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MORPHOLOGY AND HISTOLOGY OF THE DIGESTIVE GLAhTD OF Oxychilus (Drouetia) atlantic~is(MORELET & DRO~T)(: PLKMONATA)

M. LOPES, A. RODRIGUES & I. MARIGOMEZ

LOPES, M, A. RODRIGIJES& I. MARIGOMEZ. 2001. Morphology and histology of the digestive gland of Oxycliilus (Dt-ouetia) atlarzticus (Morelet & Drouet) (Gastropoda: ). Arquipélago, Life and Marine Sciences. Supplement 2(Part B): 7 1-76. Ponta Delgada. ISSN 0873-4704.

Little information exists on the histology of digestive system. This study deals with a detailed characterisation of the differeni types of cells comprising epithelium lining the digestive gland of 0,rychilus atlar~ricus.Frorn liglit and scanning electron microscope (SEM) studies, three types of differentiated cells can be identified in the digestive gland: digestive cells, cxcretory cells and calcium cells. Digestive cells are the most numerous, and are present in two forms, one believed to be absorbing food material and the other secreting material. Excretory cells are distinguished by having a large central vacuole, containing excretory granules. Calcium cells contain spherules of calcium salts. which have a characteristic birefringence.

M. Lopes, A. Rodrigues,(e-mail:[email protected]),Universidade dos Açores, Departanlerito de Biologia, Rua da Mãe de Deus 58, PT - 9501-801 Ponta Delgada, Portugal; I. Marigome:, Biologia Zeli(1arra Arloa, Zoologia eta Anirnali Zelulen Dinar7iika Saila, Euskal Herriko Unibertsitateu. 644 P. K. ES - 48080 Bilbao, Spain.

INTRODUCTION voluminous organ, which develops from the endoderm and constitutes a large part of the visceral mass (KRESS et al. 1994). This gland, Oxyclzilus (Drouetia) atlntzticus, a terrestrial also called mid-gut gland, is a much-branched zonitid gastropod endemic to São Miguel island organ ramifying through most of the visceral (Azores), has been successfully subjected to mass and ending in numerous blind, small predatory experiments on Lynuiaea truncatula, tubules. The tubules open into ductules which thus showing the carnivorous behaviour of the join to form ducts, and latter combine to form the species under laboratory conditions (CUNHA hepatic ducts which empty into the stomach 1991). Further works, concerning the analyses of (GRIEBEL 1993). stomach contents from individuals collected in According to OWEN (1966) the digestive the field, have shown the omnivorous tendency of gland is thought to perform the following this group in natural conditions (LOPES et al. functions: absorption of ingested food material, 1997). extracellular and intracellular digestion, secretion, Some studies have been undertaken excretion and osmoregulation. concerning the alimentary preferences within the Histological studies on the gastropod digestive family Zonitidae (Boyco~r1934; FROMMING gland have shown that cells structure and function 1954; RONDELAUD 1975; MORDAN 1977; vary even within the sane group (KRESS et al. RONDELAUD 1977; DIDIER & RONDELAUD 1989) 1994). Accordingly, severa1 different types of but only a few on the anatomy and histology of cells have been identified and described in the the alimentary tract (RrGsu 1963; LOPES et al. digestive gland. The most common types of cells 1998). are: digestive cell, microtubule-containing cell, The digestive system of gastropods includes, mineral-containing granule cell, excretory cell besides the alimentary tract, the digestive gland, a and calcium cell (RIGBY 1963; LURY et al. 1967; WALKER 1970; KRESS et a]. 1994; GRIEBEL RESULTS 1993). The present study provides a detailed The hepatic ducts of O. atlanticus are lined by a characterisation of the different types of cells ciliated columnar epithelium laying on a thin lining the digestive gland epithelium of O. basement membrane. atlanticus and, in the process, attempts to The fresh digestive gland is generally describe the morphological organisation of the yellowish-brown and consists of a single layered gland. epithelium, supported by a thin basement membrane bound by connective tissue and muscle MATERIAL AND METHODS fibres. According to structural features, three types of cells are identified within the digestive gland Specimens of Oxychilus atlanticus were collected epithelium: Cell type I, Cell type I1 and Cell type at Abelheira near Ponta Delgada (São Miguel I11 (Figure 1). Island). For light microscopy, specimens were dissected and the digestive gland removed and Cell type I (Figure 2, A) fixed in Bouin. Following fixation, the material was dehydrated and then, embedded in paraplast The Cell type I is the most abundant in the and stained with Haematoxylin/Eosin (MARTOJA digestive gland, corresponding to 82% of the & MARTOJA-PEARSON 1970). For the detection of lining epithelium. They are columnar in shape calcium, material was fixed in Carnoy and subject with apical walls either level or round in contour. to the Stoeltzener process (MARTOJA & The cells with level apical surfaces possess a well MARTOJA-PEARSON 1970). The blocks of developed brush border, in contrast with the ones paraplast were cut to the thickness of 7 pm. that have rounded apical surfaces, where the same For scanning electron microscope (SEM) structure is less conspicuous. These cells average examination, a particular procedure was followed. 50 pm in height and 17 pn in basal width. Nuclei The material was fixed in Bouin and embedded in are basal and oval, and nuclear chromatin can be paraplast, a method usually followed for routine observed as small granules. Much of the cell optical microscopy. Following this procedure, 60 volume is filled with granules that give a range of pm sections were cut, producing blocks with colours, according to their stain reaction. regular surfaces. The blocks were then submitted to a dehydration procedure and finally immersion in hexamethyldisilazane. The liquid phase of the Cell type I1 (Figure 2, B) hexamethyldisilazane evaporates in a few minutes, leaving cells whose ultrastructure is These cells are globular, averaging 30 pm in comparable to that of the cells dried by critical- height and 13 pm in basal width. Much of the cell point drying (GOLDSTEIN et al. 1992). For volume is taken up by a spherical central vacuole. stereology studies, in order to obtain the relative Within this, there are excretory granules, which frequency of the different cells types, was used either occur singly or are joined in small groups. the reticule Weibel Multipurpose Test System The free surface of the cell presents a well M168 (WEIBEL 1979). The reticule was defined brush border. The nucleus is basal when superimposed on the image of the digestive gland the cell reaches the mature stage, corresponding tubules using a drawing-tube adapted to a to the vacuole maximum volume. The cytoplasm microscope Leitz Laborlux S (Leica). The presents granules that give a similar stain reaction sections of each portion of the digestive gland as the ones found in the cell type I. Cell type I1 were randomly selected and viewed at 100x. corresponds to 10% of the lining epithelium. Figure 1. Digestive gland epithelium. C I, Cell type I; C 11, Cell type 11; C 111, Cell type 111. Scale bar 10 ~m.

Cell type I1 (Figure 2, B) continuously produced secretion flows out of it (VAN WEEL 1950). These cells are globular, averaging 30 pm in The digestive gland epithelium of O. height and 13 pm in basal width. Much of the cell atlarzticus is composed of three distinct types of volume is taken up by a spherical central vacuole. cells: Cell type I, Cell type I1 and Cell type 111. Within this, there are excretory granules, which Following the structural description of the either occur singly or are joined in small groups. digestive gland of Agriolimax reticulatus The free surface of the cell presents a well (WALKER 1970), the cell types found in 0. defined brush border. The nucleus is basal when atlanticus correspond, respectively, to digestive, the cell reaches the mature stage, corresponding excretory and calcium cells. Therefore, our results to the vacuole maximum volume. The cytoplasm are in contrast wjth the two types of cells presents granules that give a similar stain reaction described by RIGBY (1963) for 0. cellarius, as the ones found in the cell type I. Cell type I1 digestive and excretory cells. Other authors have corresponds to 10% of the lining epithelium. also described the same three types of cells for various pulmonates (VAN WEEL 1950; GUPTA 1977; DMITRIADIS& HONDROS 1992). DISCUSSION AS suggested by DIMITRIADIS& HONDROS (1992) for Helix lucorum, in 0. atlanticus the The hepatic ducts are responsible for the transport digestive cells constitute the major part of the of materials to and from the digestive gland. This lining epithelium. function is assisted by features of its lining The range of colours observed in the digestive epithelium, where the movement of the cilia cells cytoplasm of 0. atlanticus could be creates a current that aids the transport of explained, according to KRESS et al. (1994), by particles into the digestive gland, whereas a food vacuoles at different stages of digestion. Fig. 2. Digestive gland epithelium (S.E.M.). A (scale bar 10 ym), Digestive cell; B (scale bar 5 ym), Excretory cell; C (scale bar 16 ym), Calcium cell. L, Lumen; * Vacuole; * Brush border; ) calcium spherules.

On account. of their structural features, the well developed brush border, while the other, digestive cells can be divided in two different mainly involved in secretion, presents a rounded forms: one, believed to be absorbing food npical surface with a less conspicuous brush material, has a leve1 apical surface that possess a border. The presence of two digestive cell forms has been referred to in other pulmonates Científica. Universidade dos Açores, Ponta (DAGUZAN 1985; WALKER 1970). Delgada. The similar reaction of the granules found in DAGUZAN, J. 1985. Principales caractéristiques de digestive and excretory cells of 0. atlanticus I'appareil digestif, de Ia nutrition et de Ia digestion chez les mollusques. Année Biologique 4: 367-406. suggests a close affinity between both types of DAS, S., K.K. MISRA& K.C. GHOSE. 1992. The cells. This reaction was also noted for other digestive gland of gastropods eith different feeding species by WALKER (1970) and DAGUZAN habits. Proceedings of the Ninth Eurupean (1985), who considered that excretory cells are Malacological Congress: 117- 124. derived from digestive cells. DIDIER, B. & D. RONDELAUD. 1989. Les Calcium cells are characterised by the caractéristiques des proies consommées par le presence of calcium salts, giving a strong positive mollusque Zonitoides nitidus Muller et leur reaction to the Stoeltzener test. The function of dynamiqye en Juin, Juillet et Aofit. Birlletiii de lu the calcium reserves is not yet certain but, Socic5té d 'Histoire Natrirelle 125: 1 11- 117. according to the literature, it is believed to DIMITRIADIS, V.K. & D. HONDROS. 1992. Effect of starvation and hibernation on the fine structural participate in many important calcium dependent morphology of the digestive gland cells of the snail metabolic reactions, such of shell construction Helix lucoriiiii. Malacologia 34 (1-2): 63-73. and repair, pH regulation at the digestive tract, FROMMLNG,E. 1954. Uiologie der mitteleuropaischen and in the reproductive process (WALKER 1970; landgus~ropoden.Duncker & Humblot. Berlin. 404 CURA1977; SIMKISS & MASON 1983; IRELAND PP. & MARIGOMEZ 1992). GOLDSTEIN,J.I.. D.E. NEWBURY. P. ECHLIN, D.C. JOY, The use of special procedures to prepare SEM A.D. ROMINGJr, C.E. LYMAN, C. FIORI& E. material provided clear cuts (see figure 2, A, B LIFSHIN.1992. Scatinitig electron microcopy and and C) for 3-D observation and interpretation. X-ray iiiicroarinlysis (2nd ed.).Plenum Press, New- York-London. 820 pp. Besides, they could be compared with GRIEBEL, R. 1993. Fine structure of the three ceii types histological sections for light microscopy, which found in the digestive gland of Elysia viridis were used as templates. (Opisthobranchia: Sacoglossa). The Veliger 36 (2): 107-1 14. GUPTA, A.S. 1977. 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