HISTOCHEMISTRY AND ULTRASTRUCTURE OF THE CRYPT CELLS IN THE DIGESTIVE GLAND OF APLYSIA PUNCTATA (CUVIER, 1803) Nadira Taïeb, Nardo Vicente

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Nadira Taïeb, Nardo Vicente. HISTOCHEMISTRY AND ULTRASTRUCTURE OF THE CRYPT CELLS IN THE DIGESTIVE GLAND OF APLYSIA PUNCTATA (CUVIER, 1803). Journal of Mol- luscan Studies, Oxford University Press (OUP), 1999, 65 (4), pp.385-398. ￿10.1093/mollus/65.4.385￿. ￿hal-03024757￿

HAL Id: hal-03024757 https://hal.archives-ouvertes.fr/hal-03024757 Submitted on 26 Nov 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. J. Moll. Stud. (1999), 65, 385–398 © The Malacological Society of London 1999

HISTOCHEMISTRY AND ULTRASTRUCTURE OF THE CRYPT CELLS IN THE DIGESTIVE GLAND OF APLYSIA PUNCTATA (CUVIER, 1803)

NADIRA TAÏEB and NARDO VICENTE Centre d’Etude des Ressources Animales Marines. Faculté de St Jérôme, Case 341. 13397 Marseille Cedex, France (Received 29 December 1997; accepted 15 November 1998)

ABSTRACT 1968; Schmekel & Wechsler, 1968a,b; Griebel, 1993; Kress et al, 1994) and many prosobranchs The crypt cells lining the Aplysia punctata digestive (Mason & Nott, 1981) are known to accumu- tubules comprise of three types of cell; calcium, late inorganic salts under normal conditions. excretory, and thin cells. The calcium cells play a role This process of bioaccumulation of mineral in osmoregulation, mineral storage, exocrine secre- salts occurs in specialized cells of the digestive tion, iron detoxification, and excretion processes. They possess well- developed microvilli and a basal tubules, called crypt cells (calcium cells) which labyrinth, suggesting a role in absorption. The Golgi have different ultrastructural characteristics apparatus is involved in the production of two and functions in different molluscan species. main components of calcium spherules; the fibrillar To date, there is no available ultrastructural material and mineralized granules. Golgi complex, information for the sea hare Aplysia punctata rough endoplasmic reticulum (RER), ribosomes, and from the Mediterranean. In this classic osmo- altered mitochondria are involved in the formation adjusting species (Nicol, 1967), calcium of calcium spherules. Secretory activity is indicated appears as a major factor in its biology, for the by the formation of dense granules containing iron nervous system, toxic secretions released from and calcium salts. Lipofuscin pigment has been found in large concretions which may arise from cyto- purple and opaline glands against predators, plasmic areas surrounded by endoplasmic reticulum, muscles and eggs. If the mineral salt store RER and Golgi tubules. There are three stages of hypothesis is true, it can be predicted that the excretory cells, called early, mature, and post- digestive gland of A. punctata, would contain excretory cells. This study traces the development of inorganic salts. This prediction has been tested granulofibrillar vacuoles up to the formation of the by using histochemical methods for calcium lipofuscin concretions and shows that excretory cells and iron. The goal of this study is to analyse are in fact degenerating calcium cells. The fine struc- and identify the crypt cells and to provide ture of thin cells suggests that they are young calcium preliminary information on their relationships. cells. The morphology and functions of these cells are investigated in this ultrastructural analysis.

INTRODUCTION MATERIAL AND METHODS The digestive gland of Aplysidae has been the subject of numerous chemical investigations Aplysia punctata and Plocamium cartilagineum (a (Stallard & Faulkner, 1974; Quinoa et al., red alga), were collected during the months of 1989), however, little is known of the histology March–April, 1993-1994, from a fishing-port in and ultrastructure of the digestive gland of Vallon des Auffes (Marseille). Animals were kept in marine snails. There is only a light-microscopi- aquaria with recirculating filtered sea water at 15- cal account of the digestive gland of Aplysia 17°C with a photoperiod of 11h light and 13 h dark. All animals received daily P. cartilagineum and sam- punctata (Cuvier, 1803) (Howells, 1943) and ples were histologically examined every 5 days. For recently, an ultrastructural study of the diges- routine light microscopy, the digestive glands of tive gland of Aplysia californica (Coelho et al., numerous sea hares were removed and fixed in 1998). Many pulmonates (Abolins-Krogis, Bouin’s Holland, Zenker’s fixative and neutral for- 1961, 1970b; 1975), all opisthobranchs (Taylor, malin, then paraffin embedded, sectioned at 6 ␮m 386 NADIRA TAÏEB & NARDO VICENTE and stained by Heindenhain’s azan. For histo- the cytoplasm; they react positively with the chemical studies the following methods, as detailed tests for AMPS, calcium and lipofuscin and in Ganter & Jolles (1970) were performed: alcian show slight or negative reaction with the Prus- blue (2.5 pH) for acid muco-polysaccharides (AMPS), sian blue test. the Prussian blue reaction for iron, the Von Kossa method for calcium and the Schmorl test for lipo- fuscin. For electron microscopy, small pieces of tissue Excretory cells: these are globular in shape and were fixed for 1 h at 4°C in chilled 2% glutaralde- possess more than one large vacuole in a supra- hyde, buffered at 7.4 with Sorensen’s buffer. Speci- nucleolar position. Some of them contain mens were post-fixed in 1% OSO4 in 0.1M sodium vacuoles with concretions which are histo- phosphate buffered for 1-2h at 4°C. The tissue was chemically similar to those of calcium cells; dehydrated through a series of alcohols and embed- while others exhibit large vacuoles with AMPS ded in Epon 812 (Luft, 1961). For light microscopy, fibrillar material. the semi-thin sections were stained with azure blue. Thin sections were cut with an LKB ultra-microtome Thin cells: these are narrow but extend to the V and mounted on copper grids. These were double stained with uranyl acetate and lead citrate full height of the epithelium. The cytoplasm (Reynolds, 1963) and examined with a Philipps 400T contains small vacuoles which react negatively electron microscope. to all tests.

Electron microscopy. Crypt cells (Fig. 1: A-E) are inter-connected apically by desmosomes RESULTS and septate junctions and lined by a layered basal lamina. Nerve fibres in association with Light microscopy. the digestive tubules are smooth muscles fibres and glial cells are numerous and loosely bound together by con- observed in the inter-tubular space. Haemo- nective tissue which is devoid of muscle fibres cytes in the process of phagocytosis are present and haemocytes. The tubules are made up of in the connective tissue. four types of cells; digestive, calcium, excretory and thin cells. Calcium, excretory and thin cells Calcium cells (Fig. 2; Fig. 3: A-F; Fig. 4: A-F; (called basiphilic cells), occur in small groups, Fig. 4: A) have a broad base containing a large usually in the corners of the tubules, where chromatin-rich nucleus and are filled with digestive cells predominate. The basiphilic cells rough and smooth endoplasmic reticulum, of this typical organisation are designated crypt mitochondria, calcium spherules and ribo- cells. Some tubules do not possess digestive somes (Fig. 3: A). The basement membrane cells (Taïeb, 1996). Thin cells are few in num- exhibits deep infoldings (Fig. 3: B). The rough ber and usually situated either side of calcium endoplasmic reticulum (RER) and basal cells, but also between excretory cells. Excret- plasma membrane surround areas of cyto- ory and thin cells are rarely seen between plasm. The RER is associated with granulo- digestive cells. In this work, only the crypt cells fibrillar vacuoles and calcium spherules. Free have been studied. ribosomes and polysomes, altered mitochon- dria and Golgi apparatus are present between Calcium cells: these are often pyramidal in calcium spherules of various sizes (Fig. 3: C, shape and contain a large ovoid nucleus with a D). The matrix of the altered mitochondria is distinct nucleolus and situated usually in the dense and contains ferritin-like particles. The basal region. They are characterized by calcium calcium spherules of the final developmental spherules which occur throughout the cyto- stage are characterized by a great number of plasm, except at the apex of the cells. Some alternately dense and less-dense concentric calcium spherules show internal concentric layers around a core. Sometimes the centre is rings which stain black by the Von Kossa missing, probably having been torn out during method; others contain fibrillar material or a sectioning. The calcium spherules of the early small granule which stains pink-metachromatic developmental stage show a central granule in azure blue and gives a positive reaction with and granulofibrillar material randomly dis- the tests for AMPS. The cytoplasm contains persed in a large matrix (Fig. 3: E). In the crystals and numerous small granules which are course of continued growth, calcium spherules accumulated particularly in the apical region fuse and form a large spherule with several and stained intensively with Von Kossa and rings. Occasionally, fusion between two periph- Prussian blue tests. Sometimes one to three erical layers occurs (Fig. 3: F). The Golgi large concretions (yellow granules) are seen in apparatus is composed by two or three dictyo- CRYPT CELLS IN APLYSIA PUNCTATA DIGESTIVE GLAND 387

Figure 1. Aplysia punctata. A-B. Supra-nuclear region of the crypt cells lining the digestive tubules. A. Thin cell (tc); excretory cell (ec); concretion (cr); calcium cell (cc); calcium spherules (cs); lumen (L); digestive sphere (ds); residual bodies (arrow); cilia (arrow-head); scale bar ϭ 1␮m. B. Apical junctional complex between two crypt cells; desmosome (D); septate junction (SJ); scale bar ϭ 0,6␮m. C. Basal region of a calcium cell. Basal lamina (arrow); circular fibres (cf); scale bar ϭ 1␮m. D-E. Connective tissue. D. muscle fibres (mf); glial cell (arrows); nerve fibre (nf); scale bar ϭ 1␮m. E. Haemocyte. Pseudopodia (p); phagocytotic material (arrow); scale bar : 1␮m. 388 NADIRA TAÏEB & NARDO VICENTE

Figure 2. Diagram illustrating the fine structure of calcium cell of Aplysia punctata (Cuvier, 1803) somes in synchronous secretory activity. Each empty their content in bodies of various densi- dictyosome consists of 5 to 7 parallel curved ties. Sometimes, Golgi saccules of the concave cisternae (Fig. 4: A, B ). The cisternae of the side encircle small areas of cytoplasm con- mature face are thin, uniformly grey, except at taining secretory granules. Presumably, after the ends where highly osmophilic material destruction of the enclosed Golgi bodies and occured. In the vesicles formed by the blebbing the inner membranes, inclusions are formed. of the end of the Golgi membranes, osmophilic These are indistinguishable from the granules material appears as two main types, with originating from the Golgi saccules and dense crystal shapes or with granular form. On the bodies of undetermined origin. The Golgi concave side of the Golgi saccules, numerous membranes of cis side, are dilated and frag- vesicles of various sizes are observed. The mented into large vacuoles which contain small vesicles (0.4 ␮m) probably fuse and/or osmophilic granules and fibrillar material, simi- CRYPT CELLS IN APLYSIA PUNCTATA DIGESTIVE GLAND 389

Figure 3. Aplysia punctata. Broad base of a calcium cell. A. Ergastoplasm (er); mitochondria (m); calcium spherules (cs). B. Basal labyrinth. Infoldings of basal plasma membrane (arrows) surrounding mitochondria (m), ergastoplasm (er) and calcium spherules (cs); scale bar ϭ 1␮m. C. Relationship between ergastoplasm (er) and granulofibrillar vacuoles (GV); scale bar ϭ 1␮m. D. Numerous mitochondria (m), ergastoplasm (er) and ribosome between calcium spherules (cs); altered mitochondria with ferritinlike particles (arrows); calcium spherule of final developmental stage (csf) )with peripheral rings of electon-dense particles (arrow), separated by a pale space; scale bar ϭ 1␮m. E. Close contact between calcium spherules and Golgi apparatus (G). Calcium spherule of early developmental stage with large matrix (ma) containing dispersed fibrils, peripheral (p) and central (short arrow) accumulation of granulofibrillar material; continuity between two calcium spherules (arrow-head); scale bar ϭ 1␮m. F. Fusion (arrow) between the peripheral layers of two calcium spherules of final developmental stage (csf); scale bar ϭ 1␮m. 390 NADIRA TAÏEB & NARDO VICENTE CRYPT CELLS IN APLYSIA PUNCTATA DIGESTIVE GLAND 391 Figure 4. Aplysia punctata. A-B. Broad base of a calcium cell. A. Golgi apparatus (G); secretory vesicles (v) containing a dense product in granular form (arrow) or in crystallized shape (cr); granulofibrillar vacuoles (GV); one granule (g) formed by fusion of vesicles is encircled partially by Golgi tubules (Gt); dense body (db) with grey content (arrow); altered mitochondria (m) with granular deposit (arrows); scale bar ϭ 1␮m. B. Some of Golgi tubules are fragmented (arrows); scale bar ϭ 1␮m. C-D-E-F. Apical region of a calcium cell. C. The fine-grained material (arrow) observed in the autophagic vacuoles (av) is similar to that of the dense body (db); scale bar ϭ 1␮m. D. Dense bodies (db) of various shapes and densities accumulated in the apical region; scale bar ϭ 1␮m. E. Apical plasma membrane (arrow) showing contact with a granule (g) prior to secretion; microvilli (MV); scale bar ϭ 1␮m. F. Two dense bodies (db) in close contact; releasing of dense product (arrows) in the lumen (L); scale bar ϭ 1␮m.

lar to that of RER and calcium spherules. in the supra-nuclear region. We have distin- Areas of cytoplasm, surounded by endoplasmic guished three types of thin cells: reticulum and bodies containing material of Thin cell of a-Type. Islets of dense granules different densities are present in the supra- occur throughout the cytoplasm and are always nucleolar region (Fig. 4: C). The sub-apical seen in contact with the granulofibrillar cytoplasm is crowned with bodies of various vacuoles (Fig. 5: B). densities, forms and sizes (Fig. 4: D-E). It is Thin cell of b-Type. Mitochondria exhibit a Y impossible to distinguish these bodies from the or U form. The granulofibrillar vacuoles, irreg- granules formed through pinching off the ends ular in shape, show continuity between them; of the Golgi saccules. The small bodies, are their matrix contain dense granules (probably probably iron granules and crystals and the glycogen) similar to those of the islets (Fig. 5: large bodies are the lipofuscin concretions seen C). They apparently increase in size by fusion at light microscope level. The apical plasma and incorporation of dense granules which membrane bears well-formed microvilli and become more and more dense (Fig. 4: D). An occasionally a single flagellum. Before their amorphous dense material, resulting from coa- discharge, the apical granules present an lescence of dense granules is progressively electron-dense rim along their limiting mem- arranged in a ring around a moderately dense brane, then fuse individually with the plasma centre. The membrane limiting the vacuole membrane (Fig. 4: E). This fusion contributes exhibits invaginations, suggesting an incor- to the formation of a secretion pit, with the poration of cytoplasmic material. Occasionally granule membrane added. The core of the crystallized needles are seen in some vacuoles. granule escapes from the cell, breaks apart and Thin cell of c-Type. The vacuoles are round in dissipates in the lumen of the tubule (Fig. 4: F). shape and disposed like a ceiling-rose (Fig. 5: This secretory product, probably takes part in E). Some of them have a uniform fine-grained the extracellular digestion. Sometimes, intra- structure, while others are granulofibrillar and cellulary, dense granules fuse with one another exibit a peripheral dense material, originating producing characteristic profiles of mineralized from the Golgi membranes. contents. Occasionally, coated vesicles of finely granular material are observed in association Excretory cells. Three stages of excretory cells with the apical plasma membrane. Some are found: early excretory, mature excretory calcium cells are lysed and calcium spherules and post-excretory cells (Fig. 6: A-E). with mitochondria and cell debris are seen in The mature excretory cells (A) contain a basal the lumen of the tubules (Fig. 5: A). nucleus and electron-dense membrane-bound bodies of various which correspond to the lipo- Thin cells (Fig. 5: B-E) have a brush border of fuscin concretions. The apical surface bears slender microvilli and bear one long flagellum well developed microvilli and the basement which presumably assists in the circulation of membrane exhibits few invaginations. ERG is the fluid contents of the tubules. The RER is scarce and mitochondria are numerous in the scarce but the Golgi complex is well developed apical cytoplasm. The Golgi apparatus has bro- in the basal region. The Golgi saccules elabo- ken down and instead, there are short curved rate dense granules and granulofibrillar tubules which encircle small areas of cytoplasm vacuoles, similar to those of calcium cells. The and show contacts with the membrane of the cytoplasm contains numerous free ribosomes, inclusions. islets of dense granules, a basal nucleus and Early excretory cells (Fig. 6: B-E) The cyto- mitochondria which tend to be more numerous plasm of these cells contains altered mitochon- 392 NADIRA TAÏEB & NARDO VICENTE CRYPT CELLS IN APLYSIA PUNCTATA DIGESTIVE GLAND 393 Figure 5. Aplysia punctata. A. Degenerated calcium cell. Calcium spherule (cs) and mitochondria (m) in the lumen (L) of a tubule; scale bar: 1␮m. B. Thin cell type a (tca); short ciliary root (arrow); Golgi apparatus (G); granulofibrillar vacuoles (GV) and islets of granules (i) throughout the cytoplasm; mitochondria (m); calcium cell (cc); scale bar ϭ 1␮m. C-D. Thin cell type b (tcb). C. Apical cytoplasm with numerous mitochondria; Islets of granules (i) (probably glycogen); granulofibrillar vacuoles (GV) in conctact; continuity between two granulo-fibrillar vacuoles (long arrow) irregular in shape; granules (short arrow) similar to those of the islets within the matrix of the granulofibrillar vacuoles; scale bar ϭ 1␮m. D. Thin crystallized needles (short arrows) within a granulofibrillar vacuole showing a layer of a dense material around a nucleus of grey density (long arrow); invagination (I) of the granulofibrillar vacuole membrane; scale bar ϭ 1␮m. E. Thin cell-type c; the vacuoles (V), round in shape, are disposed like a ceiling-rose islet of granules (ig); coated vesicles (short arrow); one vacuole shows granulofibrillar material and a dense peripheral zone (p), while onother has a fine- grained structure (long arrow); scale bar ϭ 1␮m. dria, osmophilic bodies of various origin and columnar thin cells (undifferentiated cells) Golgi apparatus. These organelles are fre- have been described (Sumner, 1965a; Walker, quently observed in close contact with mem- 1970; Arni, 1974). Das et al. (1992) described brane-bound granules (B). The latter contain four to five types of cell in five gastropod mol- aggregates of osmophilic granules, concentric luscs and suggested that different physiological layers of a dense amorphous material and phases of a single cell type may exist. In occasionally, myelin-like figures (C) or a con- Aplysia punctata, four type of cells are identi- centric striation, suggesting mineral deposits fied within the digestive gland epithelium. (D). Apparently, the large concretions are Here, only crypt cells are described: calcium, formed by the aggregation of the small, dense excretory and thin cells. granules (E). Calcium cells present all the features (well Post-excretory cells (Fig. 6: A). The cytoplasm developed microvilli, coated vesicles, basal of these cells contains few mitochondria and labyrinth and numerous mitochondria) of cells generally a large granulofibrillar vacuole in a which involved in the trans-epithelial move- supranucleolar position. This vacuole exhibits ment of ions (Rolan-Cornejo, 1986). The high an electron lucent matrix with fibrillar material concentrations of calcium and magnesium in and membrane debris. Lysed excretory cells the haemolymph of A. punctata (Nicol, 1967), are observed at the base of the tubules and may be resorbed by the deep infoldings of occasionally, the concretions, fibrillar vacuoles basement membrane and deposited within the and cell debris are seen in the lumen. spherules situated in the broad base of the calcium cells. The digestive gland of many species of invertebrates has been found to accumulate metals (Nott, 1991; Almedros & DISCUSSION Porcel, 1992; Brooks & White, 1995) to concen- trations which would be toxic if free the cyto- Controversy exists about the histology, nature plasm (Taylor et al., 1988). Excess metals are and related nomenclature of the gastropod accumulated in two types of granules in the digestive gland cells and about the nature of digestive gland of terrestrial invertebrates the different inclusions (Schmekel & Wechsler, (Hopkin, 1989). Type A granules, also known 1968a,b, 1979; Mukherjee et al., 1995). Various as calcium granules, possess the ability to incor- authors have discussed the possible number porate calcium in the form of concentrically and different functions of the digestive cell layered, structured, membrane-bound vesicles types in opisthobranchs. One type of cell has in the calcium cells. Type B granules are been demonstrated in Tritonia hombergi excreted sometimes as residual bodies folowing (Thompson, 1976), two in Haminea hydatis and lysosomal action. The calcium spherules of A. Haminea zelandica (Fretter, 1939; Rudman, punctata, correspond to the type A granules 1971, 1972a) Alderia modesta and Elysia and concretions which contain lipofuscin are Chlorotica (Graves et al., 1979), three in Acteon probably the equivalent of the type B granules. tornatilis and Philine aperta (Fretter, 1939), Due to an inadequate knowledge of their Aglaja cylindrica (Rudman, 1972a, b), Elysia chemical composition and physiological signifi- viridis (Griebel, 1993), Aplysia californica cance, the calcium spherules were assigned (Coelho et al., 1998) and four in Runcinia coro- different functions. Similar granules have been nata and Runcinia ferruginea (Kress et al., described in the digestive gland of Trinchesia 1994). Other cell types, called stem, narrow or granosa (Schmekel & Weschsler, 1968a), 394 NADIRA TAÏEB & NARDO VICENTE

Figure 6. Aplysia punctata. Excretory cells. A. Excretory cells lining a digestive tubule. Mature excretory cell (Mec); post-excretory cell (Pec); dense tubules (arrow) surrounding areas of cytoplasm and showing contact with one large concretion (cr); microvilli (Mv); lumina (L); cilia (short arrows); scale bar ϭ 1␮m. B-C-D-E. Series of formative stages of concretions in early excretory cells (Eec). B. Granulofibrillar vacuoles (GV) with unequal concentration of dense material around a fibrillar center (long arrow); dense bodies (short arrows), interpreted as altered mitochondria in close contact with membrane vacuoles; islet of granules (ig); scale bar ϭ 1␮m. C. Myelin Òlike-figure (mf) and concentric layers of a dense material (dm) within a granulofibrillar vacuole; Golgi complex (G); scale bar ϭ 1␮m. D. Concentric striation interpreted as a mineral deposit (arrow); nucleus (N); scale bar ϭ 1␮m. E. Formation of a large concretion (cr) by aggregation of osmiophilic granules (OG); dense body (db); dense tubules (dt) surrounding cytoplasmic areas; scale bar ϭ 1␮m . CRYPT CELLS IN APLYSIA PUNCTATA DIGESTIVE GLAND 395 Runcinia sp. (Kress et al., 1994) and Aplysia However, we failed to detect iron in calcium californica (Coelho et al., 1998). However, spherules, probably because the latter are often Coelho et al. (1998) did not detect calcium in dissolved and lost after fixation. Iron may be these spherules which they called rhodoplast absorbed by pinocytosis into calcium cells with digestive vacuoles (RDV), because the latter food and/or resorbed from the haemolymph by contain phycoerythrin (a red algal photo- the basal labyrinth. Aplysia punctata is an synthetic pigment). Prince et al. (1998) pro- osmoadjuster and herbivorous, so the accumu- posed that the defensive ink pigment of A. lation of calcium and iron in the digestive californica (phycoerythrobilin), is cleaved from gland, seems to be related in some way to the its protein in RDV and carried in the haemo- environmental concentrations of these ele- lymph to the ink gland. In A. punctata, the cal- ments in the area from where animals and alga cium spherules like other calcifiable systems were taken. are composed of an organic matrix (AMPS) in Calcium cells, like basiphilic cells of bivalves which inorganic salts are deposited around a (Sumner, 1966c, Owen, 1972), are pyramidal nucleus. Across their growth, the calcium in shape and possess a conspicuous nucleus, spherules fused and formed a large spherule in well-developed RER and an extensive Golgi the final development stage. So the concentric complex. The secretory activity is controlled by layers observed in the calcium spherules are the release of small granules in the tubular not the characteristic lamelli of thylakoid lumen. Such secretion resembles the exocrine membranes in different stages of digestion as is secretion of the atrial gland cells (AGC) of suggested for A. californica (Coelho et al., 1998). Aplysia californica (Beard et al., 1982). Secre- Furthermore, the spherules occupy positions tion in AGC involves the fusion of granules identical to those of the calcium spherites and intracellulary and the discharge of a large bolus phagocytosis of chloroplasts has not been of secretory product. In A. punctata, fusion of observed in calcium cells. The relationships small granules with one onother occurs in the between Golgi complex, RER, ribosomes, apical region of calcium cells, however, only altered mitochondria and calcium spherules fusion of individual dense granules with the lead us to postulate that all these organelles are plasma membrane has been observed. Chestkov involved, to some extent, in the formation of et al. (1998) isolated ‘reserve’ of granules the calcium spherules. The Golgi complex is (exocytotic vesicles) from sea urchin eggs and entirely involved in the production of the two determined under which conditions these main components of calcium spherules, fibrillar granules will fuse. They demonstrated that iso- material (AMPS) and mineralized granules. lated reserve granules, laking soluble cofactors, The capability of AMPS to bind metallic ions support calcium-dependent membrane fusion is well known (Simkiss & Tyler, 1958). The in vitro. In Aplysia, the secretory products fibrillar material has its origin from the RER probably participate in ion regulation at whole vesicles as well as from Golgi vesicles. The digestive tissue level and contain digestive initial deposition of mineralized material has enzymes. No more than three large concretions been observed within cisternae of endoplasmic (like those found in the excretory cells), occur reticulum (Fain Maurel et al., 1973), Golgi vesi- in the calcium cells. They contain calcium, lipo- cles (Waku & Sumimoto, 1974), mitochondria fuscin and little iron. Lipofuscin, is an insoluble (Wasserman & Kallfelz, 1970) and vacuoles of pigment which is accumulated in lysosome-like undetermined origin (Turbeck, 1974). The structures (Essner & Novikoff, 1960). Viarengo function of Golgi saccules in absorption of (1985) found that the accumulation of metals iron, has been shown by Roche (1953) who within lysosomes can stimulate lipid peroxida- postulated that absorption occured when iron tion so increasing the quantity of lipofuscin metabolism is important. The osmophilic granules available to trap toxic metals. Fleming material is first observed within the Golgi & Joshi (1987) suggested that ferritin partici- membranes crystallized in secretory vesicles. It pates directly in the detoxification of several seems quite probable that the crystals and metals. Abolins-Krogis (1961) noticed that small granules which contain iron components, during their ascension, the secretory granules are the secretory vesicles. This crystallisation of were transformed into residual bodies. In iron, probably occurs when the concentration calcium cells, the concretions may also arise level of this metal is very high. It is evident that from cytoplasmic areas surrounded by endo- the nucleus of the calcium spherules originates plasmic reticulum, RER and Golgi tubules as from the Golgi vesicles as well as from the has been reported for other molluscs (Ruiz- Golgi saccules surrounding areas of cytoplasm. Buitrago et al., 1980). Degeneration and death 396 NADIRA TAÏEB & NARDO VICENTE of calcium cells are suggested by the presence It seems possible that during the physiological of calcium spherules within mitochondria, cycle of calcium cells, the calcium spherules are concretions and cell debris in the lumen. The transformed into lipofuscin concretions and the cellular damage may result from metabolic Golgi complex is replaced by short curved sensitivity of these cells but, may also represent tubules. In the post-excretory cells, granulo- a route of calcium and iron release in times of fibrillar vacuole profiles, reflect the last stages need. Iron is an essential element in the biology of excretion. The fine structure of excretory of the sea hares. It is likely that iron would dis- cells is completely consistent with the view that tributed in the body at points where there was they are degenerated calcium cells. The nerve a major demand for oxygen, such as, the mus- fibres present in the connective tissue, suggest culature. hormonal stimuli, responsible for the triggering of ion release and the control of their deposi- Thin cells: Sumner (1965a) described narrow tion in calcium cells and probably in glial cells columnar thin cells (undifferentiated cells) and which have been proposed to function as a suggests that they may give rise to digestive or calcium store (Keicher et al., 1991). secretory cells. The features of these cells have From this study, it would appear that the not yet been described for any molluscan diges- crypt cells represent different physiological tive gland. We identify three types of thin cells phases of calcium cell. This cell shows numer- (a, b, c) which correspond to various stages of ous parallels with the calcium cell of pulmon- development. This study shows a similarity ates (Luchtel et al., 1997), however, there are between thin and calcium cells in the posses- some differences as well. Besides its probable sion of a single flagellum, a secreting Golgi role as reservoir of ions needed in reproduc- apparatus and granulofibrillar vacuoles. The tion, mucus, toxic secretions, nervous system, profile of granulofibrillar vacuoles of the thin shell and muscles, the calcium cell is involved cells is similar to that of granules ‘type b’ in secretion, osmoregulation, iron detoxifica- which are involved in the process of calcifica- tion and excretion processes. tion (Abolins-Krogis, 1970b). They apparently increase in size by the incorporation of dense granules (probably glycogen), Golgi vesicles, REFERENCES mitochondria and ribosomes. 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