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Ultrastructural organization, ontogenesis, and regeneration of the periostracum in the slug Deroceras reticulatum (Mollusca)

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Page 1: Ultrastructural organization, ontogenesis, and regeneration of the periostracum in the slug               Deroceras reticulatum               (Mollusca)

Ultrastructural organization, ontogenesis, and regeneration of the periostracum in the slug Deroceras reticulatum (Mollusca)

JEAN F O U R N I ~ AND LOUISE ZYLBERBERG EquiPe Formution et destruction des tissus calcrfies et Equipe Formation squelettique.~, Centre National de la recherche scientijique

UA 04.1137 Laboratoire d'anatomie Compare'e, Universite' Paris VII, 2 , plac-e Jussieu, 7525 1 Paris CEDEX 05, France

Received November 6 , 1986

FOURNII?, J., and ZYLBERBERG, L. 1987. Ultrastructural organization, ontogenesis, and regeneration of the periostracum in the slug Deroceras reticulatum (Mollusca). Can. J. Zool. 65: 1935-1941.

Electron microscopy studies show that the periostracum of the internal reduced shell in Deroceras reticulatum is composed of two layers: an inner dense layer and an outer fibrous one. The inner dense layer, the thicker part of the periostracum, consists of stacked lamellae formed in the Golgi apparatus of all the mantle edge cells. The structure and the synthesis of the inner dense layer support the hypothesis that the reduced shell in Deroceras may be considered a typical molluscan shell. The outer fibrous layer appears as a peculiar characteristic of this species and it seems to be synthesized by the dorsal epithelium of the shell sac. Shell regeneration begins with a rapid restoration of the periostracum which forms an uninterrupted sheet enclosing a mineralization chamber. Rapid synthesis is related to the involvement of not only the mantle edge cells but also of all the mantle cells, indicating that the mantle cells that produce only the mineralized layers during shell ontogenesis can form the periostracal units also. This potentiality is expressed only when the closed mineralization chamber must be restored in a short time.

FOURNII?, J . , et ZYLBERBERG, L. 1987. Ultrastructural organization, ontogenesis, and regeneration of the periostracum in the slug Deroceras reticulatum (Mollusca). Can. J. Zool. 65 : 1935- 194 1 .

L'Ctude ultrastructurale montre que le periostracum de la coquille interne de Deroceras reticulatum comprend deux couches : une couche interne dense aux electrons et une couche externe fibreuse. La couche interne constitue la majeure partie du periostracum; elle est composee de lamelles accolCes apparaissant dans les saccules golgiens de toutes les cellules du bourrelet palleal. La structure et le mode de formation de la couche interne dense incitent a considkrer la coquille interne et reduite de Deroceras reticulatum comme une coquille typique de Mollusque. La couche externe fibreuse semble Ctre une particularit6 de cette espece et serait synthCtisCe par I'epithelium dorsal du sac de la coquille. La regeneration d'une nouvelle coquille commence par la reconstitution rapide du periostracum qui forme une lame continue delimitant une chambre de mineralisation close. Cette synthese rapide est assuree non seulement par les cellules du bourrelet palleal, mais par I'ensemble des cellules du manteau. Ces dernieres qui, dans les conditions normales de developpement produisent uniquement les couches calcifiees de la coquille, ont, cependant, garde la potentialit6 de synthetiser les lamelles periostracales. Cette potentialite ne s'exprime que pendant la rCgenCration lorsqu'une nouvelle chambre de mineralisation doit Ctre reconstituee le plus rapidement possible.

Introduction The periostracum is an organic layer covering the outer

surface of the molluscan shell; it is thought to be a protective layer against chemicals and (or) to provide a base for the initial deposition of calcium carbonate crystallites. Previous studies devoted to its structure were carried out in molluscs with a well-developed external shell. In these species, the perios- tracum is produced by the cells of the mantle edge area and is composed of quinone-tanned proteins associated with carbo- hydrates and lipids. However, the fine structure of this unmineralized layer varies according to the species (review in Saleuddin 1979; Saleuddin and Petit 1983).

In some species of land gastropods, the shell lies within the sac formed by the shell gland, which does not evaginate during the early ontogenesis as it does in species with a well-developed external shell (Kniprath 1979, 198 1). The slug Deroceras reticulatum shows an internal shell inserted in a dorsal sac located beneath the cephalic shield; preliminary studies (Fournie 1979) have shown that the calcified layers are covered with a thin, transluscent, organic layer inserted along the mantle edge. This organic layer shows some characteristics of a periostracum but further studies are necessary to consider it as such. The aim of this work is to compare the structure of the periostracum of the inner reduced shell of the slug Deroceras reticulatum with that of the well-developed external shell of other molluscs.

Numerous experiments on shell regeneration have been carried out in molluscs with an external shell to analyze the sequential events of the processes of shell formation. In all species, after the removal of a small piece of shell, an organic

membrane closing the injured area is rapidly synthesized; this closed area seems necessary for the mineral deposition (Beed- ham 1965; Abolins-Krogis 1968; Saleuddin and Chan 1969; Saleuddin and Wilbur 1969; Saleuddin 1970, 197 1).

In Deroceras, regeneration experiments have been per- formed by removing the whole shell (Fournie 1979; Fournie and Zylberberg 1982). In these conditions, a complete new shell is restored. Furthermore, the regenerated shell is entirely covered with an organic layer inserted along the mantle edge; this layer is similar to that observed in a normal shell. Thus, if this layer is a true periostracum, the question arises as to how it can be completely restored by the mantle edge after shell removal.

Material and methods The structure of the fully formed periostracum was observed on

small pieces of the central area of the shell of adult Deroceras reticulatum collected in the field and raised in glass dishes on wet filter paper at 15OC.

The ontogenesis of the periostracum was studied on small samples containing the mantle edge, the mantle, and the newly formed periostracum of young individuals (1 month old) hatched in the laboratory. Shell regeneration was initiated in adults by removing the whole inner shell through a small transverse incision of the cephalic shield. Thin pieces of the mantle edge and of the mantle epithelium were removed from the regenerating animals 30 min, 1 h, and 4 days after the removal of the shell.

All samples were fixed in a mixture containing 2.5% glutaraldehyde and 2% paraformaldehyde in cacodylate buffer (0.1 M, pH 7.2) for 2 h, then washed in 0.1 M cacodylate buffer containing 5% sucrose, and postfixed in 0.1% osmium tetroxide in the cacodylate buffer. Some specimens were demineralized for at least 24 h at 4OC in 0.1 M EDTA added to the aldehyde fixative. Ruthenium red was used as an anionic

Printed In Canada 1 lmprime au Canada

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Page 2: Ultrastructural organization, ontogenesis, and regeneration of the periostracum in the slug               Deroceras reticulatum               (Mollusca)

CAN. J . ZOOL. VOL. 65, 1987

FIG. 1. Diagram of a cross section of the shell sac in an adult slug Deroceras reticulatum showing the anatomical relationships of the shell with the surrounding tissues.

FIG. 2. Histological transverse section through the shell sac after the removal of the shell. cs, cephalic shield; de, dorsal epithelium of the shell sac; m, mantle; me, mantle edge; vm, visceral mass. x250.

electron microscopic stain (Luft 197 1 a , 197 1 b ) ; it was added at 0.05% in the aldehyde fixative with EDTA, the wash buffer, and the osmium tetroxide solution. The fixed samples were dehydrated in a graded series of ethanol and embedded in Epon under vacuum at 60°C. Semithin sections were stained with a toluidine blue solution (pH 4.2) for light microscopic examination. Thin sections of selected areas were obtained with the particular orientation of the block surface suitable for sectioning hard fibrous tissues (Allizard and Zylberberg 1982). The thin sections were double stained with uranyl acetate and lead citrate (Reynolds 1963) and viewed in a Philips EM 300 electron microscope at 80 kV.

Results Mantle and shell relationship in Deroceras reticulatum

The shell of D . reticulatum is located wi.thin the shell sac (Fig. 1). The central area of the ventral epithelium of the shell sac is composed of flattened cells producing the calcified layers of the shell. It is outlined by a peripheral fold of taller cells (Fig. 2) producing .the periostracum. Thus, the central area of the ventral epithelium is homologous with the dorsal mantle epithelium of gastropods carrying an external shell, and the

FIG. 3 . Cross section of a mature periostracum in an adult. The dark inner homogeneous layer (il) is the main part of the periostracum; it is lined by the ostracum (0s) and by an outer fibrous layer (01). ~ 9 0 0 0 .

peripheral fold is homologous with the mantle edge; they will be considered as such in the present study.

Ultrastructure of the periostracum in adults In adult Deroceras reticulatum, the mature periostracum is

composed of two layers (Fig. 3). The innermost one lies in contact with the ostracum and its thickness is about 75% of that of the whole periostracum. It is composed of an electron-dense lamellated material. The outer layer is formed by closely packed fibrils separated by gaps that are more or less developed.

Ultrastructure of the mantle edge and ontogenesis of the periostracum

The mantle edge of young and adult slugs is composed of

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Page 3: Ultrastructural organization, ontogenesis, and regeneration of the periostracum in the slug               Deroceras reticulatum               (Mollusca)

columnar cells of about 25 Fm in height. The nucleus lies in the basal part of the cell (Fig. 2). On its external side, the mantle edge epithelium ends in a faint groove (Fig. 2). On its internal side, this epithelium is separated from that of the mantle by a definite transitional area. In electron micrographs, all the cells of the mantle edge show similar cytological features. The apices of the cells are ornamented with microvilli (0.5- 1 Fm long), the tips of which contain electron-dense plaques (Fig. 4). The cells of the mantle edge have a well-developed rough endoplasmic reticulum (RER); the Golgi areas are scattered in the cells. Each dictyosome is composed of three to five flattened saccules. Each saccule contains a thin lamella (Fig. 5); these lamellae are also found in secretory vesicles (Fig. 4); a vesicle may contain two, three, or more superimposed lamellae. The lamellae are extruded out of the cell by exocytosis (Fig. 6). In the vicinity of the apices of the cells, they are arranged in parallel sheets and are then closely packed forming a homogeneous dense layer.

Thus, in both young and adult slugs, the cells of the whole mantle edge which are of only one cell type produce the inner layer of the periostracum. In the young slugs, the newly formed periostracum has only one layer which resembles the inner dense layer of the mature periostracum of adults; the outer fibrous layer is not yet formed (Fig. 7). However, a fibrous material similar to that of the outer layer of the mature periostracum coats the apical surface of the dorsal epithelium of the shell sac (Fig. 7).

Regeneration of the periostracum Thirty minutes after removal of the shell, the epithelium

thickens in the mantle edge and in the mantle. The intercellular spaces are often dilated (Fig. 9). The basal plasma membrane shows numerous well-developed infoldings (Fig. 16). The epithelial cells of the mantle edge and the mantle show a very well-developed RER; ribosomes are also very abundant. The Golgi areas are more numerous and more developed than in the control animals. Compared with the cells of the normal mantle (Fig. 8), the mantle cells of the regenerating animals are characterized by the presence of lamellae within the Golgi saccules (Fig. 10). The lamellae are observed in the Golgi saccules 30 min after shell removal, when vesicles containing several lamellae are found in the mantle cells (Fig. 1 I). The lamellae are extruded by exocytosis at the apical surface of the mantle cells (Figs. 1 1 and 12). One hour after shell removal, the lamellae are arranged in parallel and form a thin sheet (Fig. 13).

Four days later, groups of parallel lamellae form a loose continuous sheet similar to that observed in the early stages of the normal ontogenesis of the periostracum. The closely packed lamellae then form an electron-dense layer identical with the inner layer of the mature periostracum. At the outer surface of this newly regenerated periostracum, a fibrillar material is organized in a loose network (Fig. 15) and it may be compared to the outer layer of the mature periostracum (Fig. 3). In the Golgi saccules of the mantle cells, however, no lamellae are observed but numerous lysosomes are present (Fig. 14) whereas lamellae are still observed in the Golgi saccules of the mantle edge cells.

Discussion Structure and ontogenesis of the periostracum

The mature periostracum in Deroceras reticulatum is com- posed of two layers: an inner thick dense layer and an outer loose fibrous layer. Since the thick dense layer ensuring the protective properties of the periostracum is found in every molluscan shell, its presence may be considered as a general characteristic. In

Deroceras, this main dense layer is produced by the cells of the mantle edge which appear to be of the same type.

The data reported in other pulmonates have pointed out differences in the structure and the formation of the periostracum between land and aquatic species. Indeed, the periostracum is composed of two layers in the basommatophoran Lymnaea (Kniprath 1972), Helisoma (Saleuddin 1975), and Physa (Jones and Saleuddin 1978). The outer surface of the main periostracal layer is lined by a membrane-like lamella. Specialized cells located deep in the connective tissue open into the periostracal groove by long cell processes and produce the membrane-like lamella and the initial components of the main dense layer. The mantle edge (mantle edge gland or supramarginal ridge) is responsible for the thickening of this dense layer. In the land snail Helix (Saleuddin 1976), the periostracum has only the thick dense layer which is secreted by the periostracal gland; this gland is composed of one cell type and is embedded in the connective tissue; it opens into the periostracal groove.

In Deroceras reticulatum, the ultrastructural study indicates that the periostracal lamellae of the thick dense layer are Golgi mediated as in the formation of the dense layer in the other land species studied, Helix (Saleuddin 1976). In the aquatic species Helisoma and Physa, the Golgi apparatus is involved in the secretion of the membrane-like lamella (Saleuddin 1975; Jones and Saleuddin 1978).

The microvilli located at the apices of the mantle edge cells show distal electron-dense plaques. They are thought to-ensure the alignment of the periostracal units as suggested in Helisoma (Saleuddin 1975) and Physa (Jones and Saleuddin 1978).

Thus, from a functional point of view, the mantle edge of Deroceras reticulatum, which is composed of one cell type ensuring the synthesis of the thick dense layer, might be compared to the periostracal gland of Helix. However, from an anatomical point of view, the mantle edge of D. reticulatum shows a location similar to that in the Basommatophora. Nevertheless, in these latter species, the mantle edge cells do not initiate the formation of the dense layer, but are involved only in its thickening.

Our data concerning the structure and the formation of the thick dense periostracal layer in Deroceras as well as the presence of a polyphenoloxidase in the mantle edge cells (unpublished data) support the hypothesis that the inner reduced shell of this slug has a typical molluscan structure.

The outer fibrous layer of the periostracum of Deroceras differs in its structure and in its origin from the outer membrane-like lamella of the basommatophoran periostracum, and from the fibrous layer described in the bivalvia Astarte (Saleuddin 1974), where it lines the inner surface of the dense layer.

The late formation of the fibrous layer, which appears when the inner layer is well-developed, and the absence of differen- tiated cells at the outer margin of the mantle edge suggest that this fibrous layer might not be produced by the mantle edge. The presence of a fibrous material coating the outer surface of the dorsal epithelium of the shell sac might indicate that the dorsal epithelium would be involved in the production of the outer fibrous layer. Hence, the whole epithelium of the shell sac, which originates from the shell gland, could ensure the shell formation.

The functional significance of this outer fibrous layer is not known; however, it might be surmised that because of its loose structure it has mechanical properties which permit the sliding of the shell within the shell sac without injuries to the dorsal epithelium.

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1938 CAN. J. ZOOL. VOL. 65, 1987

FIGS. 4-7. Ontogenesis of the periostracum in young slugs. Fig. 4. Apical microvilli of the mantle edge cells. Note the presence of electron-dense plaques at the tips of the microvilli (arrows). Vesicles containing superimposed lamellae are present in the apical part of the cells (arrowheads). x40 000. Fig. 5. Detail of a dictyosome in a mantle edge cell. Note that at this stage, each cisterna of the Golgi apparatus contains only one lamellar unit (arrows). x 50000. Inset: Detail of lamellae located within Golgi cisternae. x 130 000. Fig. 6. Detail of the apical part of the mantle edge cells. The lamellae are extruded by exocytosis (arrows); they are arranged in parallel in the newly formed inner periostracal layer (il) which appears later as an electron-dense homogeneous layer (Fig. 3). x 50 000. Fig. 7. Apical part of the cells of the dorsal epithelium ( d e ) . Note the presence of a fibrous material coating the apical plasma membrane (arrows). The inner layer (il) appears as a dense layer. x 50 000.

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FOURNIE AND ZYLBERBERG 1939

FIG. 8. Mantle cells in an adult slug. X 15 000. Inset: Detail of a dictyosome. Saccules and vesicles are electron lucent and they do not contain any lamella. es, extrapallial space. x 50000. FIGS. 9 to 13. Regeneration of the periostracum in adult slugs. Fig. 9. Aspect of the mantle cells 30 min after removal of the shell. Note the presence of a dilated intercellular space (is), the development of the Golgi areas (arrows), and the presence of lamellae in a saccule (stars). Electron-dense plaques appear at the tips of the microvilli (arrowheads). es, extrapallial space. x 15 000. Fig. 10. Detail of a dictyosome in a mantle cell 1 h after removal of the shell. Note the presence of a periostracal unit in each saccule (arrows). x 40000. Fig. 11. Detail of the apical part of the mantle cells 30 min after removal of the shell. Exocytosis of superimposed lamellae located in a vesicle in the vicinity of the apical plasma membrane. x 30000. Fig. 12. Detail of the apical part of the mantle cells 30 min after removal of the shell. Note the parallel orientation of the lamellae extruded out of the cells and located among the microvilli. x 40 000. Fig. 13. Apical part of the mantle cells and newly formed periostracum (p) 1 h after removal of the shell. Note that the lamellar units of the periostracum are arranged in parallel and form a thin continuous sheet. Dense plaques are located at the tips of the microvilli (arrows). X 20 000.

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1940 CAN. J. ZOOL. VOL. 65, 1987

FIGS. 14-16. Regeneration of the periostracum in adult slugs. Fig. 14. Mantle cells 4 days after removal of the shell. Lamellae are no longer observed in the mantle cells. Note the presence of lysosomes (1) and the swollen aspect of mitochondria. The tips of the microvilli do not show very electron-dense plaques. x 15 000. Fig. 15. Cross section of the regenerated periostracum 4 days after removal of the shell. Note the presence of two layers: the inner layer (if) is composed of lamellar units the condensation of which will form the inner electron-dense and homogeneous layer; the outer layer (of) is made up of fibrillar material. os, ostracum. X 30 000. Fig. 16. Detail of the basal part of the mantle cells 4 days after removal of the shell. Note the abundance of the infoldings of the basal plasma membrane. bm, basement membrane. X 15 000.

Regeneration In Deroceras reticulaturn, shell regeneration begins with the

rapid formation of a complete new periostracum. Other studies on shell regeneration in molluscs with a well-developed outer shell indicate that mineral deposits occur only after the forma- tion of a closed mineralizing space (Wilbur 1973; Saleuddin 1979; Watabe and Blackwelder 1980) outlined by an organic membrane produced by the mantle cells. In Helix, a new periostracum covers the shell regenerate only if the damaged area is in contact with the mantle edge (Saleuddin and Chan 1969). In Bivalvia, the mantle cells regenerate the different layers of the shell including the periostracum; in Anodonta, the regenerated periostracum shows the histochemical characteris- tics of the normal one (Beedham 1965), and in Mytilus the regenerated periostracum has different microtopographical pro- perties (Meenakshi et al. 1973; Wilbur 1973). In Deroceras, the rapid regeneration of a complete periostracum is ensured by the immediate involvement of the mantle cells in the synthesis of

rapid restoration of an uninterrupted layer anchored in the periostracal groove along the mantle edge. The cytological processes of the synthesis of the periostracal lamellae in the mantle cells during regeneration appear to be similar to those observed in the mantle edge cells during ontogenesis; this explains why the thick and dense regenerated periostracal layer shows the same structure as in the control.

Four days after shell removal, a closed space is restored; no more lamellae are produced in the mantle cells, which show functional changes as suggested by the presence of numerous lysosomes. Mantle cells have probably retained the potentiality to function as mantle edge cells and express it when the need arises. This fact may indicate that one important function of the periostracum is to ensure the formation of a closed mineraliza- tion space within which the mineralized layers of the shell can be formed.

Acknowledgments the periostracal units. The synthesis occurring both in the The authors are grateful to Dr. A. Kovoor (Centre national mantle edge cells and in the mantle cells is responsible for the de la recherche scientifique UniversitC Paris VII) for reviewing

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FOURNIE AND ZYLBERBERG 1941

the English manuscript; the efficient technical assistance of F r a n ~ o i s e Allizard is acknowledged. The electron microscopic observations were performed in the Centre d'accueil d e micro- scopie electronique, CNRS, and Universite Pierre et Marie Curie, where the micrographs were printed in the Service de photographie. This work was supported by a grant from the CNRS (UA 04 1 1 37).

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FOURNI~, J., and ZYLBERBERG, L. 1982. MorphogCnese normale et rCgCnCrative du pCriostracum chez un Mollusque : Deroceras reticulatum (GastCropode, PulmonC). Biol. Cell. 45: 3 19.

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SALEUDDIN, A. S . M., and PETIT, H. P. 1983. The mode of formation and the structure of the periostracum. In The Mollusca. Vol. 4. Edited by A. S. M. Saleuddin and K. M. Wilbur. Academic Press, New York. pp. 199-234.

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WATABE, N., and BLACKWELDER, P. L. 1980. Ultrastructure and calcium localization in the mantle epithelium of the freshwater gastropod Pomacea paludosa during shell regeneration. In The mechanisms of biomineralization in animals and plants. Edited by M. Omori and N. Watabe. Tokai University Press, Tokyo, Japan. pp. 131-144.

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