, P. 2016. Comparative studies on histology and histochemistry of pancreas between Labeo calbasu (Hamilton, 1822) and Mystus gulio (Hamilton, 1822). Iranian Journal of Ichthyology 3(4): 251-265.

Introduction The pancreas of fishes is a soft, lobulated endodermal In lower vertebrates particularly in fishes, the organ having both exocrine and endocrine tissues. pancreas exhibits an immense diversity in structural The exocrine units are known as acini whereas the modification and is greatly variable being distributed endocrine units are known as islet of Langerhans. in the liver, spleen, intestinal wall or the mesenteries. The exocrine part is a serous gland that synthesizes

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and secretes digestive enzymes into the gut, while the organic detritus matter including diatoms, green endocrine compartment regulates the production and algae, desmids etc (Singh & Singh 2000). Mystus secretion of peptide hormones into the bloodstream gulio (Siluriformes, Bagridae) primarily brackish (Fortin et al. 2015). Variations with respect to water catfish; inhabits estuaries, tidal rivers and morpho-anatomical and cytoarchitectural pattern of lakes, ascending to freshwater, often entering the sea pancreas in teleosts have been investigated by several (Talwar & Jhingran 1991). Its food mainly consists workers (Sheybani & Adibmoradi 2002; Naguib et of crustaceans and insects (Pandian 1968; Hossain et al. 2009; Nejedli & Gajger 2013; Faccioli et al. 2014; al. 2015). The aim of the present investigation is to Mokhtar 2015; Ghosh et al. 2016). The pancreas as a assess the structural and functional aspects of the definite gland was believed to be absent in fishes pancreatic tissue in L. calbasu and M. gulio having until Legouis (1873) reported its existence in a different feeding habits by histological and number of teleosts. He mentioned three types of histochemical analysis. pancreas in teleosts: compact pancreas, diffuse pancreas and disseminated pancreas. A separate and Materials and Methods discrete pancreas is found in Wallago attu Tissue collection: Mature specimens of L. calbasu (Chakrabarti & Ghosh 2012), Mystus aor, Clarias (ranged 26.41±3.81cm in total length; n=10) were batrachus and Heteropneustes fossilis (Khanna obtained from the local freshwater body of Burdwan 1963). The pancreatic tissue however, is present in a (23.2333°N, 87.8667°E) and M. gulio (ranged 16.62 diffuse state and lies dispersed within the lobes of the ±2.34 cm in total length; n=10) were procured from hepatic tissue and mesenteries to form Regional Research Centre of Central Institute of hepatopancreas (Geyer et al. 1996; Petcoff et al. Freshwater Aquaculture, Kalyani Field Station 2006; Sayrafi et al. 2011). In some species, the (22°58'60N, 88°28'60E), West Bengal, India. Fishes pancreatic tissue gradually invades the spleen, were deeply anesthetized with an aqueous solution of collectively known as spleenic pancreas (Khaksar tricaine methone-sulphonate (0.1% MS 222, Sigma Mahabady et al. 2012; Chakrabarti & Ghosh 2015). Aldrich) and sacrificed following the guidelines Comprehensive investigations and information given by the Institutional Ethical Committee. The of endocrine pancreas of teleosts are well specimens were dissected out through longitudinal documented using immunocytochemical techniques incision in the abdominal wall. Small pieces of the (Klein & Van 1978; Abad et al. 1986; Elbal et al. pancreas and some tissues suspected to contain gland 1988; Al-Mahrouki & Youson 1999; Youson et al. associated with liver, spleen and intestine were 2001; Fortin et al. 2015). Within the bony fishes, four removed and processed for respective studies. cell types (A, B, D and F cells) have been recognized Fixation was done immediately after the death of the in islet of Langerhans which produce glucagon, fish to avoid cell autolysis. insulin, somatostatin and pancreatic peptides (PP) Histological analysis: The pancreatic tissues were respectively, but the density of the cells, distribution kept in aqueous Bouin’s fixative for 16-18 hrs. After and localization within the islet tissues variegate that the tissues were washed thoroughly in 70% considerably among teleosts. ethanol, dehydrated with graded ascending series of Still lacunae exist on the morpho-histological ethanol and cleared in xylene. Then the tissues were details including histochemical nature of the infiltrated in paraffin wax of 56-58°C under a pancreatic tissues in relation to feeding adaptations thermostat vacuum paraffin-embedding bath for a among Indian freshwater fishes. Labeo calbasu period of 1 hr. Serial paraffin sections were cut at 4 (Cypriniformes, Cyprinidae) is freshwater m thickness using a rotary microtome (Weswox herbivorous, bottom feeder and feeds mainly on MT-1090A). After routine histological process 252 Ghosh and Chakrabarti- Cellular organization of pancreas in Labeo calbasu and Mystus gulio

Fig.1. Showing the position of pancreatic tissue in Labeo calbasu and Mystus gulio. (A) Entire structure of the whole fish, Labeo calbasu, (B) entire structure of the whole fish, Mystus gulio, (C) distribution of hepatopancreas (HP), spleenpancreas (SP) and adipose tissue pancreas (arrows) in between the coils of the intestine (IC) in L. calbasu and (D) Pancreatic tissue (arrows) attaches to the wall of stomach (S) in M. gulio (I indicates intestine). deparaffinized sections were stained with Delafield’s compound microscope. Haematoxylin-Eosin (HE), (Suvarna et al. 2013), Chrome alum haematoxylin phloxine (CAHP) Results (Gomori 1941) Mallory’s triple (MT) (Mallory Histology 1936), Aldehyde fuchsin (AF) (Halami 1952) and Labeo calbasu: In L. calbasu (Fig. 1A), the exocrine Heidenhain’s azan (HA) stain (Kiernan 2008). pancreas occurs as a diffuse state in the three Histochemical analysis: Tissue samples for different regions viz., in the liver as hepatopancreas, histochemical technique were fixed in 10% neutral in the spleen as spleenpancreas and in the adipose formalin for 18 hrs. After proper dehydration in tissue located in between the coils of intestine (Fig. ascending series of ethanol followed by clearing in 1C). xylene, the tissues were embedded in paraffin wax at The pancreatic tissue dispersed within the lobes 52-54°C. Paraffin blocks were cut in 8 m thick of the hepatic tissue and mesenteries to form the sections and subjected to Dimethylaminobenz- hepatopancreas. The hepatopancreas is a large, aldehyde (DMAB)-Nitrate method for detection of compressed, dark-brownish body, located ventro- Tryptophan (Adams 1957). laterally to the alimentary canal (Fig. 1C). It is made The staining slides were mounted with DPX, up of a typical ground hepatic tissue in which patches observed and photographed under LEICA EC3 of various sized pancreatic tissue irregularly

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Fig.2. Photomicrographs of histoarchitecture of hepatopancreas in L. calbasu stained with Delafield’s Haematoxylin- Eosin (HE), Mallory’s triple (MT) and Chrome alum haematoxylin phloxine (CAHP) stain. (A) Oval or triangle shaped pancreatic tissue (PT) containing acinar cells (AC) scattered among ground hepatic tissue (HT). Note the presence of blood vessels (BV) and bile ducts (broken arrows) (HE 100X). (B) Exocrine pancreatic tissue (PT) consists of AC packed with dense zymogen granules (ZG) in the apical part and conspicuous nuclei (N) at the basal part. Note the presence of BV adjacent to the bile ducts (broken arrows). HT indicates hepatic tissue (HE 400X). (C) Pyramidal or cuboidal AC of pancreatic tissue (PT) distinguishes from HT by septa of connective tissue (solid arrows). BV encompass by AC arranged in multiple series. Note prominent nucleolus in the nuclei (arrow heads) and diffuse ZG in AC (MT 400X). (D) Higher magnification exhibits typical AC of pancreatic tissue arranged in several rows encircling BV. AC contains basally located prominent nuclei (N) and numerous phloxinophilic ZG (CAHP 1000X). scattered (Fig. 2A). The hepatic tissue is comprised pancreatic ductules run (Fig. 2B). The acinar cells are of hexagonal or polygonal hepatic cells having more or less pyramidal or cuboidal in contour, granular cytoplasm with rounded nuclei. The hepatic arranged in several layers and located close tissue is supplied with a close network of hepatic proximity to the blood vessels (Figs. 2B, C, D). A ductules or capillaries. The pancreatic patches seem typical exocrine cell contains a broad basal as rounded, oval or elongated in fashion, are mainly homogenous portion and a narrow apical portion. made up of exocrine acinar cells which are densely The basal region is provided with a large spherical packed together and bounded by connective tissue nucleus having distinct nucleolus. The apical portion through which numerous blood capillaries and is comprised of numerous zymogen granules which 254 Ghosh and Chakrabarti- Cellular organization of pancreas in Labeo calbasu and Mystus gulio

Fig.3. Photomicrographs of the histological sections of spleenpancreas in L. calbasu stained with Delafield’s Haematoxylin-Eosin (HE), Mallory’s triple (MT) and Chrome alum haematoxylin phloxine (CAHP) stain. (A) Spleenpancreas illustrates distribution of exocrine acinar cells (AC) surrounding blood vessels (BV). Pancreatic tissue (PT) separates from spleenic tissue (ST) by thin connective tissue septa (arrows) (HE 100X). (B) Dispersed PT containing cluster of AC distinguishes from ST by delicate connective tissue (solid arrows). BV indicates blood vessel (MT 100X). (C) Higher magnification showing densely packed AC around BV contains prominent nucleolus in nuclei (arrow heads) and acidophilic ZG (CAHP 400X). are heavily deposited and relatively larger in size showing in close association with blood capillary and (Figs. 2C, D). an apical part having profuse zymogen granules. The The exocrine cells of the pancreatic acini are phloxinophilic zymogen granules are small in size present within the spleenic tissue to form and few in numbers. Acinar cells contain nuclei with spleenpancreas. The spleen pancreas is reddish- central nucleolus located towards the base and their brown lengthen organ situated on the mid region of cytoplasm is basophilic in nature (Fig. 3C). The digestive tract, ventrally to swim bladder (Fig. 1C). endocrine pancreas containing islet of Langerhans The acinar cells are distributed in the form of could not be identified in the hepatopancreas and numerous clusters, encircling the blood vessels. The spleenpancreas. pancreatic acinar cells are distinguished from the Structural analysis also showed that exocrine as spleenic part by thin septa of connective tissue (Figs. well as endocrine pancreatic tissues are distributed 3A, B). Pyramidal acinar cell comprises a basal area, widely as dissipated condition within the adipose

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Fig.4. Photomicrographs of cellular architecture of pancreas in L. calbasu stained with Mallory’s triple (MT) and Aldehyde fuchsin (AF) stain. (A) Distribution of pancreatic tissue within the adipose tissue (arrows) in between the coils of the intestine (IC) (MT 40X). (B) Langerhan’s islet (IL) surrounded by thin capsule (solid arrows) and blood vessels (broken arrows) are present in between the acinar cells (AC) of pancreatic tissue (PT). ADT marks adipose tissue (AF 100X). (C) Islet of Langerhans (IL) is enclosed by a thin connective tissue (solid arrows) and consists of α, β and δ (arrow heads) cells. Note the presence of AC packed with zymogen granules (ZG) and blood vessel (broken arrow) (AF 400X). (D) Higher magnification of IL shows AF positive β cells adjacent to BV, peripherally located α cells and scattered δ cells (arrow heads). Solid arrows indicate septa in between endocrine component and AC. Note prominent nucleus (N) and coarse ZG of AC (AF 1000X).

256 Ghosh and Chakrabarti- Cellular organization of pancreas in Labeo calbasu and Mystus gulio

Fig.5. Photomicrographs of section of pancreas in M. gulio showing cellular organization stained with Mallory’s triple (MT), Aldehyde fuchsin (AF) and Heidenhain’s Azan (HA) stain. (A) Pancreatic tissue (PT) containing islet of Langerhans (IL) which surrounded by connective tissue (solid arrows) and blood vessel (arrow) adjacent to the stomach wall (SW) (MT 40X). (B) IL encircles by cluster of acinar cells (AC) having conspicuous nuclei (arrow heads). BV indicates blood vessel. Note also scattered blood vessels (broken arrows) among AC and within IL (MT 400X). (C) IL consists of α, β and δ cells (arrow heads) separated from exocrine AC by connective tissue septa (solid arrows) (AF 400X). (D) Higher magnification of IL showing α cells at the periphery, β cells (broken arrows) in central position and few δ cells (arrow heads). Solid arrows indicate reticular connective tissue septa in between IL and AC (HA 1000X). tissue in between the coils of intestine (Figs. 1C, 4A, tissue and consists of α, β and δ cells, interspersed B). Close association between the adipose tissue with blood vessels. Various cell types in the containing pancreatic patches and intestinal wall is pancreatic islets are recognized on the basis of their observed in some areas. Pancreatic patches occurring architecture, distribution, staining intensity and in adipose tissue is much less in number than those cytoplasmic ground substances. The position of α of hepatopancreas. The cellular architecture of the cell is confined to the periphery of islet, characterized acinar cells in exocrine pancreas located in the with scanty cytoplasm and large distinct oval nucleus adipose tissue is more or less harmonious to those of (Figs. 4C, D). Aldehyde fuchsin positive β cells are hepatopancreas, but the concentration of acidophilic capacious, polyhedral in appearance and more in the zymogen granules occurs in less magnitude. center of islet of Langerhans, contact with blood The endocrine part of the pancreas, islet of cells. These cells bear spherical nucleus and granular Langerhans is enclosed by a delicate connective cytoplasmic mass. δ cells are less abundant, solitary,

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Fig.6. Photomicrographs of sections of hepatopancreas (Figs. A-B), spleenpancreas (Figs. C-E) and adipose tissue pancreas (Fig. F) in L. calbasu showing histochemical test for trypotophan (Trypt). (A) Intense tryptophan reaction in the acinar cells of exocrine pancreatic tissue (arrows) and weak reaction in hepatic tissue (HT) (Trypt 40X). (B) Maximum reaction of tryptophan in the zymogen granules (ZG) of acinar cells (AC) in the pancreatic tissue. Note weak reaction in ground HT (Trypt 400X). (C) Strong tryptophan reaction in the AC of pancreatic tissue and weak reaction in spleenic tissue (ST). Note moderate reaction in blood vessel (broken arrow) (Trypt 40X). (D) Spleenpancreas shows acute tryptophan reaction in ZG of AC and weak reaction in ST. Note moderate reaction of tryptophan in blood vessels (BV) (Trypt 100X). (E) Intense localization of tryptophan in ZG of AC encircling BV of exocrine pancreatic tissue. Note feeble reaction in ST. Broken arrow marks spleenic duct (Trypt 400X). (F) Strong intensity of reaction for tryptophan in ZG of AC and weak reaction in adipose tissue (ADT) (Trypt 400X). 258 Ghosh and Chakrabarti- Cellular organization of pancreas in Labeo calbasu and Mystus gulio

variable in architecture having lightly stained nuclei Cytoplasm containing copious zymogen granules of and scattered anywhere in the pancreatic islet (Figs. the pancreatic acinar cells stains deeply for 4C, D). tryptophan histochemical test. However, hepatic Mystus gulio: In M. gulio (Fig. 1B), the pancreas is tissue itself shows weak reaction. separate and discrete in nature but relatively more Regarding the localization of tryptophan organized in its structural architecture. The endocrine content, the pancreatic part of spleenpancreas islets are present within the ground exocrine exhibits a puissant staining activity which is similar pancreas. The pancreatic mass is found to be adhered to hepatopancreas. The spleenic tissue furnishes a to outer layer of the stomach (Figs. 1D, 5A). The moderate to weak reaction for this histochemical test. exocrine part is composed of a number of clusters of The blood vessels containing blood cells encircled by acinar cells which are closely compressed. Blood acinar cells shows pronounced reaction for vessels and pancreatic ducts are found to occur here tryptophan (Figs. 6C, D, E). and there within the ground pancreatic tissue (Fig. The acinar cells together with zymogen granules 5B). However, acinar cells are more or less located in the adipose tissue are positively stained for pyramidal in shape although oval or columnar types tryptophan histochemical test (Fig. 6F). However, are found occasionally. They are full of zymogen adipose tissue exhibits very weak reaction. The granules. The granules are relatively smaller in size concentrations of the zymogen granules in the and number than that of L. calbasu and occupy almost pancreatic acini of hepatopancreas are abundant and the entire area of the cell concerned. heavily loaded in comparison to spleenpancreas and Endocrine units of pancreas are closely adipose tissue pancreas. embedded within exocrine mass. Round or oval Mystus gulio: Exocrine pancreatic tissue of M. gulio shaped islet tissues containing 3 cell types are displays a strong positive result for tryptophan. The surrounded by fine fibers of the reticular connective coarse zymogen granules of the acinar cells together tissue which separate from serous acini (Figs. 5C, D). with the nuclei stain darkly with this histochemical The islets are richly supplied with blood vessels (Fig. test confirming the presence of tryptophan in the said 5B). Predominating α cells are generally located region (Figs. 7A, B). Moderately deposited zymogen outside in the islet of Langerhans. They are oval in granules exist as dispersed condition within acinar shape and contain dense nuclei and acidophilic cells. The blood vessels in between the acinar cells homogenous cytoplasm. Relatively larger β cells are also exhibit positive reaction for tryptophan. found in group, towards the center of islets. These cells are stained purple with aldehyde fuchsin (AF). Discussion By Heidenhain’s azan (HA) stain, β cell shows Fish exhibits great diversity in their food and feeding orange coloured flocculent cytoplasm and large habits. The structure of the digestive tract along with round central nucleus (Fig. 5D). δ cells are few in associated organs also becomes modified number, having faintly stained nuclei and found accordingly. As a notable digestive gland and also as throughout the islets (Figs. 5C, D). a principal endocrine organ, the pancreas is of Histochemistry: Tryptophan universal adventure among vertebrates. In the higher Labeo calbasu: In general, the exocrine pancreatic vertebrates, the pancreas is a compact and lobulated tissue shows relatively higher staining reaction for organ whereas in the lower once, it is highly variable Dimethylaminobenzaldehyde (DMAB)-Nitrate test. in nature. Among the teleosts, the pancreas shows a Intense staining reaction for tryptophan showing great diversity in its structure, morphology and deep blue colour has been detected in the anatomy. In the present study, the pancreas has been hepatopancreas of L. calbasu (Figs. 6A, B). detected in L. calbasu and M. gulio either in a diffuse 259 Iran. J. Ichthyol. (December 2016), 3(4): 251-265

primitive diffuse condition which exists in intrahepatic, intraspleenic and in the adipose tissues occurring among the loops of the intestine. Intrahepatic exocrine pancreatic tissue were also evidenced in Oreochromis niloticus, associated to afferent vases (Vicentini et al. 2005) and in Dicentrarchus labrax, the diffused pancreatic tissue was observed surrounding the digestive tract (Beccaria et al. 1992). In the present work, while in M. gulio, the pancreas is discrete in nature, adhered to the stomach wall and relatively more organized. Similar findings were also reported in Glossogobius giuris (Shrivastava & Chourasia 1976) Mystus vittatus (Dwivedi & Pandey 1982) and Wallago attu (Chakrabarti & Ghosh 2012). In the present investigation, the exocrine acinar cells in the hepatopancreas and spleen pancreas of L. calbasu are organized into several rows encircling central blood vessel. Many fish possesses a diffuse pancreas which has an intimate association and admixture of cells with the spleen or liver known as spleenpancreas or hepatopancreas respectively (Khaksar Mahabady et al. 2012). In L. calbasu, the zymogen granules in the acinar cells are numerous and larger in size in comparison to M. gulio. In herbivorous, L. calbasu the intestine is enormously coiled showing adaptation for retention of food materials for a large period of time and the only source and copious amount of digestive enzymes are Fig.7. Photomicrographs of sections of pancreas in M. gulio showing histochemical test for trypotophan needed for effective of enzyme is the hepatopancreas (Trypt). (A) Intense localization of tryptophan in the digestion of food materials. The pancreatic juices acinar cells (AC) of exocrine pancreatic tissue. Note along with bile juice after being secreted from the moderate reaction in blood vessels (solid arrow) and weak reaction in the wall of stomach (SW) (Trypt hepatopancreas is collected in the gall bladder and 40X). (B) Showing acute tryptophan reaction in the ultimately emptied in the alimentary canal. Yamane zymogen granules of AC and also positive reaction in blood vessels (BV) (Trypt 400X). (1973) gave an account of the presence of amylase in the cytoplasm of the acinar cells in diffuse pancreas condition and/or as a compact gland. Gonzalez et al. and confirmed that these cells were the centre of (1993) reported the presence of diffuse exocrine amylase production in the carp. The zymogen pancreas, which expanded through the mesentry and granules of acinar cells contain proenzymes forms islets in the connective tissue around some responsible for the digestion of proteins, digestive organs or disseminates within the carbohydrates, fats and nucleotides (Alboghohobeish intraperitoneal adipose tissue in Serranus cabrilla. In & Khaksar Mahabady 2005; Mokhtar 2015). L. calbasu, the pancreas generally occurs as a Moreover, the magnitude of the zymogen granules in 260 Ghosh and Chakrabarti- Cellular organization of pancreas in Labeo calbasu and Mystus gulio

the acinar cells of hepatopancreas in L. calbasu dominant, β cells are polyhedral and they grouped in appears to be higher than that of the spleenpancreas small clusters while δ cells are small, fusiform and and adipose tissue pancreas. Therefore, it may be argyrophilic cells. It has been marked that endocrine assumed that the acinar cells of hepatopancreas are cells have contacts with the blood sinuses and physiologically more active in respect to secretion of sometimes the secretory contents of the cells are digestive enzymes. Khaksar Mahabdy et al. (2012) observed into the blood vessels. Therefore, it may be recorded that the spleen pancreas of Barbus divined that after being secreted from the endocrine pectoralis performed many functions such as cells the hormones are carried out into the blood lymphatic cell production and digestion of food capillaries and thereby promoting movement of materials. In M. gulio the acidophilic zymogen glucose. In Carassius carassius, Cyprinus carpio, containing cells of the exocrine pancreas is for the Tinca tinca and Silurus glanis α, β and δ cells are production and storage of pancreatic enzymes that shoot shaped, have contacts with the capillaries and are delivered to the digestive tract through a network the hormones from the endocrine cells is carried out of ducts for effective digestion of protein-rich food via emiocytosis into the blood vessels (Iaglov 1978). materials. Field et al. (2003) reported that the The most significant hormones secreted by the islet exocrine pancreatic tissue produces digestive of Langerhans are insulin from β cell and glucagon enzymes, such as trypsin, amylase and from α cells. Both perform a momentous role in carboxypeptidase A for effective digestion of food in proper metabolism of sugars and starches in the fish zebra fish (Danio rerio). body. Insulin promotes the movement of glucose and The endocrine pancreatic tissue exists as other nutrients out of the blood and into cells. When Langerhan’s islets and is surrounded by exocrine blood glucose rises, insulin released from the β cells acinar cells in L. calbasu and M. gulio. The islets are causes glucose to enter body cells to be used for bounded by thin capsule and consist of comparatively energy. Moreover, it sometimes stimulates faintly stained cords of cells having conspicuous conversion of glucose to glycogen in the liver. Other nuclei and interspersed with blood vessels. The hormone, glucagon from α cells promotes the endocrine pancreatic cells of L. calbasu are mainly movement of glucose into the blood when glucose distributed within the adipose tissue surrounding levels are below normal. It causes the breakdown of intestinal bulb, intestine and bile duct. Similar stored liver glycogen to glucose, so that the sugar findings were also observed in Salmoniformes content of blood leaving the liver rises (Sorokin et al. (Wang et al. 1986) and grass carp (Mokhtar 2015). In 1982; Plisetskaya et al. 1985, 1986). However, δ the present observation, α cells are the most cells, which secrete somatostatin, are reported to be dominant cell types and most common on the dispersed mainly in the central region, intermingled periphery that appeared ovoid structure bearing oval with β cells (Stefan & Falkmer 1980; Abad et al. nuclei and intense acidophilic cytoplasm. Centrally 1986). placed β cells are comparatively larger in size, The detection and localization of tryptophan polyhedral in shape having granular cytoplasm. δ content has been observed in the exocrine acinar cells cells are less abundant, solitary and characterized at varied intensities in L. calbasu and M. gulio. with clear cytoplasm. Shyamsundari et al. (2006) Tryptophan, the precursor of pancreatic enzymes mentioned that ovoid islets of Langerhans are usually mainly associated with the zymogen granules of with a central cluster of β cells and α cells in the acinar cells. The concentration and magnitude of periphery in lizard fish Saurida tumbill. Mokhtar zymogen granules also differ from species to species. (2015) opined that the endocrine part of In the present investigation, thickly packed acinar Ctenopharyngodon idella, the ovoid α cells are cells are characterized with densely loaded zymogen 261 Iran. J. Ichthyol. (December 2016), 3(4): 251-265

granules in herbivorous detritus feeder; L. calbasu Osteoglossomorpha (Teleostei). General and and moderately loaded zymogen granules are Comparative Endocrinology 116: 409-421. observed in the carnivorous, M. gulio. Zymogen Beccaria, C.; Diaz, J.P. & Connes, R. 1992. Effects of granules remain in an inactive form in the acinar cells dietry conditions on the exocrine pancreas of the of the exocrine pancreas. These granules secrete sea bass, Dicentrarchus labrax L. (Teleostei). Aquaculture 101: 163-176. zymin in their secretory phase which helps to digest Chakrabarti, P. & Ghosh, S.K. 2012. Comparative carbohydrate food materials (Sinha & Moitra 1975). histophysiology of pancreas in Cirrhinus mrigala Variable intensities of tryptophan content in the (Hamilton, 1822), Oreochromis nilotica (Linnaeus, acinar cells of the aforesaid fishes may be related 1758) and Wallago attu (Bloch & Schneider, 1801). with the synthesis and secretion of pancreatic Indian Journal of Fisheries 59: 93-99. enzymes in different proportions according to their Chakrabarti, P. & Ghosh, S.K. 2015. Comparative requirement relating to their feeding habits. histological and histochemical studies on the Further, transmission electron microscopical and pancreas of Labeo rohita (Hamilton, 1822), Mystus immunocytochemical studies on the pancreas of vittatus (Bloch, 1790) and Notopterus notopterus L. calbasu and M. gulio are recommended to identify (Pallas, 1769). International Journal of Aquatic the cellular components and their proper functional Biology 3: 28-34. Dwivedi, A.K. & Pandey, D.N. 1982. Observation on the significance. pancreas of a freshwater fish, Mystus vittatus (Bloch). Reading in Zoology 1: 48-49. Acknowledgments Elbal, M.T.; Lozano, M.T. & Agulleiro, B. 1988. The The authors are grateful to Dr. S. Ray, Head, endocrine cells in the gut of Mugil saliens Risso, Department of Zoology, The University of Burdwan, 1810 (Teleostei): an immunocytochemical and for providing essential laboratory facilities and also ultrastructural study. General and Comparative delightful to Dr. P.P. Chakrabarti, Scientist-in- Endocrinology 70: 231-246. Charge, Regional Research Centre of ICAR-Central Faccioli, C.K.; Chedid, R.A.; Bombonato, M.T.S.; Institute of Freshwater Aquaculture, Kalyani Field Vicentini, C.A. & Vicentini, I.B.F. 2014. Station, Santalpara for providing the fish specimens. Morphology and histochemistry of the liver of carnivorous fish Hemisorubim platyrhynchos. International Journal of Morphology 32: 715-720. References Field, H.A.; Si Dong, P.D.; Beis, D. & Stainier D.Y.R. Abad, M.E.; Agulleiro, B. & Rombout, J.H. 1986. An 2003. Formation of the digestive system in zebra immunocytochemical and ultrastructural study of fish. ii pancreas morphogenesis. Developmental the endocrine pancreas of Sparus auratus L. Biology 261: 197-208. (Teleostei). General and Comparative Fortin, J.S.; Santamaria-Bouvier, A.; Lair, S.; Dallaire, Endocrinology 64:1-12. A.D. & Benoit-Biancamano, M. 2015. Anatomical Adams, C.W.M. 1957. p-dimethylaminobenzaldehyde and molecular characterization of the endocrine nitrate method for the histochemical demonstration pancreas of ateleostean fish: Atlantic wolfish of tryptophan and related compounds. Journal of (Anarhichas lupus). Zoological Studies 54: 21. Clinical Pathology 10: 56-62. Geyer, H.J.; Nel, M.N. & Swanepoel, J.H. 1996. Alboghohobeish, N. & Khaksary Mahabady, M. 2005. Histology and ultrastructure of the hepatopancreas Histological study of liver and pancreas in of the tigerfish, Hydrocynus forskahlii. Journal of Ctenopharyngodon idella. Journal of School of Morphology 227: 93-100. Veterinary Medicine, Shahid Chamran University, Ghosh, S.K.; Barun, S. & Chakrabarti, P. 2016. A Ahvaz 11: 25-34. comparative study of the histoarchitecture of Al-Mahrouki, A.A. & Youson, J.H. 1999. Ultrastructure endocrine pancreas in Labeo bata (Hamilton, and immunocytochemistry of the islet organ of 262 Ghosh and Chakrabarti- Cellular organization of pancreas in Labeo calbasu and Mystus gulio

1822), Sperata aor (Hamilton, 1822) and Chitala the grass carp (Ctenopharyngodon idella). chitala (Hamilton, 1822). International Journal of European Journal of Anatomy 19: 145-153. Aquatic Biology 4: 17-24. Naguib, S.A.; Rizkalla, W. & Abd EI-Ghafar, F.A. 2009. Gomori, G. 1941. Observations with differential stains on Comparative histological and ultrastructural studies human islets of Langerhans. American Journal of on the liver and pancreas of Schilbe mystus and Pathology 17: 395. Labeo niloticus. Egyptian Journal of Aquatic Gonzalez, G.; Crespo, S. & Brusle, J. 1993. Histo– Biology and Fisheries 13: 107-127. cytological study of the liver of the cabrilla sea Nejedli, S. & Gajger, I.T. 2013. Hepatopancreas in some bass, Serranus cabrilla (Teleostei, Serranidae) an sea fish from different species and the structure of available model for marine fish experimental the liver in teleost fish, common Pandora, Pagellus studies. Journal of Fish Biology 43: 363-373. erythinus (Linnaeus, 1758) and whiting, Halami, N.S. 1952. Differentiation of the two types of Merlangius merlangus euxinus (Nordmann, 1840). basophils in the adenohypophysis of the rat and the Veterinarski Arhiv 83: 441-452. mouse. Stain Technology 27: 61. Pandian, T.J. 1968. Feeding habits of the fish Megalops Hossain, M.Y.; Islam, R.; Hossen, M.A.; Rahman, O.; cyprinoides in the Cooum backwaters, Madras. Hossain, M.A.; Islam, M.A. & Alam, M.J. 2015. Journal of the Bombay Natural History Society 65: Threatened fishes of the world: Mystus gulio 569-580. (Hamilton, 1822) (Siluriformes: Bagridae). Petcoff, G.M.; Diaz, A.O.; Escalante, A.H. & Goldenberg, Croatian Journal of Fisheries 73: 43-45. A.L. 2006. Histology of the liver of Oligosarcus Iaglov, V.V. 1978. Morphology of the endocrine portion jenysii (Ostariophysi, Characidae) from Los Padres of the teleost pancreas. Arkhiv Anatomii, Gistologii Lake, Argentina. Iheringia Série Zoologia 96: 205- I Embriologii 74: 111-115. 208. Khaksary Mahabady, M.; Morovvati, H.; Arefi, A. & Plisetskaya, E.M.; Pollock, H.G.; Rouse, J.B.; Hamilton, Karamifar, M. 2012. Anatomical and J.W.; Kimmel, J.R. & Gorbman, A. 1985. histomorphological study of spleen and pancreas in Characterization of coho salmon (Oncorhynchus Berzem (Barbus pectoralis). World Journal of Fish kisutch) insulin. Regulatory Peptides 11: 105-116. and Marine Sciences 4: 263-267. Plisetskaya, E.M.; Pollock, H.G.; Rouse, J.B.; Hamilton, Khanna, S.S. 1963. On the pancreas of some freshwater J.W.; Kimmel, J.R. & Gorbman, A. 1986. Isolation teleosts. Journal of Zoological Society of India 3: and structures of coho salmon (Oncorhynchus 1-19. kisutch) glucagon and glucagon-like peptide. Kiernan, J.A. 2008. Histological and Histochemical Regulatory Peptides 14: 57-67. Methods: Theory and Practice. 4th Edition. Scion Shyamasundari, K.; Raja Kumari, V.J.V. & Hanumantha Publishing, Bloxham, UK. Rao, K. 2006. Observation on the pancreas of the Klein, C. & Van, N.S. 1978. Use of immunocytochemical marine lizard fish Saurida tumbil (Bloch). Journal staining of somatostatin for correlative light and of Fish Biology 21: 449-454. electron microscopic investigation of D cells in the Sayrafi, R.; Najafi, G.; Rahmati-holasoo, H.; Hooshyari, pancreatic islet of Xiphophorus helleri H. A; Akbari, R.; Shokrpoor, S. & Ghadam, M. 2011. (Teleostei). Cell and Tissue Research 194: 399- Histological study of hepatopancreas in Hi Fin 404. Pangasius (Pangasius sanitwongsei). African Legouis, P. 1873. Recherches sur les tubes de weber et sur Journal of Biotechnology 10: 3463-3466. le pancréas des poissons osseux. Annales des Sheybani, M.T.; Adibmoradi, M. 2002. Histological study Sciences Naturelles-Zoologie et Biologie Animale of the liver and pancreas and their ducts in 17: 1-107. Acipenser stellatus. Journal of Faculty of Mallory, F.B. 1936. The aniline blue collagen stain. Stain Veterinary Medicine, University of Tehran 57: 19- Technology 11: 101. 23. Mokhtar, D.M. 2015. Histological, histochemical and Shrivastava, S. & Chaurasia, R.C. 1976. Pancreas in ultrastructural characterization of the pancreas of certain freshwater teleosts. Current Science 45: 263 Iran. J. Ichthyol. (December 2016), 3(4): 251-265

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264 Iran. J. Ichthyol. (December 2016), 3(4): 251–265 Received: September 03, 2016 © 2016 Iranian Society of Ichthyology Accepted: November 18, 2016 P-ISSN: 2383-1561; E-ISSN: 2383-0964 doi:10.7508/iji.2016.02 http://www.ijichthyol.org

مطالعه مقایسهای بافتشناسی و هیستوشیمی پانکراس (Labeo calbasu (Hamilton, 1822 و (Mystus gulio (Hamilton, 1822

* ساروج کومار قوش1 ، پادمانابها چاکرابارتی2

1گروه جانورشناسی، بجوی نارایان ماهاویدیاالیا، ایتاچونا، هوقلی712147، بنگال غربی، هندوستان. 2آزمایشگاه شیالت، گروه جانورشناسی، دانشگاه بوردوان، بوردوان 713104، بنگال غربی، هندوستان.

چکیده: ساختار سلولی و طبیعت هیستوشیمی سلولهای آسینار ترشح کننده آنزیم لوزالمعده در دو گونه ماهی Labeo calbasu و Mystus gulio با عادات غذایی متفاوت مورد بررسی قرار گرفت. تحلیل بافتشناسی نشان داد که در گونه L. calbasu بافتهای ترشحی برونریز لوزالمعده در داخل پارانشیمهای کبدی بهصورت کبدی-لوزالمعدی و در طحال بهصورت طحال- لوزالمعدی پراکنده شده است. همچنین تحلیل ساختار نشان داد که بافتهای ترشحی برونریز و درونریز لوزالمعده به صورت چسبیده به بافتهای چربی بین مارپیچ روده گونه L. calbasu دیده میشود. در گونه M. gulio، بافت مجزای لوزالمعده به دیواره معده چسبیده است و مجموعهای از آسینی پراکنده شده در نواحی کوچک از جزایر النگرهانس و رگهای خونی را شکل میدهد. سلولهای آسینار ترشحی برونریز لوزالمعده با هستههای واضح و گرانولهای زیموژن اسیدوفیلی به شدت متراکم هستند. بخش درونریز لوزالمعده، جزایر النگرهانس بهوسیله یک بافت پیوندی نازک و متشکل از سلولهای β ،α و δ محاط شده است. انواع سلولهای متفاوت در جزایر عمدتاً براساس شکل، موقعیت، شدت رنگ آمیزی و تراکم گرانولهای ترشحی سیتوپالسمی تمایز مییابند. سلولهای α حاوی سیتوپالسم بهصورت نازک گسترده شده و هستهای بهصورت متراکم رنگآمیزی شده است و بهطور معمول در پیرامون جزایر النگرهانس واقع شدهاند. سلولهای β مثبت به رنگآمیزی فوشین آلدهید، هسته کروی و سیتوپالسم گرانوالر داشته و در مرکز جزایر النگرهانس متصل به رگهای خونی قرار دارند. سلولهای δ کم تعداد و با هسته روشنتر در رنگآمیزی بوده و جزیره لوزالمعدی پراکنش یافتهاند. تشخیص هیستوشیمی نشان میدهد که گرانولهای زیموژن سلولهای ترشحی آسینار برونریز تراکم رنگآمیزی واکنش تریپتوفان را نشان میدهند که به پیش مادههای اولیه آنزیمهای لوزالمعدی فراوان که احتماالً به نوع غذا و عادات غذایی ماهی مرتبط هستند، مربوط میباشند. گرانولهای زیموژن از نظر اندازه بزرگتر هستند و بهشدت در سلولهای آسینار هپاتوپانکراس گونه L. calbasu در مقایسه با گونه M. gulio تجمع یافتهاند که ممکن است با رفتار تغذیه گیاهخواری در پاسخ به ترشح آنزیمهای گوارشی مرتبط باشد. کلماتکلیدی: ساختار بافتشناسی، تریپتوفان، پانکراس، تحلیل ساختار، کپورماهیان، باگریده.

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