Histology and ultrastructure of the mantle epidermis of the equilateral Venus, Gomphina veneriformis (Bivalvia: Veneridae).

ABSTRACT The histochemical characteristics and ultrastructure of the mantle of Gomphina veneriformis, were described using light and electron microscopy. When the mantle was distinguished into marginal, middle, and apical zones, the marginal mantle was also divided into 4-folds (inner-inner, inner-outer, middle, and outer fold), making it similar to the typical marginal mantle of Veneridae. The mantle thickness displayed a trend of reduction from the marginal zone to the apical zone. However, epidermal thickness was the thickest in the middle zone, with a thicker epidermal layer in the outer epidermis than the inner epidermis. The inner epidermis of the transitional area between the marginal and middle mantle is a simple columnar layer with well-developed microvilli on the free surface. Although the outer epidermis is also a simple columnar layer, few microvilli are present. The inner epidermis of the middle and apical zone is simple cuboidal layer, whereas the outer epidermis is a simple columnar layer. Ciliated cells are columnar in shape, with development of microvilli and cilia on the free surface. In the apical cytoplasm, numerous mitochondria were observed along with granules of low electron density. The distribution of mucous cells was found to be higher in the marginal mantle than those in the apical mantle. The proportion of mucous cells in the inner and outer epidermis in the marginal zone was 20.3% and 2.8%, respectively, with a high ratio in the inner epidermis. The result of this study illustrates that there are three types (A, B, and C) of secretory cells in the mantle, all of which are unicellular glands. Furthermore, in type A cells, the most ubiquitous of the three types and a typical goblet cell, the mucous substance contained in these cells was a weakly acidic or neutral carboxylated mucopolysaccaride. A periostracal groove was located between the middle fold and the outer fold, where the periostracum originates. Intermediate cells and basal cells were observed in the transitional area between the middle fold and the outer fold. Intermediate cells are elliptical with numerous vacuoles of various sizes. Basal cells have microvilli on the free surface, and there are membrane-bound granules between the microvilli and immature periostraeum. The periostracum, as it moves further from the periostracal groove, displays higher electron density and gets thicker. Mature periostracum is composed of a homogeneous layer and a fibrous layer. The homogeneous layer appeared darker than the fibrous layer because of higher electron density.

KEY WORDS: Gomphina veneriformis, mantle, periostracum, ultrastructure

INTRODUCTION

The integumentary system of bivalves is composed of a hard shell, which is the equivalent of an external skeleton composed mainly of calcium compound and mantle inside the shell. The shell plays a role in primary defense for protection of the organ system of the mantle cavity, mainly against physical and mechanical stimuli. The mantle conveys the external stimuli to internal organs, protects the organ system within the mantle cavity, and performs an important biological function in shell formation (Neff 1972b, Bubel 1984, Morrison 1993).

Various unicellular glands exist in the bivalve mantle. With regards to the types and characteristics of gland, microscopic structure and functions are broadly being reported according to taxon. In addition, it is believed that these characteristics would differ in accordance with environmental conditions and habitat (Bubel 1973, Vitellaro-Zuccarello 1981, Garcia-Gasca et al. 1994). The epithelial ceils of mantle expediently respond to various environmental stimuli. As such, investigations of the conditions of these cells could be used in assessing the physiological conditions of organisms.

Gomphina veneriformis used in this research belongs to Veneridae of Bivalvia, had a size range of 2 ~ 4 cm in shell length, and inhabited sandy sediment at a depth of 1 ~ 2 m. These are dominant species in the eastern coastal waters of the Korean peninsula, and are important commercial clams in this region (Yoo 1988).

The purpose of the present study is to describe the ultrastructure of the mantle of Gomphina veneriformis and to provide fundamental information that will enable comparison of species with different habitats and as biological data for future research on structural changes in the mantle according to changes in the environment.

MATERIALS AND METHODS

Gomphina veneriformis, was collected in the eastern coastal waters off the Korean peninsula (37[degrees]50' 20" N, 128[degrees]52' 50" E) (Fig. 1). Twenty adult clams with shell lengths of 35.0 ~ 40.0 mm were used in this study (Fig. 2). The sampled individuals were measured, the shells were removed, and the mantle tissue was fixed after it had been divided into the marginal, middle, and apical zones (Fig. 3).

For light microscopy, the tissues were fixed in aqueous Bouin solution and the preparations were made after paraffin methods. Embedded tissue were sectioned at 4 ~ 6 [micro]m thickness. H-E double stain, Masson trichrome stain, PAS reaction, AB-PAS (pH 2.5) reaction, alcian blue (pH 1.0) reaction, and AF-AB reaction were used for sections.

For transmission electron microscopy, the specimens were fixed in 2.5% glutaraldehyde solution (pH 7.2, buffered in 0.1M phosphate buffer) for 2 ~ 4 h at 4[degrees]C and postfixed in 1% osmium tetroxide (Os[O.sub.4]) solution for 2 h at 4[degrees]C. After fixation, the specimens were washed with 0.1 M phosphate buffer and dehydrated with ethanol step-by-step, and were finally embedded in Epon 812. Ultrathin sections (70 nm in thickness) were put on the copper grids (200 mesh) and double-stained with uranylacetate and lead citrate, and were examined using the transmission electron microscope (JEM-1200EXII. JEOL).

[FIGURE 1 OMITTED]

Stain affinity of the mucous cell was determined by using the Pantone Formula Guide (Pantone Inc., USA) as standard, and its unique code was indicated in parenthesis. Image analyzer (IMT, Visus, USA) was used to quantify the histological characteristics of epidermal thickness and mucous cell area (Fig. 4).

RESULTS

Light Microscopic Structure

Mantle of Gomphina veneriformis is a thin membrane with structure in which the simple epidermis is wrapped on the top and bottom of the centrally located connective tissue in the cross section. When the mantle is distinguished into the marginal, middle, and apical zones, the marginal mantle is composed of inner (inner-inner and inner-outer), middle, and outer folds. Among these, the inner fold was most developed (Fig. 5A). Mantle epidermis could be divided into inner epidermis facing the mantle cavity and outer epidermis directed to the shell (Fig. 5A). The shape of mantle epithelium differed depending on its position. Epithelial cells forming the inner fold of the marginal mantle were columnar (Fig. 5B), whereas they were mostly cuboidal in the middle fold and columnar in the outer fold. A periostracal groove could be observed between the middle fold and the outer fold, where the periostracum originates (Figs. 5A, C). The inner epidermis of the transitional area between the marginal and middle mantle is a simple columnar layer with a well-developed striated border on the free surface (Fig. 5D). Although the outer epidermis was also a simple columnar layer, the striated border was meek (Fig. 5E). The inner epidermis of the middle and apical zones was a simple cuboidal layer, whereas the outer epidermis was a simple columnar layer. Development of the striated border in both the inner and outer epidermis was meek (Figs. 5F, G).

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Mantle thickness was the thickest at the marginal zone (Fig. 5A). However, the epidermal thickness of the mantle was the highest in the middle zone, and that in the outer epidermis was thicker than that in the inner epidermis (Fig. 6).

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Mucous cells of the mantle were developed mainly from epidermis to connective tissue. The distribution and stain affinity of mucous cells showed differences in each mantle area. Distribution of mucous cells was found to be higher in the marginal mantle than those in the apical mantle. The ratio of mucous cells in the inner and outer epidermis in the marginal mantle was 20.3% and 2.8%, respectively, with a high ratio in the inner epidermis (Fig. 7).

[FIGURE 6 OMITTED]

In the results of the AB-PAS (pH 2.5) reaction. two types of mucous cells were observed in the inner fold of the marginal mantle. One type of cell reacts with a red color (2405C) and the other responds with a blue color (285C). Much higher numbers of blue-colored mucous cells were observed in this region (Fig. 8A). Pronounced activation of mucous cells reacted with a blue color (292C) in the transitional area between the marginal and middle mantle (Fig. 8B). Distribution of mucous cells in the middle and apical mantle was substantially low in comparison with that observed in the marginal mantle (Figs. 8C, D). In the result of the AF-AB reaction, mucous cells displayed a blue color (299C) caused by a reaction with alcian blue.

The connective tissue layer was loose, and it composed of mainly of collagen fiber bundles, muscle fiber bundles, and hemolymph sinuses. The distribution of muscle fiber bundles and hemolymph sinuses illustrated a tendency of reduction from the marginal zone to the apical zone (Fig. 5A).

Electron Microscopic Structure

From TEM observation, it was possible to distinguish epithelium, ciliated cells, and secretory cells in the mantle epidermis.

Epithelia displayed differences in shape and structure for each position. Columnar cells were observed in the outer epidermis of the marginal mantle. The free surfaces of the cells were covered with microvilli. Nuclei were oval with clearly visible heterochromatin, euchromatin, and nucleoli. Numerous mitochondria were observed in the apical cytoplasm, and were connected to the neighboring ceils with zonula adherens at the apico-lateral cell membrane (Fig. 9A). Epithelium that composed the outer epidermis of the middle and apical mantle was made up of columnar cells. The nuclei of these cells were elliptical, occupying approximately 50% of the cell (Fig. 9B). The inner epidermis of the middle zone was mainly composed of cuboidal epithelia. The free surfaces were covered with microvilli, as were the epithelial cells of other zones. The nuclei were irregular, oval-shaped, and existed in the basal cytoplasm. Significant numbers of mitochondria were found in the apical cytoplasm compared with epithelial cells of other zones. The lateral cell membranes were convoluted, forming membrane interdigitations with the neighboring cells (Fig. 9C). The inner epidermis of the apical zone was mainly composed of columnar epithelia. Nuclei were round with a diameter of approximately 4 [micro], and they were located in the center of the cell. Similar to the epithelial cells found in other zones, there were numerous mitochondria in the apical cytoplasm, and the free surfaces were covered with microvilli (Fig. 9D).

[FIGURE 7 OMITTED]

Ciliated cells were more pronouncedly distributed in the marginal zone than in other zones, and more of these cells were observed in the inner epidermis than the outer epidermis. Ciliated cells are columnar in shape, with development of microvilli and cilia on the free surfaces. Nuclei are circular and are located in the basal area. In the apical cytoplasm, there were numerous mitochondria along with granules of low electron density. They are connected to the neighboring cells with zonula adherens at the apico-lateral surface (Fig. 9A).

Secretory cells are unicellular glands and can be divided into three types (A, B, and C) depending on the cell shape and characteristics of secretory granules. Type A secretory cells exists mainly in the epithelial layer, with typical goblet form and a length of approximately 7 [micro]m. The electron density of secretory granules was low, with the lowest distribution level among the three types of secretory cells (Fig. 9D). The type B secretory cell has a mussel-like shape with a length of approximately 12 [micro]m, occupying areas from the epithelial layers to the connective tissue layer. The nucleus is circular with heterochromatin of high electron density in the karyoplasms. Membrane-bounded secretory granules are distinguished into two types, namely, one with high electron density and one with low electron density. Mitochondria and Golgi complex were observed around the nuclei (Fig. 10A). Type C secretory cells are rectangular with a length of approximately 17 [micro]m, and they develop in areas from the epithelial layers to the connective tissue layer, as is the case with type B secretory cells. The cytoplasm was filled with secretory granules of high electron density (Fig. 10B).

The middle fold of the periostracal groove where the periostracum begins is composed of squamous epithelia. The outer fold aspect is composed of cuboidal epithelia. Intermediate cells and basal cells were observed in the transitional area between the middle fold and the outer fold. Intermediate cells are elliptical with numerous vacuoles of various sizes (Fig. 11A). Basal cells have microvilli on the free surface, and membrane-bound granules exist between the microvilli and immature periostracum. Numerous tubular mitochondria and small granules of various electron density are observed in the cytoplasm of basal cells (Fig. 11 B). The epithelial layer adjacent to the mature periostracum is composed of squamous cells with a height of 5 ~ 6 gm and a width of 10 [micro]m. The nuclei of these cells are circular, and are centrally located in the cytoplasm (Fig. 11C). Numerous granules of high electron density and approximately 200 nm in diameter, fiber bundles, and mitochondria are observed in the cytoplasm. Microvilli develop on the free surfaces of these cells (Fig. 11D).

[FIGURE 8 OMITTED]

Periostracum, as it gains distance from the periostracal groove, displays higher electron density and becomes thicker. Mature periostracum is composed of a homogeneous layer and fibrous layer. The homogeneous layer appeared darker than the fibrous layer because of higher electron density. The homogeneous layer appeared as an obscure outer layer with low electron density in the areas near the fibrous layer. However, as it gained distance from the fibrous layer, the electron density increased (Fig. l1D). Numerous microfilaments were arranged parallel to the homogeneous layer in the fibrous layer, with the arrangement being quite loose (Fig. 11E). Cells adjacent to the fibrous layer of the periostracum are cuboidal cells of approximately 15 [micro]m in height with circular nuclei in the middle cytoplasm. Development of mitochondria was clearly pronounced in the cytoplasm, and some rough endoplasmic reticular, microsomes, and granules of various electron density were observed (Fig. 11E). Microvilli on the free surfaces of these cells developed into the fibrous layer of the periostracum, and membrane-bounded granules of various sizes were distributed near the microvilli (Fig. 11F).

DISCUSSION

The mantles of bivalves and gastropods are involved in purification of the mantle cavity and shell formation through mucous secretion. Purification of the mantle cavity is accomplished mainly by the inner epidermis, whereas the shell is formed by the outer epidermis. Although the bivalve mantle is similar to that of the gastropod in that it is basically composed of an epithelial layer and a connective tissue layer, the cell types and ultrastructure of the epithelial layer and connective tissue layer are quite diverse according to the species and mantle area (Kawaguti & Ikemoto 1962a, Kawaguti & Ikemoto 1962b, Bubel 1973, Saleuddin 1974, Morrison 1993).

The marginal zone of the mantle of Pinctada mazatlanica, which belongs to Pteridae, is divided into the inner fold, middle fold, and outer fold. Among these folds, the inner fold is the largest in its size, and the outer fold is the smallest (Garcia-Gasca et al. 1994). The marginal mantle of Veneridae is generally composed of 4-folds (Eble 2001). In this study, the marginal mantle of Gomphina veneriformis was also divided into 4-folds, making it similar to the typical marginal mantle of Veneridae.

Structural differences in the cells composing the epithelial layer of the mantle illustrate the differences in the chemical characteristics of these cells. Differences in the thickness of the epithelial layer according to the zone are associated with secretory activities (Beedham 1958a). Although four types of cells were distinguished in the outer fold of the mantle of Astarte castanea, only one type was observed in the middle fold (Saleuddin 1974). Fluid within the extrapallial cavities of Mytilus edulis and Lamellidans marginalis contain amino acid, protein, mucopolysaccharides, organic acid, lipids, and carbon dioxide. These components are associated with shell formation and are secreted by the mantle (Wilbur & Saleuddin 1983). Although ciliated cells and microvilli are well developed in the inner epithelial layer of the mantle of Crassostrea virginica, the level of the distribution of secretory cells in the outer epithelial layer is high with minute development of ciliated cells and microvilli (Morrison 1993).

[FIGURE 9 OMITTED]

The thickness of the mantle and thickness of the epithelial layer for each mantle zone illustrated the trend of reduction from the marginal zone to the apical zone in Mytilus edulis, Cardium edulis, Nucula sulcata (Bubel 1973), Scapharca broughtonii (Lee 2002), and Tegillarca granosa (Ma and Lee 2003). In this study, the mantle thickness of Gomphina veneriformis displayed a trend of reduction from the marginal zone to the apical zone, a similar result to those seen in earlier investigations. However, epidermal thickness was the highest in the middle zone, with a thicker epidermal layer in the outer epidermis than the inner epidermis.

Histologically, the molluscan integument epidermis is a single layer on a basement membrane that overlies a thin connective tissue layer. Three types of cells that compose the epithelial layer include the epithelial cell, gland cell, and ciliated cell (Bubel 1984).

It has been reported that the shape and height of the epithelial cells that compose the mantle are different in the inner epidermis and outer epidermis. For epithelial cells of the mantle in Mercenaria mercenaria of Veneridae, the outer epidermis is mostly composed of cuboidal cells, whereas mostly columnar cells are present in the inner epidermis (Neff 1972a, Neff 1972b).

Although there are slight differences in the height of epithelium that composes the marginal and pallial zones of the mantle in Pinctada mazatlanica, all cells are columnar. However, although the inner epidermis of the central zone is composed of cuboidal cells, the outer epidermis is composed of short columnar cells. Furthermore, epidermis in the mantle isthmus zone, which is an apical zone, is composed of long columnar cells with a height of 25 ~ 50 [micro]m (Garcia-Gasca et al. 1994).

Although the inner epidermis of the mantle of Scapharca broughtonii of Arcidae is composed of columnar cells, cells that compose the outer epidermis are squamous or cuboidal cells (Lee 2002). In this research, the format and height of epithelial cells that compose the mantle of Gomphina veneriformis illustrated differences in each zone, demonstrating a similar result as those found in earlier studies of other bivalves.

[FIGURE 10 OMITTED]

Ciliated cells of mantle epidermis were also reported in Mytilus edulis, Cardium edulis, Nucula sulcata (Bubel 1973), Crassostrea virginica (Morrison 1993), and Scapharca broughtonii (Lee 2002). Cilia on the free surface of the mantle epidermis perform the function of discharging foreign materials entering the mantle cavity along with moving mucous substance to the outside of the mantle cavity (Galtsoff 1964, Machin 1977). Ciliated cells that developed on the inner epithelial layer of the mantle were observed in Fabulina nitidula (Kawaguti & Ikemoto 1962a), in addition to M. edulis, C. edulis, N. sulcata, and C. virginica, as mentioned earlier. These ciliated cells contained numerous tubular mitochondria in their apical cytoplasm. In this study, as found in other reports, the presence of numerous mitochondria in the apical cytoplasm of the ciliated cells distributed in the mantle epidermis of Gomphina veneriformis was confirmed. Such development of mitochondria is deemed necessary for supplying energy sources in the functioning of the cilia of these ciliated cells as sensory cilia or kinocilia.

The characteristics of mucous secreted by the gland cells, a group of cells that compose the mantle epithelial layer, are reported to differ slightly from species to species and in the mantle zone. Although the chemical composition of mucous substances generated in the mantle varies greatly, they are composed mainly of mucopolysaccharides, glycoproteins, and carbohydrates (Prezant 1981). Secretory cells, a type of typical mucous cell, play an important role in shell formation in Mercenaria mercenaria (Hillman 1968) and Helisoma duryi eudiscus (Kapur & Gibson 1968). In the case of Lymnaea stagnalis (Timmermans 1969), mucous substances secreted from these cells contribute to lubrication action of the mantle. Two types of goblet-shaped secretory cells were found in the epidermis surrounding the mantle and the mantle cavity of Crassostrea virginica. These cells, in their reaction to aldehyde fuchsin-alcian blue, indicated the presence of carboxylated mucopolysaccaride in their reaction to alcian blue (Morrison, 1993). Four types of secretory cells were reported in the mantle epidermis of the pearl oyster, Pinctada mazatlanica. The first was a large secretory cell that was lightly basophilic, containing carbohydrate, acidic protein, sulfated mucopolysaccaride, and calcium granules. The second, a small secretory cell with highly basophilic stainability, was distributed mainly in the middle fold and secreted acidic mucopolysaccaride. The third, an acidophilic secretory cell, was distributed mainly in the periostracal groove and was involved in protein synthesis. The fourth, a large acidophilic secretory cell, was distributed mainly in the central zone and was involved in the synthesis of glycogen (Garcia-Gasca et al. 1994). Four types of secretory cells (A, B, C, and D) were observed in the mantle of Scapharca broughtonii. Among these, the mucous substance of the type A secretory cell differed slightly depending on the distribution area of the cell. Secretory cells in the inner epidermis contain neutral sulfated mucopolysubstance. However, secretory cells in the outer epidermis contain acidic sulfated mucopolysaccaride and carboxylated mucopolysaccaride (Lee 2002).

In general, exocrine glands are classified into unicellular and multicellular glands according to the number of composition cells, and they can be divided further into holocrine glands and merocrine glands depending on their patterns of secretion (Kurosumi et al. 1984). Based on such standards, all secretory cells observed and reported in Fabulina nitidula (Kawaguti & Ikemoto 1962a), Musculus senhousia (Kawaguti & Ikemoto 1962b), Mercenaria mercenaria (Neff 1972a), Mytilus edulis, Cardium edulis, Nucula sulcata (Bubel 1973), Crassostrea virginica (Morrison 1993), Scapharca broughtonii (Lee 2002), and Tegillarca granosa (Ma & Lee 2003) were unicellular glands. It was presumed that these were merocrine glands, as cell death and cellular components were not observed in the lumen. However, multicellular glands were observed in the marginal mantle of Lyonsia and Entodesma belonging to Eyonsiidae (Prezant 1981). The result of this study illustrates that there are three types of secretory cells in the mantle of Gomphina veneriformis, all of which are unicellular glands. Furthermore, in the type A cells, the most ubiquitous of the three types and a typical goblet cell, the mucous substances contained within were weakly acidic or neutral carboxylated mucopolysaccarides.

[FIGURE 11 OMITTED]

The shell formation of the bivalves depends on the growth of the marginal mantle (Owen 1953). Periostracum is composed of mucopolysaccaride, lipid, and protein, and functions to prevent corrosion of the outermost layer of the shell by acidic sub stances (Brown 1952, Beedham 1958b, Saleuddin 1974). Inner epithelial cells of the outer fold in the marginal zone of the bivalve mantle secrete a substance that forms the periostracum (Bubel 1973). In the majority of bivalves, the periostracum originates between the middle fold and outer fold of the marginal mantle (Saleuddin 1974). Hillman (1961) and Neff (1972a) discussed the role of the different cell types in the formation of the periostracum of Mercenaria mercenaria. Epithelial cells in the middle fold of the mantle play a role in supporting the periostracum (Neff 1972a), whereas the gland cells in the outer fold are involved in the synthesis of proteineous membrane found in the periostracum (Hillman 1961). The periostracum of Astarte castanea is composed of a homogeneous layer and a fibrous layer. The homogeneous layer is further distinguished into a dark homogeneous layer and an outer membrane layer. In addition, inner epithelia between the fibrous layer and outer fold contain microvilli. Glycogen granules are located between these microvilli (Saleuddin 1974). Periostracum of Gomphina veneriformis originates at the periostracal groove, and the electron density and thickness of periostracum increases as its distance from the periostracal groove increases. Mature periostracum is composed of a homogeneous layer and a fibrous layer. Microscopic structures of the cells adjacent to the fibrous layer were similar to those of the periostracum of Astarte castanea. In the present study, the epithelial layer of the mantle of Gomphina veneriformis is deemed to have the structural characteristics associated with purification of the mantle cavity and shell formation as discussed in earlier reports.

The connective tissue layer of the mantle of Gomphina veneriformis was composed of loose connective tissue with a developed matrix, collagen fibers, muscle fibers, and hemolymph sinuses. Similar structures were reported in many bivalves, including Crassostrea virginica (Morrison 1993), the pearl oyster, Pinctada mazatlanica, (Garcia-Gasca et al. 1994), and Scapharca broughtonii (Lee 2002). Although such loose connective tissue is deemed to have an appropriate structure for activation of secretory cells and expansion of hemolymph sinus, further investigation and discussion are necessary on this issue.

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JUNG SICK LEE, * JA YOUNG JOO AND JUNG JUN PARK

Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Republic of Korea

* Corresponding author. E-mail: [e-mail]