Electron microscopic studies of the hepatopancrcatic tubule revealed four distinct cell type . The Embryonic cells were undifferentiated with a large nucleus and cytoplasm surrounding it. Golgi complex and free ribosomes wcre abundant. Fibrillar (F-cells) contained extensive rough endoplasmic reticulum with ribosome - studded cisternae. The golgi cisternae were always dilated with numerous zymogen grnnules. Secretory (B-cel1s) showed many vesicles and a large vacuol

Hemambika, M

Paulraj, R

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'nll.: Fourth Indian Fj):heries Forum Proceedings 24-28. November. 19%. Kochi p. 291·294. Published in 1999. An Ultrastructural Study of the Hepatopancreas of Indian White Prawn Penaeus indicus H. Milne Edwards M. HEMAMBIKAand R. PAULRAJ* Central Marine Fisheries Research Institute, Kochi 'Madras Research Centrc o f CMFRI, 6811 Greams Road, Chcnnai - 600 006,lndia. Abstract Electron microscopic studies of the hepatopancrcatic tubule revealed four distinct cell typc.~ . The Embryonic ([-cells) were undifferentlated with a large nucleus and cytoplasm surrounding it. Golgi complex and free ribosomes wcre abundant. Fibrillar (F-cells) cont..1ioed extensive rough endoplasmic reticulum with ribosome - studded cisternae. 1llc golgi cistern:le were nlways dilmed with numerous zymogen grnnule.~. Secretory (B-cel1s) showed many vesicles and a liltgC vacuole. Microvillar border wa~ prominent. Absorptive (R-cclls) were c1mf:lcterized by the presence of lipid material. The cytoplasm showed a number of mitochondria. rough endopl;:l.~mic reticulum and golgi complex through out the ceIL 111e<;e cellular Mructural make-up reflected the complex functions of the digestive gland in food absorption. secretion of digeslh'c enzymes. storage and depot of lipids. glycogen and minerals. Introduction The crustacean hepatopancreas has attracted the interest of scientists for at least a century and a half, yet as Loizzi ( 1966) commented. " the total information accumulated on thi s organ is meagre, when compared to other areas of biology". That the hepatopancreas is a vital and major organ in the Crustacea is beyond doubt, involved in diverse metabolic activities, it is primarily rcsponsible for the synthesis and secretion of digestive enzymes and subsequent uptake of nut rient materials, but is also implicated in excretion. the moulting cycle, the storage of inorganic reserves, and in the lipid and carbohydrate metabolism (Gibson a nd Barker, 1979). Recently cytology of the hepatopanereas of decapod crustaceans has been stud ied extensively by light and electron microscopy. Early histological studies defined two cell types but, since the classification of Hirsch and Jacobs (1928), the E-, R-, F-and B-cells have been described in many decapods. Recent studies on the Reptantia or walking fonns of decapods (Hopkin and Nott, 1979, 1980; Erri-Babu et al., 1982), have revealed further cytological charactcristics and con fimled the earlier observations that the tubule epithelium contains fo ur cell types. The Natantia or swimming forms have been largely neglected, except for the work of Pillai ( 1960) and Dall ( 1967), wherein they have identified four and three types of cell s in Caridilla laevis and Metapellaeus bel1aetlae respectively. Papathanassiou and King ( 1984) and A I-Mohan na ef al .. (1985) have distinguished fou r types by electron microscopy. The Indian wh ite prawn , Penaells illdicllS is an ecologically important and widely distributed species which has potential for economic exploitation. It is of interest, therefore to, examine the ultrastructural make up of the complex organ -hepatopancreas. 291 Materials and Methods Specimens of Penaells indicus of size tOO - 120 mOl collected from the culture ponds of Central Marine Fisheries Research Station at Narakkal were maintained in the laboratory for 2 to 3 days. Animals in their intermoult stages were selected and dissected live in ice. The gland as a whole was fixed in ice-cold 5% gl utaraldehyde solut ion in phosphate buffer of pH 7.2. A few glands dissected wcre fixed in 5% glutaraldehyde solution in buffered sodium cacodylate (PH 7.2) for I hour at 0-4"C. The glands cut into small pieces were then washed in several changes of buffer solution followed by post fixation in 0.5% Osmium tetroxide solution tor I hour at 0-4"C. Afterdchydration in graded levels of cold acetone 25%, 75%, 95%, 100%), the material was embedded in Spurr resin. Sections were cut wi th a ul tram icrotome (LKB Bromma 8800 ultrotome) and mounted on copper grids. Thc ultrathin (6OO"A) sections were then double stai ned in 30% uranyl acetate (30 minutes) followed by lead citrate (10 minutes) and viewed in a Carl Zeiss Transmission Electron Microscope t 09 R - at an accelerating voltage of 80 Kv). Electron micrographs were taken using AGFA Ortho 25 fi lm . Results and Discussions Gelleral morphology oJthe Ilepatopallcreas The reddish brown hepatopJ llc reas, is a large compact organ which occupies the greater part of the cephalothoracic cavity, posterior to the cardiac portion of the stomach. 11 has two separate lobes which are composed of compact arrays of blindended tubu les; each lobe connects ventrally with the gut at the junction of the pyloric stomach and the anterior end of the midgut. Each tubule under light microscopic studies consists of four cells types which can be classified as E-, F-, B-, and R-cells according to the scheme of Jacobs (I928). The E- cells, 'Embryonic cells' are undifferentiated and localized at the bl ind end of the tubule. F and R- cells occupy mid distal region. B cells occupy the mid region and are mostly interspersed wilh R- cells. The proximal portion of the tubule is lined predominantly by R- cells with few F- cells in between (Hemambika, 1989). There are four cell types in hepatopancreas of P. indicus as observed under electron microscope (Plates - I and 2). Cell type I : E- Cell (Embryonic Cell): These are undifferentiated cell (Plate Ib) and have high nuclear to cytoplasmic ratio. They contain few mitochondria having randomly oriented cristae. Proximal to the nucleus there are Glogi Complexes and moderate amounts of rough endoplasmic reticulum (plate Ib). The cytoplasm possess abundant free ribosomes and single membrane recticulurn. Microvilli are present on the border adjacent to the lumen (plate la,b). The E- cell at the blind ends of the tubule in decapods are generally believed to be involved in mitotic activity (G ibson and Barker, 1979) for the production of other cell types which Plate I a) Electron micrograph of hepulopancrea<; of Penacus indicus showing the different celilypes (E. F. B & R cells) x 3,000 b) F, R. E cells with rough endoplasmic reliculum. GoJgi cisternae. N-nucleus. L-lipid. g-globule, m-milOchondria x 7000. c) E. Embryonic cell with Goigi complexes (gc). rough endoplasmic recticulum in the cytoplasm and vesicles of smooth endoplasmic reticulum. d) F-Fibrillar cell with vesicles (V), golgi cisternae (gc), few metochondria (01) and a centrally located nucleus (n). Note the abundant rough endopia .. mic reticulum (rer) db-dense bodies. X 3000. 292 comprise the tubule epithelium. The E- Cell of Penoells il1dicliS as observed in the present study is similar to the E - Cell observed in other crustaceans (Van Wecl , 1955; Davis and Burnett. 1964: Bunt, 1968; Stanier et al., 1968: Gibson and Barker. 1979; Papathanassiou and King, 1984; AI-Mohanna et al .. 1985). Cell Type - /I : F-Cells (Fibrillar cell) The cell (Plate la) contain extensive rough endoplasmic reticulum with long, narrow ribosome studded cisternae. The golgi cisternae in the cells arc always dilated and the entire complex is ringed by small dense vesicles (Plate I b and d). The cytoplasm conta in s abundant free ribosomes but fewer mitochondria. The structure of these cells resemble that of the F-cclls in the Crayfish hepatopancreas (Loizzi, 1971 ). Extremely electron dense cytop lasmic inclusions arc occasionally ohservable in the fibrillar cells. Each inclusion is enclosed within a membranous vacuole and consists of granular material which is concentrated centrally to form a rather solid core surrounded by in'egular clumps of the dense material. These are the zymogen granules. Based on these structural observations in cells it may Plme 2 a) Electron micrograph showing the speciai mcmbrane of the Band R cel ls - Mv. microvilli, db - dense bodic. ... lu - lumen between the adjacl!nt cell s x 7000. b) A portion of secret ary cell (8) show in g the vesicles filled with proteinaceous substances althe periphery X 12000. e) Absorptive R cell with lipid droplets and dense inclusions and autophagic vaclloles (av). X 7000. d) R-eeli with lipid droplet (I) and mitochondrion (m) and cisternae. X 7000. be noted that they are probably designed for manufacture of digestive enzymes. Similar supporting observations have been made by Bunt (1968) and Stanier el al., (1968) in other crustaceans. It may be recalled that the sequence of cell transformation as put forth by Jacobs ( 1928) for Astacus as E-cell> F-cell>B-cell is very evident in this species, also by the increased amount of rough endoplasmic reticulum and again the secretory cells, secret ing their products into the lumen. Loizzi (1971) suggested that F-cells in cray fish are involved in synthesis of digestive enzymes because of their rich concentrations of rough endoplasmic reticulum. Here again their cisternae are filled with a nocculcnt material (plate I b). They may be actively involved in digestive enzyme synthesis which the cell sequester them in a supranuclear vacuole which enlarges by pinocytic intake of luminal nutrients and fluids. Such kinds of cells arc also reported in vertebrate pancreas (Fawcett, 1966). Within the F-cells of P. indicus also active endoplasmic reticulum and Golgi complex are seen supporting their role in enzyme secretion. Inside the Golgi vesicles are seen certain moderately electron dense, somewhat flocculent material which may be interpreted as secretory products of proteinaceous nature, again indicative of active protein synthesis. C _II T\'pe - 11/ : n -Cell, I Secretory Cells) ['he ... \.! l·eJls contain large number of ves icles, which inili:tll: ()ct:upy :l t:ollsiderahle part of the cell and eventually occlude il (Pbtc 2a and b) or sometime contain a single large vacuole enclosed by a thin shell of cy:oplasm. Separating the vacuole from the lumen is an apical complex consisting of a microvillar border, dense cytoplasm. Small mitochondria and a meagre surface enteric coat. These are the secretory cells and resemble the B-cells of Davis and Burnett (1964) and Stanier el al .. ( 1968). These cells are sometimes characterised by a single large vacuole, and may be considered to be essentially an F-cell in which the small secretory vacuoles have coalesced (Plate 2b) 10 form a single large vacuole and a compressed basal nucleus. The contents of the vacuole have obviously been affected by sol vents used during dehydration and embedding. Loizzi (1971) has attributed that in crayfish these cells are secretory in nature and 8-cell secretion involves pinching off the apical complex followed by extrusion of the enzyme-rich vacuolar contents. Some what similar components are also observed in the 8-cell ofP. indicus indicating their functional significance in secretion. Cell Type-IV: R-Cell (Absorptive Cell) These cells are characterized by the presence of lipid material (Plate 2c and d). The cytoplasm contains a number of mitochondria, some of which are concentrated below the apical plasma membrane (Plate I aJ. The nucleus is centrally or basally 293 located and nucleolus is present. The apical membrane of the cells have microvilli, the surface of which are covered by short filaments. Rough endoplasmic reticulum and golgi complex occur through out the cell. Smooth en?oplasmic reticulum is restricted to the basal region of the cell. Apically, the cell resembles a vertebrate intestinal absorptive epithelium with a dense brush border and an organelle - free region lying below the microvilli. Autophagic vacuoles containing material of different configurations with low electron density are present in the cytoplasm (Plate 2d). These cells arc similar to those described as R-cells in the Crayfish (Loizzi, 1971). The function of lipid storage may be attributed to this cell , because orthe presence of striated apical border, believed to be involved in food absorption. Again light microscopic observation ofhistochemicaltcsls reported dense concentration of lipids in membrane bound vacuoles. Hence, their role in storage and metabolism of glycogcn and lipids. Gopalakrishnan ( 1957) also noted si milar cells in P. illdieus under light microscopic observations. However, he could only distinguish secretory, storage and basal cells. In the present investigation. ultrastructural details could be well delineated on account of higher resolution and this lead to the identification of four type of cell in the tubular epithelium ofhepatopancreas. This findi ng support the work of Papathanassiou and King (1984), AI-Mohanna el al., (1985) on Palaemon serratus and Paellae"s sel1lisu/cattlS respectively. Acknowledgments I have great pleasure in acknowledging my deep sense of gratitude to Dr.N.P. Jayashankar, Joint Director and Dr. J.J. Solomon, Senior Scientist at Central Plantation Crops Research Institute, Kayamkulam, Kerala for providing all the facilities to carry out electron microscopical studies. I am also thankful to my teacher. Dr. R. Paul Raj for his valuable guidance. The study was supported by ICAR, New Delhi. References AI-Mohanna, S.Y., Nott, J.A. and Lane, DJ.W, 1985. M -Midget cells in the hepatopanereas of the shrimp, Penaeus semisulcatus Dc Haan, (Decapods, Natantia). Cruslaeealla, 48(3): 260-268. Bunt, A.H. , 1968. An ultrastructural study of the hepatopancreas of Proeambart/s c1arkii (Giard) (Decapoda, Astacidae) Crt/slaeealla, 15: 282-288. Dall , W, 1967. The functional anatomy of the digestive tract of a shrimp Metapenaeus bennettae Racek and Dall (Crustacea; Decapoda: Penaeidae). Ausl. J. Zool., 15: 699-714. Davis, L.E. and Burnett,A.L., 1964. A study of growth and cell differentiation in the hepatopancreas of Crayfish. Dev!. Bioi , 10: 122-153. Erri Babu, D. Shyamasundari , K. and Hanumantha Rao, K. , 1982. Studies on the digestive system of the Crab, Menippe rumphii (Fabricius) (Crustacea: Brachyura) J. Exp. Mar. Bio. Eeol, 58: 175-191. Fawcett, D. w., 1966. The cell W.B. Saunders Compan y, Philadelphia. 225 pp. Gibson R. and Barker, PL, 1979. The decapod hepatopancreas. Oeeanogr. MG/: BioI. 17: 285-346. Gopalakrishnan, v., 1957. Studies on the biology of Madras Penaeids. University of Madras. Unpublished Thesis. PP. 1-95. Hirseh, G.c. and Jacobs. w., 1928. Der Arbeitrhythmus der Milleldarmdruse von ASlaells leptodactyills. Z. Vergl. Physiol., 8: 102-144. Hemambika, M., 1989. Studies on the digestive e nzymes of the Indian White Prawn Pel1aeus indiclls H. Milne Edwards. Unpublished Thesis. Hopkin, S.P. and NOll, J.A., 1980. Studies on the digestive cycle of the shore Crab Carcifllls maenas (L. ) with special reference to the B-cell in the hcpatopancrcas. J. Mat: Bioi. Assoc. u. K. , 60: 891-907. 294 Jacob, W., 1928. Undersuchungen ubcrdie cytologic def sekeret bildung in def Mitteldarm druse von Astacus leplodaetyills. Zeitsc/lI:, ZellJorseh. MikroskAllol. 8 (I): 1-62. Loizzi, R.F., 1966. Interpretation of Crayfish hepatopancreati c function based on fine structural analysis of epithelial cell lines and muscle nct work. Zell forseh. Mikroskip. AIIOI. , 113(3): 420-440. Lozzi R.F. and Petcrson , D.R. , 1971. Lipolytic sites in the crayfish hepatopanc rcas and correl ation with fine structure. Compo Bioehem. Physiol. 39B(2): 227-236. Papathanassiou, E. and King, P.E., 1984. Effects of starvation on the fine structure of the hepatopancreas in the common Prawn. Palaemoll serratw; (Pennant) . Compo Rioelzem. Physiol. , 77 A (2): 243-249. Pillai, R.S ., 1960. Studies on the shrimp Caridillo laevis (Heller). The digestive system. Jour: Mar: Assoc. India. 2: 57-74. Stanier, J.E., Woodhouse, M.A. and Griffin,R.L., 1968. The jjne structure of the Hepatopancreas of Carcinlls maenas (L.). (Decapod a : Brachyura), Crustaeealla, 14: 56-66. Van Weel, P.B. 1955. Process of secretion , restitution and resorption in the gland of midgut (Glandular media intenstini) of Atya Spinipes (Decapoda : Brachyura). Physiol. Zool. 28: 40-54. 

An ultrastructural study of the Hepatopancreas of Indian white prawn Penaeus indicus H. Milne Edwards

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An ultrastructural study of the Hepatopancreas of Indian white prawn Penaeus indicus H. Milne Edwards

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