AquaJ l.i1'ing Rcsour., 1992, 5, 287-298

Mande histology, histochem.istry and ultrastructure of the pearl oyster Pinctada rnargaritijera (L.)

Roula Jabbour-Zahab (1), Dominique Chagot (2) t, Françoise Blanc (1 \ *) and Henri Grizel (21

(1) raboraloiTe dp ,(oogéograf!/tie GénPllqne, Cnivpnilé JifonlfJPllieT 3, !J.J'. j043.3!032 Afonlpel/in Cedex J, Franle.

(2) l_abowtoire dE j>atilillogie el CéndiquE rle.1 11l1nlébréJ Afarin.l, 1FR EAU,-'!?, B.l'. 1']3, J7390 La Tremblade, FmnCf.

(*) 10 wfwm aU corre.lpolldCllle slwuld be uddrl'.lsld.

Rcceivcd Deccmber 20, 19(JI; acecptcd Oclobcr 2(j, 19~)2.

Jabbour-Zahah R., D. Chagot, F. Blanc, H. Grizel. Aquat. Ur/ng Resour., 1992,5,287-298.

Abstract Thc cdgc and isthmus of the mantlc of Pilletada margaritirera (Mol/usc, Bivalvia) were studied using light and clectron microscopy l'rom a morphological and histochemical point of vicw. Six areas with several subarcas wcre diffcrentiated in the mantle edge. One of thesc areas is similar to the epithelium isthmus, Trials to elucidate the nature of the pigment responsible for the black colour of the shcll and pearl indicate a melanin-likc material. Possible funetions of the distinctive cpithelial areas in Pinetada margaritilera mantle are discussed aceording to avaiJable data on the secretion of organie matrix of the shell, protein sc1erotization and deposition of crystals in sorne pearl-forming molluses. Three different epithelial areas in the outer mantle edge of pjllC1ada margaritiféra are probably involvcd in the scquential processes of deposition of calcite prisms, aragonite fibres and aragonite rhombs.

Keywords: Mantle, structure, ultrastrueture, pigment, Pinetada margaritilera, Mollusc, Bivalvia.

Hislologie, histoehimie el ultrastructure du manteau de l'IIUÎtr!' perlière, Pinctada margaritifera (L).

Résumé Le bord et l'isthme du manteau de Pinelada margaritifera (Mollusque, Bivalve) ont été étudiés du point de vue morphologique et histoehimique par microscopies photonique ct électronique. Six zones et plusieurs sous-zones ont été identifiées dans le hord du manteau. L'une d'entre elles est similaire cl l'épithélium de j'isthme. Les essais de détermination de la nature du pigment responsable de la wuleur noire de la coquille et de la perle, ont abouti à un type de mélaninc, Le rôle des différentes zones idcntifiées dans l'épithélium palléal de Pinctada margaritijera est discuté en fonction des données acquises chez d'autres Mollusques perliers ou non sur la sécrétion de la matrice organique de la coquille, la sc!érotisation des protéines et le dépôt des biocristaux, Cette analyse permet notamment d'attribuer à trois des zones décrites ici unc fonction dans le dépôt séquentiel des prismes de calcite, des fibres et des rhomboèdres d'aragonite.

i\1ots-c1és ; Manteau, structure, ultrastruclure, pigment, Pillctada margaritifàa, Mollusques, Bivalves.

A'lua(. Li"ing Rfsallr. 92;04 2x7 12 $ 320 /«:) IfREMER -Gauthier-Villars

AquaJ l.i1'ing Rcsour., 1992, 5, 287-298

Mande histology, histochem.istry and ultrastructure of thepearl oyster Pinctada rnargaritijera (L.)

Roula Jabbour-Zahab (1), Dominique Chagot (2) t, Françoise Blanc (1 \ *) and Henri Grizel (21

(1) raboraloiTe dp ,(oogéograf!/tie GénPllqne, Cnivpnilé JifonlfJPllieT 3,!J.J'. j043.3!032 Afonlpel/in Cedex J, Franle.

(2) l_abowtoire dE j>atilillogie el CéndiquE rle.1 11l1nlébréJ Afarin.l, 1FREAU,-'!?,B.l'. 1']3, J7390 La Tremblade, FmnCf.

(*) 10 wfwm aU corre.lpolldCllle slwuld be uddrl'.lsld.

Rcceivcd Deccmber 20, 19(JI; acecptcd Oclobcr 2(j, 19~)2.

Jabbour-Zahah R., D. Chagot, F. Blanc, H. Grizel. Aquat. Ur/ng Resour., 1992,5,287-298.

Abstract

Résumé

Thc cdgc and isthmus of the mantlc of Pilletada margaritirera (Mol/usc, Bivalvia) were studiedusing light and clectron microscopy l'rom a morphological and histochemical point of vicw. Six areaswith several subarcas wcre diffcrentiated in the mantle edge. One of thesc areas is similar to theepithelium isthmus, Trials to elucidate the nature of the pigment responsible for the black colour ofthe shcll and pearl indicate a melanin-likc material. Possible funetions of the distinctive cpithelialareas in Pinetada margaritilera mantle are discussed aceording to avaiJable data on the secretion oforganie matrix of the shell, protein sc1erotization and deposition of crystals in sorne pearl-formingmolluses. Three different epithelial areas in the outer mantle edge of pjllC1ada margaritiféra areprobably involvcd in the scquential processes of deposition of calcite prisms, aragonite fibres andaragonite rhombs.

Keywords: Mantle, structure, ultrastrueture, pigment, Pinetada margaritilera, Mollusc, Bivalvia.

Hislologie, histoehimie el ultrastructure du manteau de l'IIUÎtr!' perlière, Pinctada margaritifera (L).

Le bord et l'isthme du manteau de Pinelada margaritifera (Mollusque, Bivalve) ont été étudiés dupoint de vue morphologique et histoehimique par microscopies photonique ct électronique. Six zoneset plusieurs sous-zones ont été identifiées dans le hord du manteau. L'une d'entre elles est similaire cll'épithélium de j'isthme. Les essais de détermination de la nature du pigment responsable de la wuleurnoire de la coquille et de la perle, ont abouti à un type de mélaninc, Le rôle des différentes zonesidcntifiées dans l'épithélium palléal de Pinctada margaritijera est discuté en fonction des donnéesacquises chez d'autres Mollusques perliers ou non sur la sécrétion de la matrice organique de lacoquille, la sc!érotisation des protéines et le dépôt des biocristaux, Cette analyse permet notammentd'attribuer à trois des zones décrites ici unc fonction dans le dépôt séquentiel des prismes de calcite,des fibres et des rhomboèdres d'aragonite.

i\1ots-c1és ; Manteau, structure, ultrastruclure, pigment, Pillctada margaritifàa, Mollusques, Bivalves.

A'lua(. Li"ing Rfsallr. 92;04 2x7 12 $ 320 /«:) IfREMER -Gauthier-Villars

288

INTRODUCTION

The black pearl oyster Pinctada margaritif'era (Linné, 1758), widespread in French Polynesia, has been harvested since the nineteenth century for its nacreous matter and, more recently, cultured for the production of pearls. This species produces a striking amount of black pearls in addition to white or grey pearls.

ln ail molluscs, the mantle is directIy responsible for shel1 synthesis. In Bivalve molluscs, the mantle tissue covcrs the visceral mass and adheres to the inner surface of the valves. The two mantle lobes are usually unattached ventrally and laterally, but are joined together dorsally along the hinge line to consti­tute the mantle isthmus. The central part of each lobe con tains a thin connective tissue layer that covers muscle fibres, nerves and blood vessels. The connec­tive tissue is limited by two epitheliai unicellular layers (Istin and Masoni, 1973).

The mantle edge as described by Dix (1973) in Pinctada maxima consists of three folds: an outer, a middle and an inner fold and the palliaI zone lying immediately inside these three free folds up to the !ine of gill attachment. The central mantle is situated between the !ine of gill attachment and the mantle isthmus.

The present study was carried out to describe the general structure of the mantle edge and isthmus of P. margaritifera. Histological and histochemical data are compared to those obtained from some other Bivalves incJuding pearl species such as P. martensii, P. maxima and P. radiata and discussed according to the potcntial raie of the mantle edge in the complex process of shcll formation (Ojima, 1952; Kawakami and Yasuzumi, 1964; Nakahara and Bevelander, 1971; Dix, 1972; Dix, 1973; Sa1euddin and Petit, 1983).

In Pinctada margaritifera and other pearl species, the shell is composed of two major crystalline forms: peripheral calcitic prisms and aragonitic rhom bs. However. un1ike al! other pearl oysters, in P. margaritij'era the calcitic prisms are total!y black while the aragonitic rhombs are pure white nacre except the very first aragonitic layer which is slightly coloured (J. P. CuiL pers. comm.).

Finally sorne assays were undertaken to determine the nature of the pigment that may be re1eascd from the mantle and be responsible for the black coloration of the shell and pearl.

MATERIAL AND METHOOS

Specimens of adult P. margaritifera were collected by diving in Takapoto, an atoll of the Tuamotu archipelago in French Polynesia. For Iight micros­copy, whole black mantles were dissected and fixed

R. Jabbour-Zahab et al.

either in buffered formaI in according to Carson et al. (1973) or in Bouin Hollande's fluid (Gabe, 1968).

Selected pieces of tissue as indicated in figure 1 werc cmbedded in paraffin. and eut into 3 ~m thick

manUe --t--t

F- ..o---~~ntral pleçeshell_--t

Figure 1. - Parts of Pil1cladu !I1urgarilijàu uscd for lhe histological sludy.

sections. The sections were stained for morphological studies with either Ehrlich's hemaroxylin-eosin, Goldner's trichrome (a modification of Masson's trichrome) or Giemsa's stain.

Histochemical studies were made using periodic­acid-Schiff (PAS) for the detection of carbohydrates and PAS combined with alcian blue (PAS-AB) for the characterization of the ditlerent mucopolysaccharides. Trials to determine the chemical nature of the black pigment of the mantle were performed using Turnbulrs blue method for detection of terrous ions, the Prussian blue method for ferric ions, and Turnbull's blue reaction modified by Tirmain and Schmeltzer for total ionic iron. Copper and magne­sium were stained with the method of Okamoto and Utamura and the magneson method respectively. Melanins were investigated using the Hueck's staining (hydrogen peroxide bleaching), potassium permanga­nate bleaching, the ferrous ion technique for melanins according to Lillie, and the hexamine-silver variant of the Masson-Fontana method. Al! staining methods were performed according to Gabe (1968) and Pearse (1980). In addition, buffered formalin flxed tissues were used ta localize peroxidase activity in 5 /Jm thick frozen sections (Graham and Karnovsky, 1966).

For transmission electron microscopy (TEM), smal! mantle pieces were fixed for 12 h at 4T in 2.5% glutaraldehyde in 0.1 M cacodylate-HCl buffer (pH 7.6, 1000 mosm). The tissues were rinsed in buf­fer alone, then postfixed for 1 h in 1% osmium tetro­xide in the same buffer. After a final rinse in buffer, tissues were embedded in Epon 812. Semithin sections (0.5 /Jm thick) were eut using glass knives and stained with 0.5% toluidine blue in 1% sodium carbonate (pH Il.0) Ultrathin sections (60 to 90 nm) were eut

A411al. Living Resour.

288 R. Jabbour-Zahab et al.

Figure 1. - Parts of Pil1ctadu !I1urgarilijàu uscd for lhe histologicalsludy.

either in buffered formaI in according to Carson et al.(1973) or in Bouin Hollande's fluid (Gabe, 1968).

Selected pieces of tissue as indicated in figure 1werc cmbedded in paraffin. and cut into 3 ~m thick

sections. The sections were stained for morphologicalstudies with either Ehrlich's hematoxylin-eosin,Goldner's trichrome (a modification of Masson'strichrome) or Giemsa's stain.

Histochemical studies were made using periodic­acid-Schiff (PAS) for the detection of carbohydratesand PAS combined with alcian blue (PAS-AB) for thecharacterization of the ditlerent mucopolysaccharides.Trials to determine the chemical nature of the blackpigmcnt of the mantle were performed usingTurnbulrs blue method for detection of terrous ions,the Prussian bluc method for ferric ions, andTurnbull's blue reaction modified by Tirmain andSchmeltzer for total ionic iron. Copper and magne­sium were stained with the method of Okal11oto andUtamura and the magneson method respectively.Melanins were investigated using the Hueck's staining(hydrogen peroxide bleaching), potassium permanga­nate bleaching, the ferrous ion technique for melaninsaccording to Lillie, and the hexamine-silver variantof the Masson-Fontana method. Al! staining methodswere performed according to Gabe (1968) and Pearse(1980). In addition, buffered formalin flxed tissueswere used ta localize peroxidase activity in 5 /Jm thickfrozcn sections (Graham and Karnovsky, 1966).

For transmission electron microscopy (TEM), smal!mantle pieces were fixed for 12 h at 4T in 2.5%glutaraldehyde in 0.1 M cacodylate-HCl buffer(pH 7.6, 1000 mosm). The tissues were rinsed in buf­fer alone, then postfixed for 1 h in 1% osmium tetro­xide in the same buffer. After a final rinse in buffer,tissues were embedded in Epon 812. Semithin sections(0.5 /Jm thick) were eut using glass knives and stainedwith 0.5% toluidine blue in 1% sodium carbonate(pH Il.0) Ultrathin sections (60 to 90 nm) werc eut

F- ..o---~~ntral pleçeshell_--t

manUe--t--t

INTRODUCTION

MATERIAL AND METHOOS

The black pearl oyster Pinctada margaritif'era(Linné, 1758), widespread in French Polynesia, hasbeen harvested since the nineteenth century for itsnacreous matter and, more recently, cultured for theproduction of pearls. This species produces a strikingamount of black pearls in addition to white or greypearls.

ln ail molluscs, the mantle is directIy responsiblefor shel1 synthesis. In Bivalve molluscs, the mantletissue covcrs the visceral mass and adheres to theinner surface of the valves. The two mantle lobes areusually unattached ventrally and laterally, but arejoined together dorsally along the hinge line to consti­tute the mantle isthmus. The central part of each lobecontains a thin connective tissue layer that coversmuscle fibres, nerves and blood vessels. The connec­tive tissue is limited by two epitheliai unicellular layers(Istin and Masoni, 1973).

The mantle edge as described by Dix (1973) inPinctada maxima consists of three folds: an outer, amiddle and an inner fold and the palliaI zone lyingimmediately inside these three free folds up to theline of gill attachment. The central mantle is situatedbetween the !ine of gill attachment and the mantleisthmus.

The present study was carried out to describe thegeneral structure of the mantle edge and isthmus ofP. margaritifera. Histological and histochemical dataare compared to those obtained from some otherBivalves incJuding pearl species such as P. martensii,P. maxima and P. radiata and discussed according tothe potcntial raie of the mantle edge in the complexprocess of shcll formation (Ojima, 1952; Kawakamiand Yasuzumi, 1964; Nakahara and Bevelander,1971; Dix, 1972; Dix, 1973; Sa1euddin and Petit,1983).

In Pinctada margaritifàa and other pearl species,the shell is composed of two major crystalline forms:peripheral calcitic prisms and aragonitic rhom bs.However. un1ike al! other pearl oysters, inP. margaritij'era the calcitic prisms are total!y blackwhile the aragonitic rhombs are pure white nacreexcept the very first aragonitic layer which is slightlycoloured (J. P. CuiL pers. comm.).

Finally sorne assays were undertaken to determinethe nature of the pigment that may be re1eascd fromthe mantle and be responsible for the black colorationof the shell and pearl.

Spccimens of adult P. margaritifera were collectedby diving in Takapoto, an atoll of the Tuamotuarchipelago in French Polynesia. For Iight micros­copy, whole black mantles were dissected and fixed

A411al. Living Resour.

289 Mantle histology and ultrastructurc of the pearl oyster

with a diamond knife, automatically contrasted with aqueous manyl acetate and lead citrate in a LKB Ultrostainer, and examined in a Jeol 1200 SX TEM.

RESULTS

Mantle edge

On the basis of specifie cellular characteristic, thc mantle edge has been divided into six areas with several subareas From the inner side to the outer side (fig. 2; lahle 1).

Inner fold

Middle fold

Perla.tracsl groove

~=__======---c===U Figure 2. - Radial scction through the mantlc cdge of Fil/cwil" I/liIrgaririjera.

Area (1) comprises the inner fold and the inner side of the middle fold. The epithelium Iining the inner slde of the free mantlc edge and up to the tip of the middle fold is eomposed of small columnar cells that are heavily pigmented when using Bouin­Hollande's fixation. When using buffered formalin, however, these cells appear unpigmented (fig. 3). TEM shows that three cellular types are prcsent: eleetron-Iuccnt cells with vacuoles and microvilli, pig­ment containing cclls with electron dense spherical granules and microvilli, elcctron dense cells bearing microvilli and cilia tlig. 15). Intcrccllular dilated spa­ces are sometimes observed near the inner fold. Neu­tral and acid glycoprotein-containing rnucous cells (PAS and PAS-AB positive) are very abundanl in the inner side epitheliurn of the inner fold and of the palliaI zonc. Many secretory cells containing acid and neutral glycoprotein granules are visible in the subepithelium of the inner side. The inner fald shows numerous radial and cireular muscles. In connective tissue, the intra- and extracellular granulcs are clee­tron dense.

In the middle fold, the shape of the cells is sirnilar to that found in the inner fold. Sorne of the cells arc pigrnented. From the inner lO the middle fold. the number of cells slained by the PAS and PAS-AB reactions decreases.

Area (2) is the outer side of the middle fold. Area (2 a) (fig. 4) is characterized by aeidophilic secretory cells and rare pigmented cells identieal to those of area (1). The cells are connected by desmosomes and alternate with vacuolar cells more or less empty towards the apical cytoplasm (fig. 16). Sorne pig­rnented cells have microvilli and cilia while others possess brush borders. In these latter cells. the Golgi apparatus is prominent. Ciliatcd cells apparently

Table t. - Pillclada margarili(era: Data on epithelial areas (+ presence. - absence)

Inner palliai zone and inner fold

Middle fold

Periostracal groove

Outer fo]d

Outer palliaI zone

Area Epithelium

and cellular type Mclanin-like

pigment

Columnar epithc1lum +++ Cells with mierovilli and cilia Vaeuolar œlls with microvillj

2 Cuboidal or columnar Ciliated eclls

3 P,eudostratified epithc1ium Ciliated œlls

4 Columnar epithelium + -Ciliatcd cclls with microvi]1i Non-eiliated cclls

5 Transitional epithelium +­(l'rom columnar 10 cuboidaI)

6 Cuboidal epithelium

Isthmus Cuboidal epithelium Vacuolar cells CeIls with microvilli

Secretions

Mucous neutral and acid glyeoproteins

Granular neutral glyco­proteins Neutral and acid glyco­proteins Granular and IllUCOUS

neutral and acid g]yco­protcins Mucous rare ncutral gly­coproteins Granular and mucous

neutral and acid glyeo­protcins

Granulai and mueous neutral and aeid glyco­proteins

Vol. 5, n" 4· 1992

Mantle histology and ultrastructurc of the pearl oyster

with a diamond knife, automatically contrasted withaqueous manyl acetate and lead citrate in a LKBUltrostainer, and examined in a Jeol 1200 SX TEM.

RESULTS

Mantle edge

On the basis of specifie cellular characteristic, thcmantle edge has been divided into six areas withseveral subareas From the inner side to the outer side(fig. 2; lahle 1).

Inner fold

Middle fold

Perla.tracslgroove

~=__======---c===UFigure 2. - Radial scction through the mantlc cdge of Fil/cwil"I/liIrgaririjera.

289

Area (1) comprises the inner fold and the innerside of the middle fold. The epithelium Iining theinner slde of the free mantlc edge and up to the tipof the middle fold is eomposed of small columnarcells that are heavily pigmented when using Bouin­Hollande's fixation. When using buffered formalin,however, these cells appear unpigmented (fig. 3).TEM shows that three cellular types are prcsent:eleetron-Iuccnt cells with vacuoles and microvilli, pig­ment containing cclls with electron dense sphericalgranules and microvilli, elcctron dense cells bearingmicrovilli and cilia tlig. 15). Intcrccllular dilated spa­ces are sometimes observed near the inner fold. Neu­tral and acid glycoprotein-containing rnucous cells(PAS and PAS-AB positive) are very abundanl in theinner side epitheliurn of the inner fold and of thepalliaI zonc. Many secretory cells containing acidand neutral glycoprotein granules are visible in thesubepithelium of the inner side. The inner fald showsnumerous radial and cireular muscles. In connectivetissue, the intra- and extracellular granulcs are clee­tron dense.

In the middle fold, the shape of the cells is sirnilarto that found in the inner fold. Sorne of the cells arcpigrnented. From the inner lO the middle fold. thenumber of cells slained by the PAS and PAS-ABreactions decreases.

Area (2) is the outer side of the middle fold. Area(2 a) (fig. 4) is characterized by aeidophilic secretorycells and rare pigmented cells identieal to those ofarea (1). The cells are connected by desmosomes andalternate with vacuolar cells more or less emptytowards the apical cytoplasm (fig. 16). Sorne pig­rnented cells have microvilli and cilia while otherspossess brush borders. In these latter cells. the Golgiapparatus is prominent. Ciliatcd cells apparently

Table t. - Pillclada margarili(era: Data on epithelial areas (+ presence. - absence)

AreaEpithelium

and cellular typeMclanin-like

pigmentSecretions

Inner palliai zone and innerfold

Middle fold

Periostracal groove

Outer fo]d

Outer palliaI zone

Vol. 5, n" 4· 1992

Columnar epithc1lumCelis with mierovilii and ciliaVaeuolar œlls with microvillj

2 Cuboidal or columnarCiliated eclls

3 P,eudostratified epithc1iumCiliated œlls

4 Columnar epitheliumCiliatcd cclls with microvi]1iNon-eiliated cclls

5 Transitional epithelium(l'rom columnar 10 cuboidaI)

6 Cuboidal epilhelium

Isthmus Cuboidal epitheliumVacuolar cellsCeIls with microvil!i

+++

+ -

+-

Mucous neutral and acidglyeoproteins

Granular neutral glyco­proteinsNeutral and acid glyco­proteinsGranular and IllUCOUS

neutral and acid g]yco­protcinsMucous rare ncutral gly­coproteinsGranular and mucous

neutral and acid glyeo­prolcins

Granulai and mueousneutral and aeid glyco­proteins

290

deposit electron-dense material in the periostracal groove.

Tall glycoprotein-containing epithelial and subepi­thelial mucous cells (fig. 17), and ciliated cens are abundant in the (2 h) area (fig. 5). In this area. the number of pigmented cells decreases.

The epithelium of the middle fold in the (2 c) area (fig. 6). is composed of some pigmented cens. Other cells are non-ciliated, irregularly shaped. Some of them bear microvilli (fig. 18) with apical granules of moderate electron density containing neutral glyco­proteins.

Arca (3) represents the bottom of the periostracal groove (fig. 7). This area is covered by a pseudo­stratified epithelium. Electron microscopy shows dilated intercellular spaces into which electron dense material, thought to he periostracum, aceumulates (fig. 19). The cells are eharaeterized by numerous small eleetron-Iucent vacuoles (fig. 20). A short area eomposed of ciliated cells (fig. 21) is seen immcdiately after the (2 c) area. The pcriostraeal material ean be seen between the eilia (.fig. 22). Based on histochemi­cal reactions, the periostracal material is proteina­ceous in nature and contains neutraJ and acid glyco­proteins as shown by PAS and PAS-AB positivity.

Area (4) corresponds to the two sides of the outer fold. Below area (3), the epithelium (4a) (fig. 8) is columnar, with non-ciliated eells and a sparse popula­tion of pigmented cells provided with a brush border (fig. 23). Neutral glycoprotein-containing secrelory granular cells are suhepithelial.

Towards the (4b) area the columnar epithelial eclls become less high and the intercellular spaces increase (.fig. 9). The cells contain electron dense pigment. Sorne vacuolar cells have a clear cytoplasm with mierovilli. others have a dense cytoplasm wilh miero­villi and cilia (fig. 24). The mueous and the granular subepithelial cells show PAS positivity (acid glycopro­teins).

At the outer fold tip (4 c) area, an infolded and cuboidal epithelium, withoul granular secretory cells.

R. Jabbour-Zahab et al.

is observed (fig. 10). Cuboidal cells are characterized hy basal nuc!ei with mierovilli and cilia (fig. 25). M ucous ecll containing neutral glycoproteins are suhepithelial.

In area (4d) (fig. Il). the epithelium. composed of tall columnar ce1ls and of mucous cells with microvilli and cilia, is more infolded than that of (4 cJ. Ultra­structural fcatures of cells in this area are identical to those of area (4 cl. Epithelial or subepithelial cells contain eithcr acid glycoproteins granular secretions or neutral mucous secretions.

Area (5) is a very small area limiting the outer free mantle rold. In this area, the ccII height decreases progressive1y to constitute a transition zone (fig. 26,27.28) between the columnar (4d) and cuboidal epilhelium typical of area 6. In the transition zone, the vacuolar eclls are eharaeterized by apical secretions (fig. 26). In this area, little of the neutral secretions are subepithelial.

Further in toward the shel\' the epithelial cells flat­ten in area (6) (fig. 13). They have a basal elongated nucleus with short protruding cell processes (fig. 29). The epithelial cells containing aeid glycoprotein seere­tory granules alternate with neutral glycoprotein-con­taining mucous epithelial cells. The lwo seeretory types occur in the subepithelial connective tissue of the ouler palliaI zone.

Peroxidase activity with similar intensity was detected throughout the whole mantle edgc.

Mantle isthmus

Lying inside the shell hinge line, the mantle isthmus (fig. 14) shows a dominant proportion of euboidal eells with basal nuclei and polymorphic vaeuoles (j7g. 30). These cells have microvilli (fig. 30. 31) and sometimes few eleetron dense gr<lnules (.fig. 31).

Light microscopy shows that some eclls contain mucus, and thal others have small or large secretory granules. Mucous cells and largc seeretory granule cells are positive for neutral glycoprotein reaclion.

Figures 3 to 14. - I.ighl micrographs. 3 j1111 radial parartin sections 01' the mantle edge. and isthmus of P. I1wrgariti/l'w.

3: Arca (1). cpithe1ium (ep) of the inner rold showing pigmented œlls, connective tissue (ct) and muscle lihre (1110.

4: Arca (2a) sllOwing aeidophilie secretory cells (ac) and rare pigmented œlls (pc)

5: An:a (2 h) containing tall secretory cells (sc).

6: Arca (2 c) with pigmented cells.

7: Ar~a (3). p~riostracal groov~ hottom eontaining pscudo-stratified cpithelium (pse) and vacuolar ceUs (ve). The l'orming perioslracum (P)

is c1early visihle.

8: Area (4 a). eolumnar epithelium with sparse pigmented cells (pc). neutral glycoprottin-eontaining suhepithelial cells (nSe).

9: Area (4 hl. vaeuolar ceUs (ve) with proeminent intercellular spaces (is).

Ill: Area (4 l'). infolded cuhie epitheliuOl.

Il: Arca (4 d), eolumnar epithelial ee1ls and mueous subepilheliaJ œl1s (I11Se).

12: Area (5) showing Iwo cellular types: cubie cclls (cc) and taller cells (tel.

13: Flaltened cells in "rea (6) with mueoUs cells (mc! and aeid glycoprolcin containing secretory eells (ase).

14: Mantle islhmus showing vacuolar cdls (ve) and mucolls œl1s (me).

AquaL Living ReS()UL

290

deposit electron-dense material in the periostracalgroove.

Tall glycoprotein-containing epithelial and subepi­thelial mucous cells (fig. 17), and ciliated cens areabundant in the (2 h) area (fig. 5). In this area. thenumber of pigmented cells decreases.

The epithelium of the middle fold in the (2 c) area(fig. 6). is composed of some pigmented cens. Othercells are non-ciliated, irregularly shaped. Some ofthem bear microvilli (fig. 18) with apical granules ofmoderate electron density containing neutral glyco­proteins.

Arca (3) represents the bottom of the periostracalgroove (fig. 7). This area is covered by a pseudo­stratified epithelium. Electron microscopy showsdilated intercellular spaces into which electron densematerial, thought to he periostracum, aceumulates(fig. 19). The cells are eharaeterized by numeroussmall eleetron-Iucent vacuoles (fig. 20). A short areaeomposed of ciliated cells (fig. 21) is seen immcdiatelyafter the (2 c) area. The pcriostraeal material ean beseen between the eilia (.fig. 22). Based on histochemi­cal reactions, the periostracal material is proteina­ceous in nature and contains neutraJ and acid glyco­proteins as shown by PAS and PAS-AB positivity.

Area (4) corresponds to the two sides of the outerfold. Below area (3), the epithelium (4a) (fig. 8) iscolumnar, with non-ciliated eells and a sparse popula­tion of pigmented cells provided with a brush border(fig. 23). Neutral glycoprotein-containing secrelorygranular cells are suhepithelial.

Towards the (4b) area the columnar epithelial ecllsbecome less high and the intercellular spaces increase(.fig. 9). The cells contain electron dense pigment.Sorne vacuolar cells have a clear cytoplasm withmierovilli. others have a dense cytoplasm wilh miero­villi and cilia (fig. 24). The mueous and the granularsubepithelial cells show PAS positivity (acid glycopro­teins).

At the outer fold tip (4 c) area, an infolded andcuboidal epithelium, withoul granular secretory cells.

R. Jabbour-Zahab et al.

is observed (fig. 10). Cuboidal cells are characterizedhy basal nuc!ei with mierovilli and cilia (fig. 25).M ucous ecll containing neutral glycoproteins aresuhepithelial.

In area (4d) (fig. Il). the epithelium. composed oftall columnar ce1ls and of mucous cells with microvilliand cilia, is more infolded than that of (4 cJ. Ultra­structural fcatures of cells in this area are identicalto those of area (4 cl. Epithelial or subepithelial cellscontain eithcr acid glycoproteins granular secretionsor neutral mucous secretions.

Area (5) is a very small area limiting the outer freemantle rold. In this area, the ccII height decreasesprogressive1y to constitute a transition zone(fig. 26,27.28) between the columnar (4d) andcuboidal epilhelium typical of area 6. In the transitionzone, the vacuolar eclls are eharaeterized by apicalsecretions (fig. 26). In this area, little of the neutralsecretions are subepithelial.

Further in toward the shel\' the epithelial cells flat­ten in area (6) (fig. 13). They have a basal elongatednucleus with short protruding cell processes (fig. 29).The epithelial cells containing aeid glycoprotein seere­tory granules alternate with neutral glycoprotein-con­taining mucous epithelial cells. The lwo seeretorytypes occur in the subepithelial connective tissue ofthe ouler palliaI zone.

Peroxidase activity with similar intensity wasdetected throughout the whole mantle edgc.

Mantle isthmus

Lying inside the shell hinge line, the mantle isthmus(fig. 14) shows a dominant proportion of euboidaleells with basal nuclei and polymorphic vaeuoles(j7g. 30). These cells have microvilli (fig. 30. 31) andsometimes few eleetron dense gr<lnules (.fig. 31).

Light microscopy shows that some eclls containmucus, and thal others have small or large secretorygranules. Mucous cells and largc seeretory granulecells are positive for neutral glycoprotein reaclion.

Figures 3 to 14. - I.ighl micrographs. 3 j1111 radial parartin sections 01' the mantle edge. and isthmus of P. I1wrgariti/l'w.

3: Arca (1). cpithe1ium (ep) of the inner rold showing pigmented œlls, connective tissue (ct) and muscle lihre (1110.

4: Arca (2a) sllOwing aeidophilie secretory cells (ac) and rare pigmented œlls (pc)

5: An:a (2 h) containing tall secretory cells (sc).

6: Arca (2 c) with pigmented cells.

7: Ar~a (3). p~riostracal groov~ hottom eontaining pscudo-stratified cpithelium (pse) and vacuolar ceUs (ve). The l'orming perioslracum (P)

is c1early visihle.

8: Area (4 a). eolumnar epithelium with sparse pigmented cells (pc). neutral glycoprottin-eontaining suhepithelial cells (nSe).

9: Area (4 hl. vaeuolar ceUs (ve) with proeminent intercellular spaces (is).

Ill: Area (4 l'). infolded cuhie epitheliuOl.

Il: Arca (4 d), eolumnar epithelial ee1ls and mueous subepilheliaJ œl1s (I11Se).

12: Area (5) showing Iwo cellular types: cubie cclls (cc) and taller cells (tel.

13: Flaltened cells in "rea (6) with mueoUs cells (mc! and aeid glycoprolcin containing secretory eells (ase).

14: Mantle islhmus showing vacuolar cdls (ve) and mucolls œl1s (me).

AquaL Living ReS()UL

291 Mantle histology and ultrastructurc of the pearl oystcr

'.3@J.tfl1 • ,li i\!i

•

Figures 3 to 14

Vol 5. n° 4 - 1492

Mantle histology and ultrastructurc of the pearl oystcr 291

'.3@J.tfl1• ,li i\!i

•

Figures 3 to 14

Vol 5. n° 4 - 1492

292 R. Jabbour-Zahab et al.

Figures 15 to 22. - Electronmicrographs.

15: Two main cellular types in area (1) (inner fold), pigmented cell (pc) containing spherical electron dense granules (dg) and hearing microvilli (mv); ekclron densc cells (dc) bearing microvilli and cilia (ci); nucleus (N); mitochondria (mi).

16: Arca (2a) acidophilic secretory œil (asc) containing granules (ag). vacuolar cell (vc); desmosome (d); mucous gland (mu).

17: Arca (2b) Ciliated (ci) and mucous (mu) cclls.

18: Area (2 c) Microvilli-hearing cells containing apical granules (ag).

19: Arca (3) Pseudo-stratified eprthelium into which intercellular spaces (is) are dilated and coumain periostracum (P).

20: Same area than figure 19 showing eells with numerous electron Juœnt vacuoles (Iv).

21: Ciliated (ci) cells Iying irnmediately after 2e area.

22: Periostracum (P) accurnulating between cilia (ci).

Sorne small granule secretory cells appear to contain acid mucopolysaccharides while others are devoid of glycoproteic secretion (PAS negative).

Pigment histochemistry

The histochemical reactions performed to deter­mine the nature of the black pigment in the mantle edge are presented in tahle 2. The reactions were negative for ferrous iron, ferric iron, ionic and non­ionic iron, copper, and magnesium. The hexamine sil ver staining variant for melanin and the Hueck staining were also negative. In contrast. the ferrous iron technique for melanin (Lillie's reaction. 1957) and the bleaching by the permanganate method showed positive reactions.

DISCUSSION

According to Wilbur and Saleuddin (1983) the formation of shell can be described in tenns of two major phases: 1) cellular processes of ion transport, protein synthesis and secretion and 2) a series of physicochemical processes in which crystals of CaC03 are nucleated, oriented, and grow in intima te associa­tion with a secreted organic matrix. Shell formation is a complex process involving three steps: the secre­tion of the organic matrix of the shell, protein tan­ning, and deposition of the crystals.

We tried to find a relationship between the mineral­ogical sequence occurring in shell formation and the cellular differentiation of the palliai epithelium, especially that of the outer fold. The presence of pigment in sorne epithelial areas may also indicate their possible role in black ca1citic prism deposition.

Tn Pinctada margaritifcra, the epithelium in area (1) is characterized by acid and neutral glycoprotein­filled mucous cells, and numerous pigmented cells with microvilli and cilia or with microvilli on1y. One type of these secretory cells is similar to those descri­bed in Anodonta cygnea. These cells are supposed to play a role in controlling the osmotic pressure of the hemolymph (Machado et al., 1988). Tn Pùzctada maxima, in this area. acid and neutral glycoproteic

secretions are granular (Dix, 1972) instead of mucous as in P. margaritzlera.

Our test on the chemical nature of the pigment found in areas (1), (2) up to (6) and absent in isthmus. have led to inconsistent resu1ts with respect to the presence of melanins: two assays were found positive (Lillie, KMn04 bleaching) and two were found nega­tive (Hueck, Masson-Fontana). ft must be mentioned that the first two reactions had been tested in P. maxima (Dix, 1973), another pearl-forming species. and produced the same positive result, leading Dix to assume that the pigment was indeed melanin. Tn P. margaritifèra. the who1e set of reactions regarding melanin is not positive. Considering, according to Pearse (1985). Lillie's reaction as the most specific for melanins, we consider the black pigment in P. margaritifèra to be mclanin-like.

Some metals originating from basaltic substratum. such as iron (Fe) or magnesium (Mg) have been found in lagoon waters (Serra, 1989). These metals, if selectively concentrated in Pinctada mantle, could be involved in the mantle coloration. Nevertheless, reactions for iron and magnesium were also negative.

Area (2) is characterized by ciliated cells and rare pigmented cel1s. Epithelial and subepithelial secretory cells contain acid and neutral glycoproteins like in Pinctada maxima (Dix, 1972). According to Petit (1978), Petit et al. (1980), Petit (1981) and Richardson et al. (1981), the epithe1ial cel1s lining the periostracal groove in Amhlema and Cerastoderma edule maintain and guidt: the initial periostracal material. These cells have microvilli in both species.

ln P. margaritifera. like in ail Bivalves, the perios­tracum arises from a groove located between the outer fold and the middle fold. Nascent periostracum can be seen in intercel1ular spaces at the pseudo-stratified epithelium level (area 3). These ciliated cells may be involved in guiding the initial periostracum towards the t:xit of the groove. The two epithelial layers (2 c) and (4 a), composed of cells with microvilli and which secrete neutral glycoproteins may play an important part in periostracum maturation, as suggested by the PAS-positive reaction of the periostracum. The newly formed periostracum in P. maxima is also found PAS positive (Dix, 1972).

AquaL Living Rcsour.

292 R. Jabbour-Zahab et al.

Figures 15 to 22. - Electronmicrographs.

15: Two main cellular types in area (1) (inner fold), pigmented cell (pc) containing spherical electron dense granules (dg) and hearingmicrovilli (mv); ekclron densc cells (dc) bearing microvilli and cilia (ci); nucleus (N); mitochondria (mi).

16: Arca (2a) acidophilic secretory œil (asc) containing granules (ag). vacuolar cell (vc); desmosome (d); mucous gland (mu).

17: Arca (2b) Ciliated (ci) and mucous (mu) cclls.

18: Area (2 c) Microvilli-hearing cells containing apical granules (ag).

19: Arca (3) Pseudo-stratified eprthelium into which intercellular spaces (is) are dilated and coumain periostracum (P).

20: Same area than figure 19 showing eells with numerous electron Juœnt vacuoles (Iv).

21: Ciliated (ci) cells Iying irnmediately after 2e area.

22: Periostracum (P) accurnulating between cilia (ci).

Sorne small granule secretory cells appear to containacid mucopolysaccharides while others are devoid ofglycoproteic secretion (PAS negative).

Pigment histochemistry

The histochemical reactions performed to deter­mine the nature of the black pigment in the mantleedge are presented in table 2. The reactions werenegative for ferrous iron, ferric iron, ionic and non­ionic iron, copper, and magnesium. The hexaminesil ver staining variant for melanin and the Hueckstaining were also negative. In contrast. the ferrousiron technique for melanin (Lillie's reaction. 1957)and the bleaching by the permanganate methodshowed positive reactions.

DISCUSSION

According to Wilbur and Saleuddin (1983) theformation of shell can be described in tenns of twomajor phases: 1) cellular processes of ion transport,protein synthesis and secretion and 2) a series ofphysicochemical processes in which crystals of CaC03are nucleated, oriented, and grow in intima te associa­tion with a secreted organic matrix. Shell formationis a complex process involving three steps: the secre­tion of the organic matrix of the shell, protein tan­ning, and deposition of the crystals.

We tried to find a relationship between the mineral­ogical sequence occurring in shell formation and thecellular differentiation of the palliai epithelium,especially that of the outer fold. The presence ofpigment in sorne epithelial areas may also indicatetheir possible role in black ca1citic prism deposition.

Tn Pinctada margaritifcra, the epithelium in area(1) is characterized by acid and neutral glycoprotein­filled mucous cells, and numerous pigmented cellswith microvilli and cilia or with microvilli on1y. Onetype of these secretory cells is similar to those descri­bed in Anodonta cygnea. These cells are supposed toplay a role in controlling the osmotic pressure ofthe hemolymph (Machado et al., 1988). Tn Pùzctadamaxima, in this area. acid and neutral glycoproteic

secretions are granular (Dix, 1972) instead of mucousas in P. margaritzlera.

Our test on the chemical nature of the pigmentfound in areas (1), (2) up to (6) and absent in isthmus.have led to inconsistent results with respect to thepresence of melanins: two assays were found positive(Lillie, KMn04 bleaching) and two were found nega­tive (Hueck, Masson-Fontana). ft must be mentionedthat the first two reactions had been tested inP. maxima (Dix, 1973), another pearl-forming species.and produced the same positive result, leading Dixto assume that the pigment was indeed melanin. TnP. margaritifèra. the who1e set of reactions regardingmelanin is not positive. Considering, according toPearse (1985). Lillie's reaction as the most specificfor melanins, we consider the black pigment inP. margaritifèra to be mclanin-like.

Some metals originating from basaltic substratum.such as iron (Fe) or magnesium (Mg) have beenfound in lagoon waters (Serra, 1989). These metals,if selectively concentrated in Pinctada mantle, couldbe involved in the mantle coloration. Nevertheless,reactions for iron and magnesium were also negative.

Area (2) is characterized by ciliated cells and rarepigmented cells. Epithelial and subepithelial secretorycells contain acid and neutral glycoproteins like inPinctada maxima (Dix, 1972). According to Petit(1978), Petit et al. (1980), Petit (1981) and Richardsonet al. (1981), the epithe1ial cells lining the periostracalgroove in Amblema and Cerastoderma edule maintainand guidt: the initial periostracal material. These cellshave microvilli in both species.

ln P. margaritifera. like in ail Bivalves, the perios­tracum arises from a groove located between the outerfold and tht: middle fold. Nascent periostracum canbe seen in intercel1ular spaces at the pseudo-stratifiedepithelium level (area 3). These ciliated cells may beinvolved in guiding the initial periostracum towardsthe t:xit of the groove. The two epithelial layers (2 c)and (4 a), composed of cells with microvilli and whichsecrete neutral glycoproteins may play an importantpart in periostracum maturation, as suggested by thePAS-positive reaction of the periostracum. The newlyformed periostracum in P. maxima is also found PASpositive (Dix, 1972).

AquaL Living Rcsour.

293 Mantle histology and ultrastructure of the pearl oyster

Figur~, 15 10 22

Vol. 5. n"4 - 1992

Mantle histology and ultrastructure of the pearl oyster 293

Figur~, 15 10 22

Vol. 5. n"4 - 1992

294

Figures 23 to 29. - Elcctronmicrographs (contd.).

23: Non-ciliatcd colul11nar cell bcaring microvilli in area (4 Il).

24: (4 h) area dense cytoplasrn cell with microvilli and cilia.

25: (4c) area. cuboidal cells with basal nuclei. microvilli and cilia.

26: Arca (5): cells containing vacuoles (va) and apical granules (ag).

27 and 28: Area (5): cells becoming Iess high compared to F<;urc 26.

29: Area (6) palliai zone. Fiai cclls with short ccII processcs (Cp).

Kawakami and Yasuzumi (1964) mentioned that in P. marlcnsii the periostraeum is formed by a special modilïeation of the internaI surface of the outer fold.

1n Amhlcl71a, (Petit, 1978) the periostraeal material originates l'rom interealated eells loeated in the inner side of the periostraeal groove. These cells are devoid of mierovilli and have a protruding tongue. The epi­thelial layers along the periostraeal groove secrete a glueidic coating on the two sides of the periostraeum with the help of two cell ular types similar to those we describe for P. rnargarilijera: cuboidal cclls with short mierovilli in area (2 e) and epithelial cells with high microvilli in area (4a) (Petit, 1978; Saleuddin and PetiL 1983).

1n Maerumllisla maculala (Bevelander and Naka­hara. 1967). the periostracum is first c1aborated in a Iimited cellular area at the bottom of the periostracal groove by cells bearing long microvilli, a situation different l'rom Aslarlc (Saleuddin, 1974) in whieh the periostracum origin can be found in the large epithe­liai cells of the outer fold devoid of mierovilli but containing ehannels. Their cytoplasm has eleetron dense inclusions, which are membrane-bound vesicles supposed to be periostracum precursors.

ln Cerasluderma cdule (Richardson el al., 1981), periostraeal groove cells play a role in periostracum production: these are basal cells of the outer fold and cells called accessory basal cells. A type of the latter, located between the ftrst basal cell and the first epithe­liaI cell of the outer rold. is supposed to push the periostracum towards the outside of the groove and to protect it l'rom tearing when the mantle retracts during shell c1osure.

The secretion of periostracum is aeeompanied by sclerotization of the proteins. a process presumed to be similar to quinone tanning, by polymerization of phenolic eompounds resulting l'rom oxidative degeneration of tyrosine. Thus. melanins, a group of polymerie molecules formed l'rom tyrosine (Verhecken. 1989) have been described in several spe­cies of Molluscs (Fox, 1983), and particulary in P. ma.Yinw (Dix, 1973) in the middle and inner rold epithelia of the mantle.

Enzymes of the mono- and polyphenol oxidase classes are copper proteins. The tyrosinase of the wall of the ink sac in the octopus and squid is especially rich in copper. However. in our study copper was not deteeted in any mantle tissue cclls indicating that, if

R. Jabbour-Zahah et al.

present. copper concentration is below the sensitivity of the used method (Pearse. 1985). The strong peroxi­dase activity might indieate the involvement of sueh an cnzyme in the tanning system (Waite. 1(83).

ln P. margarilllem, brown pigmented cells are spar­sely distributed in (4a) and (4h) areas of the outcr fold an in (2 a) and (2 e) arcas of the middle fold. The pigment in these areas persists partly when using buffered formalin fixation. Howevcr, the pigmcnt of others areas is dissolvcd in the same buffered fixation. Despite this differential behaviour. histochemical reactions used indieated the whole pigment to be of melanin nature.

ln Cerasloderma edl/le (Richardson el al., 1(81). the ion pump transporting calcium l'rom mantle towards the palliaI cavity. is assumed to be present in the cclls of the outer fold which arc provided with brush border and characterized by nurnerous mitochondria and large endoplasmic retieulum. These cells are similar to those deseribed in P. l71argaritijera area (4). Aeeording to Istin and Masoni (1973), in Bivalves, the number of mitochondria underlying thc epithelial border of the outer fold indicates a meta­bolic activity for such eells. This <lctivity is not linked with calcium movements but may be linked with matrix eomponent synthesis.

ln Pinelada margarilijera the columnar epithclium in area (4d) is charaeterized by ciliated cells with mierovilli. Numerous <lcid and neutral glycoprotein­filled gmnular and rnucous cells are epithelial and subepithelial.

Aeeording to Nakahara and Bevelander (1971), in P. rodiata, the prismatic layer of the shell is derived exelusively l'rom the tall columnar cells lining the outer surface of the outer rnant1c fold. Thesc cells are identical to those described in P. margarilijàa area (4d). Aecording to Petit (1978), in Al71hlel1la the middlc layer of the periostracum is involved in the prismatic layer of the shell. Unlike in Al71hlema. mantle epithelial cells loeated in the outer fold are assumed to contribute to this prismatic layer in P. radillta and in P. lIlargaritijera as weil. Furthcr­more, similar cells are described in the periostraeal or prismatic pearl sac in P. maxima (Dix, 1(73) and P. l71artensii (Machii, 1959; Aoki. 1(66).

ln area (6). flat epithelial cells are found to contain extremely abundant acidie and neutral glycoproteins secretions.

Aqual. Living Resour.

294

Figures 23 to 29. - Elcctronmicrographs (contd.).

23: Non-ciliatcd colul11nar cell bcaring microvilli in area (4 Il).

24: (4 h) area dense cytoplasrn cell with microvilli and cilia.

25: (4c) area. cuboidal cells with basal nuclei. microvilli and cilia.

26: Arca (5): cells containing vacuoles (va) and apical granules (ag).

27 and 28: Area (5): cells becoming Iess high compared to F<;urc 26.

29: Area (6) palliai zone. Fiai cclls with short ccII processcs (Cp).

Kawakami and Yasuzumi (1964) mentioned that inP. marlcnsii the periostraeum is formed by a specialmodilïeation of the internaI surface of the outer fold.

1n Amhlcl71a, (Petit, 1978) the periostraeal materialoriginates l'rom interealated eells loeated in the innerside of the periostraeal groove. These cells are devoidof mierovilli and have a protruding tongue. The epi­thelial layers along the periostraeal groove secrete aglueidic coating on the two sides of the periostraeumwith the help of two cell ular types similar to thosewe describe for P. rnargarilijera: cuboidal cclls withshort mierovilli in area (2 e) and epithelial cells withhigh microvilli in area (4a) (Petit, 1978; Saleuddinand PetiL 1983).

1n Maerumllisla maculala (Bevelander and Naka­hara. 1967). the periostracum is first c1aborated in aIimited cellular area at the bottom of the periostracalgroove by cells bearing long microvilli, a situationdifferent l'rom Aslarlc (Saleuddin, 1974) in whieh theperiostracum origin can be found in the large epithe­liai cells of the outer fold devoid of mierovilli butcontaining ehannels. Their cytoplasm has eleetrondense inclusions, which are membrane-bound vesiclessupposed to be periostracum precursors.

ln Cerasluderma cdule (Richardson el al., 1981),periostraeal groove cells play a role in periostracumproduction: these are basal cells of the outer fold andcells called accessory basal cells. A type of the latter,located between the ftrst basal cell and the first epithe­liaI cell of the outer rold, is supposed to push theperiostracum towards the outside of the groove andto protect it l'rom tearing when the mantle retractsduring shell c1osure.

The secretion of periostracum is aeeompanied bysclerotization of the proteins, a process presumed tobe similar to quinone tanning, by polymerization ofphenolic eompounds resulting l'rom oxidativedegeneration of tyrosine. Thus, melanins, a group ofpolymerie molecules formed l'rom tyrosine(Verhecken, 1989) have been described in several spe­cies of Molluscs (Fox, 1983), and particulary inP. ma.Yinw (Dix, 1973) in the middle and inner roldepithelia of the mantle.

Enzymes of the mono- and polyphenol oxidaseclasses are copper proteins. The tyrosinase of the wallof the ink sac in the octopus and squid is especiallyrich in copper. However, in our study copper was notdeteeted in any mantle tissue cclls indicating that, if

R. Jabbour-Zahah et al.

present, copper concentration is below the sensitivityof the used method (Pearse. 1985). The strong peroxi­dase activity might indieate the involvement of suehan cnzyme in the tanning system (Waite. 1(83).

ln P. margarilllem, brown pigmented cells are spar­sely distributed in (4a) and (4h) areas of the outcrfold an in (2 a) and (2 e) arcas of the middle fold.The pigment in these areas persists partly when usingbuffered formalin fixation. Howevcr, the pigmcnt ofothers areas is dissolvcd in the same buffered fixation.Despite this differential behaviour, histochemicalreactions used indieated the whole pigment to be ofmelanin nature.

ln Cerasloderma edl/le (Richardson el al., 1(81),the ion pump transporting calcium l'rom mantletowards the palliaI cavity, is assumed to be presentin the cclls of the outer fold which arc providedwith brush border and characterized by nurnerousmitochondria and large endoplasmic retieulum. Thesecells are similar to those deseribed in P. l71argaritijeraarea (4). Aeeording to Istin and Masoni (1973), inBivalves, the number of mitochondria underlying thcepithelial border of the outer fold indicates a meta­bolic activity for such eells. This <lctivity is not linkedwith calcium movements but may be linked withmatrix eomponent synthesis.

ln Pinelada margarilijera the columnar epithcliumin area (4d) is charaeterized by ciliated cells withmierovilli. Numerous <lcid and neutral glycoprotein­filled gmnular and rnucous cells are epithelial andsubepithelial.

Aeeording to Nakahara and Bevelander (1971), inP. rodiata, the prismatic layer of the shell is derivedexelusively l'rom the tall columnar cells lining theouter surface of the outer rnant1c fold. Thesc cells areidentical to those described in P. margarilijàa area(4d). Aecording to Petit (1978), in Al71hlel1la themiddlc layer of the periostracum is involved in theprismatic layer of the shell. Unlike in Al71hlema,mantle epithelial cells loeated in the outer fold areassumed to contribute to this prismatic layer inP. radillta and in P. lIlargaritijera as weil. Furthcr­more, similar cells are described in the periostraealor prismatic pearl sac in P. maxima (Dix, 1(73) andP. l71artensii (Machii, 1959; Aoki, 1(66).

ln area (6). flat epithelial cells are found to containextremely abundant acidic and neutral glycoproteinssecretions.

Aqual. Living Resnur.

295 IMllntl1' histology and ultrastructurr of the pearl oyster

1· i~urr' 23 tu 29

Vol ). 11" .f - ]LJl)2

IMllntl1' histology and ultrastructurr of the pearl oyster 295

1· i~urr' 23 tu 29

Vol ). 11" .f - ]LJl)2

296 R. Jabbour-Zahab et al.

Figures 30 and 31

Figure 30. - Mantle isthmus œlls with polymorph vacuoles (va) and brush border (mv).

Figure 31. - Same areafigure 30 showing kw electron dense granules (dg).

Table 2. - Pigment determination by using histochemical staining reactions.

Reaction Result

TurnbuIl's blue (Fe2 » Prussian blue (Fe 3 +)

TurnbuIl's blue (modified by Tirmain and Schmcltzerl (whole ionic Fe)

Okamoto and Utamura (Cu) Magneson (Mg) H ueck (peroxide bleaching) KMn04 bleaching + Lillie's (melanins) + Masson-Fontana (AgN0 3)

Area (5) may be considered as a transition zone between (4d) and (6) epithelia. However. unlike areas (4d) and (6). area (5) is found very poor in neutral as well as in acidic secretions,

Mantle isthmus cells are more similar to area (6) cells than other areas of mantle edge: cuboidal epithe­lium. no pigmented ce)]s and rich in acidic and neutral glycoproteins secretions,

As Samata et al, (1989) have pointed out. Ca­binding glycoproteins have been found very acidic in gorgonian spicules and in several Bivalve sheIls,

Thus. in P. margariti[era. areas (4d) and (6) can be related to intense activity in crystalline deposition due to special richness in acidic glycoprotein secre­tions. Conversely. area (5). found poor in neutral as weil as in acidic secretions. may be involved in a different process related ta aragonitic fibraus variety described by (Caseiro and Gauthier. in press). Fur­thermore. the morphological change noticed between (4d) and (6) areas allows us to corre1ate (4d). (5) and (6) areas ta the sequential processes of deposition of calcitic prisms. aragonitic fibres and aragonitic rhombs. Thus. epithelial and subepithelial cells in area (6) may be involved in the nacreous layer of the shell in Pinctada margaritifera. unlike in Amblema (Petit. 1978; Saleuddin and Petit. 1983).

Assuming the potential raIe of areas (5) and (6) in the deposition of aragonitic fibres and rhombs. these areas are supposed ta be convenient as graft in pearl

Aq U;Jt LIVlIlf!. ReSOLU'.

296 R. Jabbour-Zahab et al.

Figures 30 and 31

Figure 30. - Mantle isthmus œlls with polymorph vacuoles (va) and brush border (mv).

Figure 31. - Same areafigure 30 showing kw electron dense granules (dg).

Table 2. - Pigment determination by using histochemical stainingreactions.

As Samata et al, (1989) have pointed out. Ca­binding glycoproteins have been found very acidic ingorgonian spicules and in several Bivalve sheIls,

TurnbuIl's blue (Fe2 »Prussian blue (Fe 3 +)

TurnbuIl's blue (modified by Tirmainand Schmcltzer) (whole ionic Fe)

Okamoto and Utamura (Cu)Magneson (Mg)H ueck (peroxide bleaching)KMn04 bleaching +Lillie's (melanins) +Masson-Fontana (AgN0 3)

Area (5) may be considered as a transition zonebetween (4d) and (6) epithelia. However. unlike areas(4d) and (6). area (5) is found very poor in neutralas well as in acidic secretions,

Mantle isthmus cells are more similar to area (6)cells than other areas of mantle edge: cuboidal epithe­lium. no pigmented ce)]s and rich in acidic and neutralglycoproteins secretions,

Reaction ResultThus. in P. margariti[era. areas (4d) and (6) can

be related to intense activity in crystalline depositiondue to special richness in acidic glycoprotein secre­tions. Conversely. area (5). found poor in neutral asweil as in acidic secretions. may be involved in adifferent process related ta aragonitic fibraus varietydescribed by (Caseiro and Gauthier. in press). Fur­thermore. the morphological change noticed between(4d) and (6) areas allows us to corre1ate (4d). (5) and(6) areas ta the sequential processes of deposition ofcalcitic prisms. aragonitic fibres and aragoniticrhombs. Thus. epithelial and subepithelial cells in area(6) may be involved in the nacreous layer of the shellin Pinctada margaritifera. unlike in Amblema (Petit.1978; Saleuddin and Petit. 1983).

Assuming the potential raIe of areas (5) and (6) inthe deposition of aragonitic fibres and rhombs. theseareas are supposed ta be convenient as graft in pearl

Aq U;Jt LIVlIlf!. ReSOLU'.

297 Mantle histology and ultrastructure of the pearl oyster

culture. Furthermore, similar cells ta those of (5) and (6) areas in P. margaritlfera are found in the nacreous pearl sac of P. maxima (Dix, 1973).

Moreover the description of the healthy tissues can be used ta examine possible histological and histoche-

Acknowledgements

mical alterations of the mantle inducing dysfunctions in crystal or proteinaceous matI'ix production. Such dysfunctions leading ta proteinaceous hyperproduc­tian and decrease in crystal production are sometimes observed in this species.

The authors thank Dr Monique Henry and anonymous referees for eritieal reading of the manuscript. They are grateful to EVAAM for help in providing the samples of pearl oysters used in this study. This research was supported by funds from "Ministère des Départements et Territoires d'Outrc-Mer" to f". Blanc and H. Grizel (Grant CORDET XR-21O).

REFERENCES

Aoki S.. 1966. Comparative histological observations on the pearl-sac tissues forming nacreous. prismatie and periostraeal pcarls. Bull. lap. Soc. Sci. Fish, 32. 1-10.

Bevelander G .. H. Nakahara. 1967. An electron microscope study of the formation of the periostracum or Macro­callisla maculata. Ca/cif Tissue R",s .. 1. 55-67.

Carson F. L .1. H. Martin. J. A. Lynn. 1973. FOl'malin fixation for electron microscopy: a re-evaluation. Am. 1. C/in. Pa/hol.. 59, 365-373.

Caseiro J .. J. P. Gauthier. 1993. Experimental data on the shell reconstruction or the pearl oyster Pinc/ada l11argari­tifàa: 1. Damages intentionally eaused on the palliaI edge. Mar. Biol. (in press).

Dix T. G .. 197'2. Histoehemistry of mantle and pearl sac seeretory cells in Pinctada maxima (Lamellibranehia). Aust/'. 1. Zoo!.. 20..159-3611.

Dix T. G .. 1973. Histology of the mantle and pearl sac of th(; pearl oyster Pillclada maxima (Lamellibranehia) . .1. Malam/. Soc, Austral.. 2, 365-375.

Fox D. L.. In3. Bioehromy of the Mollusea. [Il: Thc Mollusca. 2, P. W. Hoehachtka Ed. Academic Press. N y, 281-303.

Gabe M., 196R. Techniques histologiques. Masson Paris. 1113 p.

Graham R. c.. M. J. Karnovsky. 1966. The early stages or absorption of injectcd horseradish peroxidase in the proximal tubuks or mouse kidney. Ultrastruetural cyto­chemistry by a new technique. l. Histo('lzem. Cytoclzem .• 14. 291-297.

Istin M .. A. Masoni, 1973. Absorption ct redistribution du Calcium dans le manteau des Lamellibranches. Calcif Tissue Res., 1L 151-160.

Kawakami 1. K .. G. Yasuzumi, 1964. Electron microscope studies on the mantle of the pearl oyster Pinctada manen­sii Dunker. Preliminary report. The fine structure of the periostracum fixed with permanganate. 1. Elec!ron. Aficrosc.. 13. 119-123.

Machado J .. F. Castilho. J. Coimbra. E Monteiro, C. Sa, M. Reis. 19811. Ultrastruetural and eytochemieal studies in the mantle of Anodon/a cygnea. Tissue Ce Il. 20, 797­807.

Vol. 5, n" 4 - 1992

Machii A.. 1959. Studies on the histology of the pearl sac. IV. On the pearl-sac of "so-called Kokuhan" bl<Kk spottcd. Bull. lI/all. Pearl Res. Lob, 5, 407-410.

Nakahara H., G. Bevelander. 1971. The formation and growth of the prismatic layer of Pillctada radialli. Calcif Tissue Res.• 7. 31-45.

Ojima Y.. 1952. Histological studies of the mantle or pearl oyster (PinCfada martellsù Dunkcr). C.J'Iologia. 2. 134­

143.

Pearse A. G. E.. 19115. Histochemistry. Theorcti(;al and applied. Churchill Livingstone. London. Fourth Ed .. 998 p.

Petit H., 1978. Recherches sur des séquences d'événements périostracaux lors de l'élaboration de la coquille d'Am­hl",ma plicilla perplica/a Conrad. 1834. Ph.D. dissertation. V.B.O.. France. 185 p. and Atlas.

Petit H., 1911 1. Survol de la minéralisation chez les Unioll­idac. Haliotis. Il. 181-195.

Petit H .. W. L. Davis. R. G. Jones. H. K. Hagler. 1980. Morphological studies on the ca1cifi(;ation process in the fresh-water mussel Amh!ema. Tissl/'" C",II. 12. 13-28.

Richardson C. A.. N. W. Runham. D. J. Crisp. 1981. A histological and ultraslructural study of the cells of the mantle edge of a marine bivalve. C",/"{/s/od",rl11a cdule. Tissue Cell, 13, 715-730.

Saleuddin A. S. M .. 1974. An electron microscopie study of the formation and structure or the periostracum in Asllirre (Bivalvia). Con 1. Zoo!., 52. 1463-1471.

Saleuddin A. S. M .. H. P. Petit, 1983. Thc mode of forma­tion and the structure or the periostracum. ln: The Mol­Il/sm. 4. K. M. Wilbur Ed .. Academie Press. N Y, 199­231.

Samata T.. R. J. Kingsley, N. Watabe, 19119. Ca-binding glycoproteins l'rom the spicules of the octocoral Lcpto­gorgia L'irglilala. CO/llp. Biocbcm. PhJ'.l'io!., 948. 4, 651-654.

Serra c.. 1989. Contribution du LESE au programme bio­géochimie des fonds de lagons d·atolls. Rapp. CEA. LESE. Papee/",. Pol. Fr .• 33 p.

Verheken A.. 1989. Th(; indole pigments of \1ollusea. Ann. Soc. R. Zool. Be/g.• 119, 181-197.

Mantle histology and ultrastructure of the pearl oyster

culture. Furthermore, similar cells ta those of (5) and(6) areas in P. margaritlfera are found in the nacreouspearl sac of P. maxima (Dix, 1973).

Moreover the description of the healthy tissues canbe used ta examine possible histological and histoche-

297

mical alterations of the mantle inducing dysfunctionsin crystal or proteinaceous matI'ix production. Suchdysfunctions leading ta proteinaceous hyperproduc­tian and decrease in crystal production are sometimesobserved in this species.

Acknowledgements

The authors thank Dr Monique Henry and anonymous referees for eritieal reading of the manuscript. They are grateful toEVAAM for help in providing the samples of pearl oysters used in this study. This research was supported by funds from"Ministère des Départements et Territoires d'Outrc-Mer" to f". Blanc and H. Grizel (Grant CORDET XR-21O).

REFERENCES

Aoki S.. 1966. Comparative histological observations onthe pearl-sac tissues forming nacreous. prismatie andperiostraeal pcarls. Bull. lap. Soc. Sci. Fish, 32. 1-10.

Bevelander G .. H. Nakahara. 1967. An electron microscopestudy of the formation of the periostracum or Macro­callisla maculata. Ca/cif Tissue R",s .. 1. 55-67.

Carson F. L .1. H. Martin. J. A. Lynn. 1973. FOl'malinfixation for electron microscopy: a re-evaluation. Am. 1.C/in. Pa/hol.. 59, 365-373.

Caseiro J .. J. P. Gauthier. 1993. Experimental data on theshell reconstruction or the pearl oyster Pinc/ada l11argari­tijàa: 1. Damages intentionally eaused on the palliaIedge. Mar. Biol. (in press).

Dix T. G .. 197'2. Histoehemistry of mantle and pearl sacseeretory cells in Pinctada maxima (Lamellibranehia).Aust/'. 1. Zoo!.. 20..159-3611.

Dix T. G .. 1973. Histology of the mantle and pearl sac ofth(; pearl oyster Pillclada maxima (Lamellibranehia) . .1.Malam/. Soc, Austral.. 2, 365-375.

Fox D. L.. In3. Bioehromy of the Mollusea. [Il: ThcMollusca. 2, P. W. Hoehachtka Ed. Academic Press. Ny, 281-303.

Gabe M., 196R. Techniques histologiques. Masson Paris.1113 p.

Graham R. c.. M. J. Karnovsky. 1966. The early stagesor absorption of injectcd horseradish peroxidase in theproximal tubuks or mouse kidney. Ultrastruetural cyto­chemistry by a new technique. l. Histo('lzem. Cytoclzem .•14. 291-297.

Istin M .. A. Masoni, 1973. Absorption ct redistribution duCalcium dans le manteau des Lamellibranches. CalcifTissue Res., 1L 151-160.

Kawakami 1. K .. G. Yasuzumi, 1964. Electron microscopestudies on the mantle of the pearl oyster Pinctada manen­sii Dunker. Preliminary report. The fine structure ofthe periostracum fixed with permanganate. 1. Elec!ron.Aficrosc.. 13. 119-123.

Machado J .. F. Castilho. J. Coimbra. E Monteiro, C. Sa,M. Reis. 19811. Ultrastruetural and eytochemieal studiesin the mantle of Anodon/a cygnea. Tissue Ce Il. 20, 797­807.

Vol. 5, n" 4 - 1992

Machii A.. 1959. Studies on the histology of the pearlsac. IV. On the pearl-sac of "so-called Kokuhan" bl<Kkspottcd. Bull. lI/all. Pearl Res. Lob, 5, 407-410.

Nakahara H., G. Bevelander. 1971. The formation andgrowth of the prismatic layer of Pillctada radialli. CalcifTissue Res.• 7. 31-45.

Ojima Y.. 1952. Histological studies of the mantle or pearloyster (PinCfada martellsù Dunkcr). C.J'Iologia. 2. 134­

143.

Pearse A. G. E.. 19115. Histochemistry. Theorcti(;al andapplied. Churchill Livingstone. London. Fourth Ed ..998 p.

Petit H., 1978. Recherches sur des séquences d'événementspériostracaux lors de l'élaboration de la coquille d'Am­hl",ma plicilla perplica/a Conrad. 1834. Ph.D. dissertation.V.B.O.. France. 185 p. and Atlas.

Petit H., 1911 1. Survol de la minéralisation chez les Unioll­idac. Haliotis. Il. 181-195.

Petit H .. W. L. Davis. R. G. Jones. H. K. Hagler. 1980.Morphological studies on the ca1cifi(;ation process in thefresh-water mussel Amh!ema. Tissl/'" C",II. 12. 13-28.

Richardson C. A.. N. W. Runham. D. J. Crisp. 1981. Ahistological and ultraslructural study of the cells of themantle edge of a marine bivalve. C",/"{/s/od",rl11a cdule.Tissue Cell, 13, 715-730.

Saleuddin A. S. M .. 1974. An electron microscopie studyof the formation and structure or the periostracum inAsllirre (Bivalvia). Con 1. Zoo!., 52. 1463-1471.

Saleuddin A. S. M .. H. P. Petit, 1983. Thc mode of forma­tion and the structure or the periostracum. ln: The Mol­Il/sm. 4. K. M. Wilbur Ed .. Academie Press. N Y, 199­231.

Samata T.. R. J. Kingsley, N. Watabe, 19119. Ca-bindingglycoproteins l'rom the spicules of the octocoral LCPfO­gorgia L'irglilala. CO/llp. Biocbcm. PhJ'.l'io!., 948. 4,651-654.

Serra c.. 1989. Contribution du LESE au programme bio­géochimie des fonds de lagons d·atolls. Rapp. CEA.LESE. Papee/",. Pol. Fr .• 33 p.

Verheken A.. 1989. Th(; indole pigments of \1ollusea. Ann.Soc. R. Zool. Be/g.• 119, 181-197.

298 R. Jabbour-Zahab et al.

Waite J. H., 1983. Quinone-Tanned Scleroproleins. The Wilbur K. M., A. S. M. Saleuddin. 1983. Shell formation. Mo!!usca L K. M. Wilbur Ed. Academie Press. N Y, The Mo!!usca 4. K. M. Wilbur Ed .. Academie Press. 446-504. N Y, 235-287.

Aqual. Living Resour.

298

Waite J. H., 1983. Quinone-Tanned Scleroproleins. TheMo!!usca L K. M. Wilbur Ed. Academie Press. N Y,446-504.

R. Jabbour-Zahab et al.

Wilbur K. M., A. S. M. Saleuddin. 1983. Shell formation.The Mo!!usca 4. K. M. Wilbur Ed.. Academie Press.N Y, 235-287.

Aqual. Living Resour.