HISTOCHEMISTRY AND ULTRASTRUCTURE OF THE CRYPT …

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HISTOCHEMISTRY AND ULTRASTRUCTURE OFTHE CRYPT CELLS IN THE DIGESTIVE GLAND

OF APLYSIA PUNCTATA (CUVIER, 1803)Nadira Taïeb, Nardo Vicente

To cite this version:Nadira Taïeb, Nardo Vicente. HISTOCHEMISTRY AND ULTRASTRUCTURE OF THE CRYPTCELLS 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�

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J. Moll. Stud. (1999), 65, 385–398 © The Malacological Society of London 1999

ABSTRACT

The crypt cells lining the Aplysia punctata digestivetubules comprise of three types of cell; calcium,excretory, and thin cells. The calcium cells play a rolein osmoregulation, mineral storage, exocrine secre-tion, iron detoxification, and excretion processes.They possess welldeveloped microvilli and a basallabyrinth, suggesting a role in absorption. The Golgiapparatus is involved in the production of two main components of calcium spherules; the fibrillarmaterial and mineralized granules. Golgi complex,rough endoplasmic reticulum (RER), ribosomes, andaltered mitochondria are involved in the formationof calcium spherules. Secretory activity is indicatedby the formation of dense granules containing ironand calcium salts. Lipofuscin pigment has been foundin large concretions which may arise from cyto-plasmic areas surrounded by endoplasmic reticulum,RER and Golgi tubules. There are three stages ofexcretory cells, called early, mature, and post-excretory cells. This study traces the development ofgranulofibrillar vacuoles up to the formation of thelipofuscin concretions and shows that excretory cellsare in fact degenerating calcium cells. The fine struc-ture of thin cells suggests that they are young calciumcells.

INTRODUCTION

The digestive gland of Aplysidae has been thesubject of numerous chemical investigations(Stallard & Faulkner, 1974; Quinoa et al.,1989), however, little is known of the histologyand ultrastructure of the digestive gland ofmarine snails. There is only a light-microscopi-cal account of the digestive gland of Aplysiapunctata (Cuvier, 1803) (Howells, 1943) andrecently, an ultrastructural study of the diges-tive gland of Aplysia californica (Coelho et al.,1998). Many pulmonates (Abolins-Krogis,1961, 1970b; 1975), all opisthobranchs (Taylor,

1968; Schmekel & Wechsler, 1968a,b; Griebel,1993; Kress et al, 1994) and many prosobranchs(Mason & Nott, 1981) are known to accumu-late inorganic salts under normal conditions.This process of bioaccumulation of mineralsalts occurs in specialized cells of the digestivetubules, called crypt cells (calcium cells) whichhave different ultrastructural characteristicsand functions in different molluscan species.To date, there is no available ultrastructuralinformation for the sea hare Aplysia punctatafrom the Mediterranean. In this classic osmo-adjusting species (Nicol, 1967), calciumappears as a major factor in its biology, for thenervous system, toxic secretions released frompurple and opaline glands against predators,muscles and eggs. If the mineral salt storehypothesis is true, it can be predicted that thedigestive gland of A. punctata, would containinorganic salts. This prediction has been testedby using histochemical methods for calciumand iron. The goal of this study is to analyseand identify the crypt cells and to provide preliminary information on their relationships.The morphology and functions of these cellsare investigated in this ultrastructural analysis.

MATERIAL AND METHODS

Aplysia punctata and Plocamium cartilagineum (ared alga), were collected during the months ofMarch–April, 1993-1994, from a fishing-port in Vallon des Auffes (Marseille). Animals were kept inaquaria with recirculating filtered sea water at 15-17°C with a photoperiod of 11h light and 13 h dark.All animals received daily P. cartilagineum and sam-ples were histologically examined every 5 days. Forroutine light microscopy, the digestive glands ofnumerous sea hares were removed and fixed inBouin’s Holland, Zenker’s fixative and neutral for-malin, then paraffin embedded, sectioned at 6 mm

HISTOCHEMISTRY AND ULTRASTRUCTURE OF THECRYPT CELLS IN THE DIGESTIVE GLAND OF

APLYSIA PUNCTATA (CUVIER, 1803)

NADIRA TAÏEB and NARDO VICENTECentre d’Etude des Ressources Animales Marines. Faculté de St Jérôme, Case 341. 13397 Marseille

Cedex, France(Received 29 December 1997; accepted 15 November 1998)

386 NADIRA TAÏEB & NARDO VICENTE

and stained by Heindenhain’s azan. For histo-chemical studies the following methods, as detailedin Ganter & Jolles (1970) were performed: alcianblue (2.5 pH) for acid muco-polysaccharides (AMPS),the Prussian blue reaction for iron, the Von Kossamethod for calcium and the Schmorl test for lipo-fuscin. For electron microscopy, small pieces of tissuewere fixed for 1 h at 4°C in chilled 2% glutaralde-hyde, buffered at 7.4 with Sorensen’s buffer. Speci-mens were post-fixed in 1% OSO4 in 0.1M sodiumphosphate buffered for 1-2h at 4°C. The tissue wasdehydrated through a series of alcohols and embed-ded in Epon 812 (Luft, 1961). For light microscopy,the semi-thin sections were stained with azure blue.Thin sections were cut with an LKB ultra-microtomeV and mounted on copper grids. These were doublestained with uranyl acetate and lead citrate(Reynolds, 1963) and examined with a Philipps 400Telectron microscope.

RESULTS

Light microscopy. the digestive tubules arenumerous and loosely bound together by con-nective tissue which is devoid of muscle fibresand haemocytes. The tubules are made up offour types of cells; digestive, calcium, excretoryand thin cells. Calcium, excretory and thin cells(called basiphilic cells), occur in small groups,usually in the corners of the tubules, wheredigestive cells predominate. The basiphilic cellsof this typical organisation are designated cryptcells. Some tubules do not possess digestivecells (Taïeb, 1996). Thin cells are few in num-ber and usually situated either side of calciumcells, but also between excretory cells. Excret-ory and thin cells are rarely seen betweendigestive cells. In this work, only the crypt cellshave been studied.

Calcium cells: these are often pyramidal inshape and contain a large ovoid nucleus with adistinct nucleolus and situated usually in thebasal region. They are characterized by calciumspherules which occur throughout the cyto-plasm, except at the apex of the cells. Some calcium spherules show internal concentricrings which stain black by the Von Kossamethod; others contain fibrillar material or asmall granule which stains pink-metachromaticin azure blue and gives a positive reaction withthe tests for AMPS. The cytoplasm containscrystals and numerous small granules which areaccumulated particularly in the apical regionand stained intensively with Von Kossa andPrussian blue tests. Sometimes one to threelarge concretions (yellow granules) are seen in

the cytoplasm; they react positively with thetests for AMPS, calcium and lipofuscin andshow slight or negative reaction with the Prus-sian blue test.

Excretory cells: these are globular in shape andpossess more than one large vacuole in a supra-nucleolar position. Some of them contain vacuoles with concretions which are histo-chemically similar to those of calcium cells;while others exhibit large vacuoles with AMPSfibrillar material.

Thin cells: these are narrow but extend to thefull height of the epithelium. The cytoplasmcontains small vacuoles which react negativelyto all tests.

Electron microscopy. Crypt cells (Fig. 1: A-E)are inter-connected apically by desmosomesand septate junctions and lined by a layeredbasal lamina. Nerve fibres in association withsmooth muscles fibres and glial cells areobserved in the inter-tubular space. Haemo-cytes in the process of phagocytosis are presentin the connective tissue.

Calcium cells (Fig. 2; Fig. 3: A-F; Fig. 4: A-F;Fig. 4: A) have a broad base containing a largechromatin-rich nucleus and are filled withrough and smooth endoplasmic reticulum,mitochondria, calcium spherules and ribo-somes (Fig. 3: A). The basement membraneexhibits deep infoldings (Fig. 3: B). The roughendoplasmic reticulum (RER) and basalplasma membrane surround areas of cyto-plasm. The RER is associated with granulo-fibrillar vacuoles and calcium spherules. Freeribosomes and polysomes, altered mitochon-dria and Golgi apparatus are present betweencalcium spherules of various sizes (Fig. 3: C,D). The matrix of the altered mitochondria isdense and contains ferritin-like particles. Thecalcium spherules of the final developmentalstage are characterized by a great number ofalternately dense and less-dense concentric layers around a core. Sometimes the centre ismissing, probably having been torn out duringsectioning. The calcium spherules of the earlydevelopmental stage show a central granuleand granulofibrillar material randomly dis-persed in a large matrix (Fig. 3: E). In thecourse of continued growth, calcium spherulesfuse and form a large spherule with severalrings. Occasionally, fusion between two periph-erical layers occurs (Fig. 3: F). The Golgi 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. Thincell (tc); excretory cell (ec); concretion (cr); calcium cell (cc); calcium spherules (cs); lumen (L); digestivesphere (ds); residual bodies (arrow); cilia (arrow-head); scale bar 5 1mm. B. Apical junctional complexbetween two crypt cells; desmosome (D); septate junction (SJ); scale bar 5 0,6mm. C. Basal region of a calciumcell. Basal lamina (arrow); circular fibres (cf); scale bar 5 1mm. D-E. Connective tissue. D. muscle fibres (mf);glial cell (arrows); nerve fibre (nf); scale bar 5 1mm. E. Haemocyte. Pseudopodia (p); phagocytotic material(arrow); scale bar : 1mm.


somes in synchronous secretory activity. Eachdictyosome consists of 5 to 7 parallel curvedcisternae (Fig. 4: A, B ). The cisternae of themature face are thin, uniformly grey, except atthe ends where highly osmophilic materialoccured. In the vesicles formed by the blebbingof the end of the Golgi membranes, osmophilicmaterial appears as two main types, with crystal shapes or with granular form. On theconcave side of the Golgi saccules, numerousvesicles of various sizes are observed. Thesmall vesicles (0.4 mm) probably fuse and/or

empty their content in bodies of various densi-ties. Sometimes, Golgi saccules of the concaveside encircle small areas of cytoplasm con-taining secretory granules. Presumably, afterdestruction of the enclosed Golgi bodies andthe inner membranes, inclusions are formed.These are indistinguishable from the granulesoriginating from the Golgi saccules and densebodies of undetermined origin. The Golgimembranes of cis side, are dilated and frag-mented into large vacuoles which containosmophilic granules and fibrillar material, simi-

Figure 2. Diagram illustrating the fine structure of calcium cell of Aplysia punctata (Cuvier, 1803)


Figure 3. Aplysia punctata. Broad base of a calcium cell. A. Ergastoplasm (er); mitochondria (m); calciumspherules (cs). B. Basal labyrinth. Infoldings of basal plasma membrane (arrows) surrounding mitochondria(m), ergastoplasm (er) and calcium spherules (cs); scale bar 5 1mm. C. Relationship between ergastoplasm(er) and granulofibrillar vacuoles (GV); scale bar 5 1mm. 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 5 1mm. 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 calciumspherules (arrow-head); scale bar 5 1mm. F. Fusion (arrow) between the peripheral layers of two calciumspherules of final developmental stage (csf); scale bar 5 1mm.



lar to that of RER and calcium spherules.Areas of cytoplasm, surounded by endoplasmicreticulum and bodies containing material ofdifferent densities are present in the supra-nucleolar region (Fig. 4: C). The sub-apicalcytoplasm is crowned with bodies of variousdensities, forms and sizes (Fig. 4: D-E). It isimpossible to distinguish these bodies from thegranules formed through pinching off the endsof the Golgi saccules. The small bodies, areprobably iron granules and crystals and thelarge bodies are the lipofuscin concretions seenat light microscope level. The apical plasmamembrane bears well-formed microvilli andoccasionally a single flagellum. Before theirdischarge, the apical granules present an electron-dense rim along their limiting mem-brane, then fuse individually with the plasmamembrane (Fig. 4: E). This fusion contributesto the formation of a secretion pit, with thegranule membrane added. The core of thegranule escapes from the cell, breaks apart anddissipates in the lumen of the tubule (Fig. 4: F).This secretory product, probably takes part inthe extracellular digestion. Sometimes, intra-cellulary, dense granules fuse with one anotherproducing characteristic profiles of mineralizedcontents. Occasionally, coated vesicles of finelygranular material are observed in associationwith the apical plasma membrane. Some calcium cells are lysed and calcium spheruleswith mitochondria and cell debris are seen inthe lumen of the tubules (Fig. 5: A).

Thin cells (Fig. 5: B-E) have a brush border ofslender microvilli and bear one long flagellumwhich presumably assists in the circulation ofthe fluid contents of the tubules. The RER isscarce but the Golgi complex is well developedin the basal region. The Golgi saccules elabo-rate dense granules and granulofibrillar vacuoles, similar to those of calcium cells. Thecytoplasm contains numerous free ribosomes,islets of dense granules, a basal nucleus andmitochondria which tend to be more numerous

in the supra-nuclear region. We have distin-guished three types of thin cells:Thin cell of a-Type. Islets of dense granulesoccur throughout the cytoplasm and are alwaysseen in contact with the granulofibrillar vacuoles (Fig. 5: B). Thin cell of b-Type. Mitochondria exhibit a Yor U form. The granulofibrillar vacuoles, irreg-ular in shape, show continuity between them;their matrix contain dense granules (probablyglycogen) similar to those of the islets (Fig. 5:C). They apparently increase in size by fusionand incorporation of dense granules whichbecome more and more dense (Fig. 4: D). Anamorphous dense material, resulting from coa-lescence of dense granules is progressivelyarranged in a ring around a moderately densecentre. The membrane limiting the vacuoleexhibits invaginations, suggesting an incor-poration of cytoplasmic material. Occasionallycrystallized needles are seen in some vacuoles.Thin cell of c-Type. The vacuoles are round inshape and disposed like a ceiling-rose (Fig. 5:E). Some of them have a uniform fine-grainedstructure, while others are granulofibrillar andexibit a peripheral dense material, originatingfrom the Golgi membranes.

Excretory cells. Three stages of excretory cellsare found: early excretory, mature excretoryand post-excretory cells (Fig. 6: A-E). The mature excretory cells (A) contain a basalnucleus and electron-dense membrane-boundbodies of various which correspond to the lipo-fuscin concretions. The apical surface bearswell developed microvilli and the basementmembrane exhibits few invaginations. ERG isscarce and mitochondria are numerous in theapical cytoplasm. The Golgi apparatus has bro-ken down and instead, there are short curvedtubules which encircle small areas of cytoplasmand show contacts with the membrane of theinclusions.Early excretory cells (Fig. 6: B-E) The cyto-plasm of these cells contains altered mitochon-

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 5 1mm. B. Someof Golgi tubules are fragmented (arrows); scale bar 5 1mm. C-D-E-F. Apical region of a calcium cell. C. Thefine-grained material (arrow) observed in the autophagic vacuoles (av) is similar to that of the dense body (db);scale bar 5 1mm. D. Dense bodies (db) of various shapes and densities accumulated in the apical region; scalebar 5 1mm. E. Apical plasma membrane (arrow) showing contact with a granule (g) prior to secretion;microvilli (MV); scale bar 5 1mm. F. Two dense bodies (db) in close contact; releasing of dense product(arrows) in the lumen (L); scale bar 5 1mm.



dria, osmophilic bodies of various origin andGolgi apparatus. These organelles are fre-quently observed in close contact with mem-brane-bound granules (B). The latter containaggregates of osmophilic granules, concentriclayers of a dense amorphous material andoccasionally, myelin-like figures (C) or a con-centric striation, suggesting mineral deposits(D). Apparently, the large concretions areformed by the aggregation of the small, densegranules (E).Post-excretory cells (Fig. 6: A). The cytoplasmof these cells contains few mitochondria andgenerally a large granulofibrillar vacuole in asupranucleolar position. This vacuole exhibitsan electron lucent matrix with fibrillar materialand membrane debris. Lysed excretory cellsare observed at the base of the tubules andoccasionally, the concretions, fibrillar vacuolesand cell debris are seen in the lumen.

DISCUSSION

Controversy exists about the histology, natureand related nomenclature of the gastropoddigestive gland cells and about the nature ofthe different inclusions (Schmekel & Wechsler,1968a,b, 1979; Mukherjee et al., 1995). Variousauthors have discussed the possible numberand different functions of the digestive celltypes in opisthobranchs. One type of cell hasbeen demonstrated in Tritonia hombergi(Thompson, 1976), two in Haminea hydatis andHaminea zelandica (Fretter, 1939; Rudman,1971, 1972a) Alderia modesta and ElysiaChlorotica (Graves et al., 1979), three in Acteontornatilis and Philine aperta (Fretter, 1939),Aglaja cylindrica (Rudman, 1972a, b), Elysiaviridis (Griebel, 1993), Aplysia californica(Coelho et al., 1998) and four in Runcinia coro-nata and Runcinia ferruginea (Kress et al.,1994). Other cell types, called stem, narrow or

columnar thin cells (undifferentiated cells)have been described (Sumner, 1965a; Walker,1970; Arni, 1974). Das et al. (1992) describedfour to five types of cell in five gastropod mol-luscs and suggested that different physiologicalphases of a single cell type may exist. InAplysia punctata, four type of cells are identi-fied within the digestive gland epithelium.Here, only crypt cells are described: calcium,excretory and thin cells.

Calcium cells present all the features (welldeveloped microvilli, coated vesicles, basallabyrinth and numerous mitochondria) of cellswhich involved in the trans-epithelial move-ment of ions (Rolan-Cornejo, 1986). The highconcentrations of calcium and magnesium inthe haemolymph of A. punctata (Nicol, 1967),may be resorbed by the deep infoldings ofbasement membrane and deposited within thespherules situated in the broad base of the calcium cells. The digestive gland of manyspecies of invertebrates has been found toaccumulate metals (Nott, 1991; Almedros &Porcel, 1992; Brooks & White, 1995) to concen-trations which would be toxic if free the cyto-plasm (Taylor et al., 1988). Excess metals areaccumulated in two types of granules in thedigestive gland of terrestrial invertebrates(Hopkin, 1989). Type A granules, also knownas calcium granules, possess the ability to incor-porate calcium in the form of concentricallylayered, structured, membrane-bound vesiclesin the calcium cells. Type B granules areexcreted sometimes as residual bodies folowinglysosomal action. The calcium spherules of A.punctata, correspond to the type A granulesand concretions which contain lipofuscin areprobably the equivalent of the type B granules.Due to an inadequate knowledge of theirchemical composition and physiological signifi-cance, the calcium spherules were assigned different functions. Similar granules have beendescribed in the digestive gland of Trinchesiagranosa (Schmekel & Weschsler, 1968a),

Figure 5. Aplysia punctata. A. Degenerated calcium cell. Calcium spherule (cs) and mitochondria (m) in thelumen (L) of a tubule; scale bar: 1mm. 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); calciumcell (cc); scale bar 5 1mm. C-D. Thin cell type b (tcb). C. Apical cytoplasm with numerous mitochondria; Isletsof 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 isletswithin the matrix of the granulofibrillar vacuoles; scale bar 5 1mm. D. Thin crystallized needles (short arrows)within a granulofibrillar vacuole showing a layer of a dense material around a nucleus of grey density (longarrow); invagination (I) of the granulofibrillar vacuole membrane; scale bar 5 1mm. E. Thin cell-type c; thevacuoles (V), round in shape, are disposed like a ceiling-rose islet of granules (ig); coated vesicles (shortarrow); one vacuole shows granulofibrillar material and a dense peripheral zone (p), while onother has a fine-grained structure (long arrow); scale bar 5 1mm.


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 contactwith one large concretion (cr); microvilli (Mv); lumina (L); cilia (short arrows); scale bar 5 1mm. B-C-D-E.Series of formative stages of concretions in early excretory cells (Eec). B. Granulofibrillar vacuoles (GV) withunequal 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 51mm. C. Myelin Òlike-figure (mf) and concentric layers of a dense material (dm) within a granulofibrillar vacuole; Golgi complex (G); scale bar 5 1mm. D. Concentric striation interpreted as a mineral deposit (arrow);nucleus (N); scale bar 5 1mm. E. Formation of a large concretion (cr) by aggregation of osmiophilic granules(OG); dense body (db); dense tubules (dt) surrounding cytoplasmic areas; scale bar 5 1mm .


Runcinia sp. (Kress et al., 1994) and Aplysiacalifornica (Coelho et al., 1998). However,Coelho et al. (1998) did not detect calcium inthese spherules which they called rhodoplastdigestive vacuoles (RDV), because the lattercontain phycoerythrin (a red algal photo-synthetic pigment). Prince et al. (1998) pro-posed that the defensive ink pigment of A.californica (phycoerythrobilin), is cleaved fromits protein in RDV and carried in the haemo-lymph to the ink gland. In A. punctata, the cal-cium spherules like other calcifiable systemsare composed of an organic matrix (AMPS) inwhich inorganic salts are deposited around anucleus. Across their growth, the calciumspherules fused and formed a large spherule inthe final development stage. So the concentriclayers observed in the calcium spherules arenot the characteristic lamelli of thylakoid membranes in different stages of digestion as issuggested for A. californica (Coelho et al., 1998).Furthermore, the spherules occupy positionsidentical to those of the calcium spherites andphagocytosis of chloroplasts has not beenobserved in calcium cells. The relationshipsbetween Golgi complex, RER, ribosomes,altered mitochondria and calcium spheruleslead us to postulate that all these organelles areinvolved, to some extent, in the formation ofthe calcium spherules. The Golgi complex isentirely involved in the production of the twomain components of calcium spherules, fibrillarmaterial (AMPS) and mineralized granules.The capability of AMPS to bind metallic ions is well known (Simkiss & Tyler, 1958). The fibrillar material has its origin from the RERvesicles as well as from Golgi vesicles. The initial deposition of mineralized material hasbeen observed within cisternae of endoplasmicreticulum (Fain Maurel et al., 1973), Golgi vesi-cles (Waku & Sumimoto, 1974), mitochondria(Wasserman & Kallfelz, 1970) and vacuoles ofundetermined origin (Turbeck, 1974). Thefunction of Golgi saccules in absorption of iron, has been shown by Roche (1953) whopostulated that absorption occured when ironmetabolism is important. The osmophilic material is first observed within the Golgimembranes crystallized in secretory vesicles. Itseems quite probable that the crystals andsmall granules which contain iron components,are the secretory vesicles. This crystallisation ofiron, probably occurs when the concentrationlevel of this metal is very high. It is evident thatthe nucleus of the calcium spherules originatesfrom the Golgi vesicles as well as from theGolgi saccules surrounding areas of cytoplasm.

However, we failed to detect iron in calciumspherules, probably because the latter are oftendissolved and lost after fixation. Iron may beabsorbed by pinocytosis into calcium cells withfood and/or resorbed from the haemolymph bythe basal labyrinth. Aplysia punctata is anosmoadjuster and herbivorous, so the accumu-lation of calcium and iron in the digestivegland, seems to be related in some way to theenvironmental concentrations of these ele-ments in the area from where animals and algawere taken.

Calcium cells, like basiphilic cells of bivalves(Sumner, 1966c, Owen, 1972), are pyramidal in shape and possess a conspicuous nucleus,well-developed RER and an extensive Golgicomplex. The secretory activity is controlled bythe release of small granules in the tubularlumen. Such secretion resembles the exocrinesecretion of the atrial gland cells (AGC) ofAplysia californica (Beard et al., 1982). Secre-tion in AGC involves the fusion of granulesintracellulary and the discharge of a large bolusof secretory product. In A. punctata, fusion ofsmall granules with one onother occurs in theapical region of calcium cells, however, onlyfusion of individual dense granules with theplasma membrane has been observed. Chestkovet al. (1998) isolated ‘reserve’ of granules (exocytotic vesicles) from sea urchin eggs anddetermined under which conditions these granules will fuse. They demonstrated that iso-lated reserve granules, laking soluble cofactors,support calcium-dependent membrane fusionin vitro. In Aplysia, the secretory productsprobably participate in ion regulation at wholedigestive tissue level and contain digestiveenzymes. No more than three large concretions(like those found in the excretory cells), occurin the calcium cells. They contain calcium, lipo-fuscin and little iron. Lipofuscin, is an insolublepigment which is accumulated in lysosome-likestructures (Essner & Novikoff, 1960). Viarengo(1985) found that the accumulation of metalswithin lysosomes can stimulate lipid peroxida-tion so increasing the quantity of lipofuscingranules available to trap toxic metals. Fleming& Joshi (1987) suggested that ferritin partici-pates directly in the detoxification of severalmetals. Abolins-Krogis (1961) noticed that during their ascension, the secretory granuleswere transformed into residual bodies. In calcium cells, the concretions may also arisefrom cytoplasmic areas surrounded by endo-plasmic reticulum, RER and Golgi tubules ashas been reported for other molluscs (Ruiz-Buitrago et al., 1980). Degeneration and death


of calcium cells are suggested by the presenceof calcium spherules within mitochondria, concretions and cell debris in the lumen. Thecellular damage may result from metabolic sensitivity of these cells but, may also representa route of calcium and iron release in times ofneed. Iron is an essential element in the biologyof the sea hares. It is likely that iron would dis-tributed in the body at points where there wasa major demand for oxygen, such as, the mus-culature.

Thin cells: Sumner (1965a) described narrowcolumnar thin cells (undifferentiated cells) andsuggests that they may give rise to digestive orsecretory cells. The features of these cells havenot yet been described for any molluscan diges-tive gland. We identify three types of thin cells(a, b, c) which correspond to various stages ofdevelopment. This study shows a similaritybetween thin and calcium cells in the posses-sion of a single flagellum, a secreting Golgiapparatus and granulofibrillar vacuoles. Theprofile of granulofibrillar vacuoles of the thincells is similar to that of granules ‘type b’ which are involved in the process of calcifica-tion (Abolins-Krogis, 1970b). They apparentlyincrease in size by the incorporation of densegranules (probably glycogen), Golgi vesicles,mitochondria and ribosomes. The crystallizedneedles observed in the vacuole matrix, proba-bly correspond to aragonitic calcium carbonate.This mineral is under the control of carbonicanhydrase and ureas which regulate the stato-cyst pH and the formation and maintenance of statoconia in Aplysia californica (Pedrozo et al., 1998). The fine structure of these cells isconsistent with that of young calcium cells.

Excretory cells: these are thought to have amainly excretory. Sumner (1965) suggestedthat they derived from secretory cells. Walker(1972) observed several cycles of yellow gran-ule excretion in digestive cells before develop-ing into excretory cells. In A. punctata, we findthree stages of excretory cells termed, early,excretory and post-excretory cells. The earlyexcretory cell exhibits some characteristics ofboth thin and calcium cells. Their vacuole system shows aggregations of osmophilic smallbodies which originate from cytoplasmic material. The myelin-like figures and concen-tric layers observed in some vacuoles, suggest,respectively, the degradation of mitochondrialphospholopids membranes and mineral deposit.This signifies that the vacuoles could developinto residual bodies or into calcium spherules.

It seems possible that during the physiologicalcycle of calcium cells, the calcium spherules aretransformed into lipofuscin concretions and theGolgi complex is replaced by short curvedtubules. In the post-excretory cells, granulo-fibrillar vacuole profiles, reflect the last stagesof excretion. The fine structure of excretorycells is completely consistent with the view thatthey are degenerated calcium cells. The nervefibres present in the connective tissue, suggesthormonal stimuli, responsible for the triggeringof ion release and the control of their deposi-tion in calcium cells and probably in glial cellswhich have been proposed to function as a calcium store (Keicher et al., 1991).

From this study, it would appear that thecrypt cells represent different physiologicalphases of calcium cell. This cell shows numer-ous parallels with the calcium cell of pulmon-ates (Luchtel et al., 1997), however, there aresome differences as well. Besides its probablerole as reservoir of ions needed in reproduc-tion, mucus, toxic secretions, nervous system,shell and muscles, the calcium cell is involvedin secretion, osmoregulation, iron detoxifica-tion and excretion processes.

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