Microscopic anatomy of foot of the spiny top shell, Batillus cornutus (lightfoot, 1786) (Gastropoda: Turbinidae).

ABSTRACT Foot structures of the spiny top shell, Batillus cornutus were investigated by light and transmission electron microscopy. The foot was composed of epithelial layer, connective tissue layer, and muscular layer from outside inward. The epithelial layer was multiple-folded and simple and was composed mostly of columnar and mucous cells. Epithelial layer thickness was approximately 40 [micro]m in the pedal body and approximately 30 [micro]m in the opercular zone. Distribution of mucous cells in the epithelial layer was found to be higher in the opercular zone than those in the pedal body. Mucous cells contained acidic material abundant in sulfate and carboxylate group, from the results of AF-AB reaction. From TEM observation, the epithelial layer is composed of epithelial cell, ciliated cell, absorptive cell, and secretory cell. Secretory cells can be classified into six types (A, B, C, D, E, F) in accordance with the shapes and ultrastructural characteristics of secretory granule. Type A secretory cell is the most abundant cell of the six types of secretory cells. The type of muscle fibers was mostly smooth muscle fibers. Muscle fibers can be divided into two types, one that is composed only of thin microfilaments and the other composed of thin and thick microfilaments.

KEY WORDS: foot, microscopic anatomy, Batillus cornutus, TEM, top shell

INTRODUCTION

The top shell, Batillus, belongs to Turbinidae of Gastropoda, and it is widely distributed throughout temperate coastal waters to tropical coastal waters including the Pacific, Indian, and Atlantic Ocean. Among these, spiny top shell, Batillus cornutus is a settled gastropod thriving in the waters of the southern part of the East Sea and coast of Cheju Island in Korea, southern coastal waters of Japan, and reef ridden coastal waters of the Yellow Sea of China. The spiny top shell is one of the most important food resources in Korea, along with abalone, placing it at a very important position industrially (Yoo 1988).

The foot of the gastropod is an organ with flexibility corresponding to the motile system, and it performs functions such as mobility, attachment, capturing of food, mating, spawning, and shell cleaning (Voltzow 1994). The foot is composed of muscular tissue and nerve bundles, and it is partially covered by an operculum (Bullock 1965).

Study on the pedal and muscular structure of the gastropod includes reports on Patella vulgate (Jones & Trueman 1970), Neritina reclivata and Thais rustica (Gainey 1976), Patella vulgate and Acmaea tessulata (Grenon & Walker 1978), Bullia digitalis (Trueman & Brown 1976, 1987), Bullia rhodostoma (da Silva & Hodgson 1987), patellid limpets (Frescura 1987; Frescura & Hodgson 1990a, 1990b), Busycon contrarium and Haliotis kamtschatkana (Voltzow 1990), and Nassarius kraussianus (Trueman & Hodgson 1990).

However, in relation to these studies, there is no biological study on the foot structure of the spiny top shell, Batillus cornutus. Therefore, this study aims to describe the ultrastructure of the foot of the spiny top shell and to provide basic information for future study on structural changes of the foot in accordance with environmental and physiological changes.

MATERIALS AND METHODS

The spiny top shells were collected by divers in the coastal waters of Modori, Cheongsanmyeon, Wandogun, Cheollanamdo Korea (N 34[degrees]13', E 126[degrees]47') (Fig. 1) May 2003. Thirty spiny top shells in shell height 60.0-69.9 mm were used in this study. The sampled individuals were measured, the shell removed, and the pedal tissue was fixed after dividing into the pedal body and opercular zone (Fig. 2).

[FIGURES 1-2 OMITTED]

For light microscopy the tissues were fixed in Bouin solution and then prepared according to the following paraffin methods: 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 in 0.1 M phosphate buffer, dehydrated by ethanol step-by-step, and finally embedded in Epon 812. Ultrathin sections (70 nm in thickness) were put on copper grids (200-mesh), double stained with uranylacetate and lead citrate, and finally examined using a transmission electron microscope (JEM-1200EXII, JEOL).

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.

RESULTS

The foot of the spiny top shell is the organ that extends from the dorsal side of the anterior portion of the body and continues onto the head. Overall color of the foot was dark brown and the portion attached to the operculum was a wrinkled light orange- colored muscular mass.

Histological structure of the foot was composed of an epithelial layer, connective tissue layer, and a muscular layer from outside inward. The epithelial layer was a simple multiple-folded layer composed mostly of columnar and mucous cells (Figs. 3, 4).

[FIGURES 3-4 OMITTED]

The epithelial layer was approximately 40 [micro]m thick in the pedal body and approximately 30 [micro]m in the opercular zone (Fig. 5). A well-developed striated border was formed on the free surface of the epithelial layer (Fig. 3B). Mucous cells were observed between columnar epithelial cells in the epithelial layer, and these cells exhibited vacuoles in H-E stain and Masson triple stain (Figs. 3A, B; 4A, B).

[FIGURE 5 OMITTED]

Mucous cells reacted weakly in PAS reaction in the pedal body (Fig. 3C) and opercular zone (Fig. 4C). However, mucous cells revealed a blue color (300 C) in AB-PAS (pH 2.5) reaction (Figs. 3D; 4D) and AF-AB reaction (pedal body: 2995 C; opercular zone: 306 C) (Figs. 3F; 4F). Distribution of mucous cells in the epithelial layer was found to be higher in the opercular zone than those in the pedal body (Fig. 6).

[FIGURE 6 OMITTED]

The connective tissue layer appeared very weak between the epithelial and muscular layer (Fig. 1B).

The muscular layer is composed of connective tissue and muscular fiber bundles with the presence of hemolymph sinus. Muscular fiber bundles were distributed regularly in horizontal and vertical directions, with higher density in the opercular zone than in the pedal body (Fig. 4B).

From TEM observation, the epithelial layer is composed of epithelial, ciliated, absorptive, and secretory cells.

Epithelial cells were columnar and approximately 30 [micro]m in height both body and opercular zone of the foot (Figs. 7A, C). In addition, microvilli of approximately 2 [micro]m height were developed commonly on the free surface of epithelial cells, and tight junctions of apical lateral aspect and membrane interdigitations were found between the epithelial cells (Fig. 7B). However, these columnar epithelial cells displayed differences in shapes and positions of the nucleus. In the columnar epithelial cell of the pedal body, the shape of the nucleus was irregular and oval-shaped and located in the middle or basal portion of the cell (Fig. 7A). In comparison, the nucleus of the columnar epithelial cell in the opercular zone had an elongated oval-shape with length equivalent to 2/3 of the cell length (Fig. 7C).

[FIGURE 7 OMITTED]

Ciliated cells were better developed in the epithelial layer of the body than those of opercular zone of the foot. These ciliated cells have well-developed cilia on the free surface. The length of these ceils was approximately 45 [micro]m and the cilia length was approximately 4 [micro]m. These cells have a rectangular-shaped nucleus of approximately 17 [micro]m in the basal portion (Fig. 8A). Tubular mitochondria are clustered in the upper portion of the cytoplasm, and the ciliary rootlet is connected to mitochondria (Fig. 8B). Cross section of the cilia showed "9+2" microtubular structure of approximately 200 nm in diameter (Fig. 8C).

[FIGURE 8 OMITTED]

Cells with absorptive function that is rarely observed in the epithelial layer have microvilli on the free surface; and several pinocytotic vesicles, multivesicular bodies, and mitochondria were found in the upper portion of cytoplasm (Fig. 8D).

Secretory cells can be classified into 6 types (A, B, C, D, E, F) in accordance with the shapes and ultrastructural characteristics of secretory granule.

The type A secretory cell (Fig. 9A) is the most abundant cell of the six types of secretory cells. These cells have membrane-bounded secretory granules, and the granules had higher electron density in comparison with the secretory granules of other secretory cells (Fig. 9B). Furthermore, numerous rough endoplasmic reticula were found in the basal cytoplasm of these cells (Fig. 9A).

[FIGURE 9 OMITTED]

The type B secretory cell is circular. Electron density of the secretory granule was low, and the internal space of the secretory granule was filled with fine granular substances. The distribution of these cells was lower than the type A secretory cell but higher than all other secretory cells (Fig. 9C).

The type C secretory cell is clavate shaped. The nucleus of these cells was irregularly shaped and smooth endoplasmic reticula were scattered in the cytoplasm. Shape of the secretory granules was diverse, and the electron density was low (Fig. 9D). The type D secretory cell is columnar with the nucleus located in the basal portion. Secretory granule of the cells did not have a membrane, and the electron density was the lowest among the six types of secretory cells (Fig. 9E).

The type E secretory cell is circular. Rough endoplasmic reticula and secretory granules with diverse and lower electron density were scattered in the cytoplasm (Fig. 9F).

The type F secretory cell is oval-shaped. The distribution of these cells was the lowest among the six types of secretory cells. The secretory granule contained vacuoles with various electronic densities (Fig. 9G).

Numerous collagen fibers, fibrocytes, some muscle fibers, and hemocytes were observed in the connective tissue layer (Figs. 10A, B).

[FIGURE 10 OMITTED]

Some collagen fibers were observed in the muscular layer. In the longitudinal section, although the electron density was low, some transverse striations were distinguished in the collagen fibers (Fig. 10C).

Several types of muscle fibers were arrayed in various directions in the muscular layer, and they were mostly smooth muscle fibers. Muscle fibers can be divided into two types, one that is composed only of thin microfilaments and the other composed of thin and thick microfilaments. The former showed high-level distribution. The density of microfilament in the muscle fiber illustrated slight difference (Fig. 10D).

Muscle fibers composed of thin filament were categorized into two types. One type was composed only of muscle fibers with same electron density, and the other type was composed of muscle fibers with higher electron density in the cortex and lower electron density in the medulla (Fig. 10E). Thin filament muscle fibers were composed mainly of microfilaments approximately 20 nm in diameter. These have tubular mitochondria and a small number of sarcoplasmic reticula around the sarcolemma and some glycogen granules in the sarcoplasm (Fig. 10F).

In the muscle fiber in which thin and thick filament were mixed, the thin filament was approximately 10 nm in diameter, whereas that of the thick filament was 80 nm. Distribution of mitochondria and sarcoplasmic reticula in these muscle fibers was lower than the muscle fiber that was composed only of thin filament (Fig. 10G).

In addition, nerve cells were found between collagen fibers of the muscular layer (Fig. 10H).

DISCUSSION

The foot of the gastropod is composed of the epithelial layer, connective tissue layer, and muscular layer. The epithelial layer is simple and composed of columnar epithelial and secretory cells. The connective tissue layer is relatively thin. In addition, the muscular layer is composed mainly of collagen fibers and smooth muscle fibers (Bullock 1965, Grenon & Walker 1978, da Silva & Hodgson 1987, Trueman & Hodgson 1990).

In this study, it was also found that the foot of the spiny top shell, Batillus cornutus was composed of epithelial layer, connective tissue layer, and muscular layer from exterior inward in cross section. The epithelial layer was simple and composed of columnar and mucous cells. Well-developed circular muscle, longitudinal muscle, and hemolymph sinus was observed in the muscular layer.

Generally, mucous cells in the epithelial layer of the mollusc foot contain acidic glycosaminoglycans within the sulfate and carboxylate group (Eble 2001). This study confirmed that the mucous cells contained acidic material abundant in the sulfate and carboxylate group from the results of AF-AB reaction.

Among gastropods, limpet, Acmaea tessulata had six types of secretory glands in the foot. The secretory substance is weakly acidic mucopolysaccharide and correlates with mobility function. Patella vulgata on the other hand has nine types of glands. Among these, six types are distributed in the foot, and contain weak acidic mucopolysaccharide and correlates with mobility function. The other three types are sole glands with highly viscous acidic mucopolysaccharide and attachment function (Grenon & Walker 1978).

In this study, 6 types (A, B, C, D, E, F) of secretory cells were distinguished in the epithelial layer of the foot. Among these, C; D; and E type were observed mostly in the opercular zone, enabling one to predict that these cells are associated with operculum formation.

Although the muscle fiber of molluscs, in comparison with the vertebrates; is limited, transverse striations are found in some smooth muscle cell (Lowy & Vibert 1967; Millman & Bennett 1976; Sobieszek 1973). Characteristics of muscle fiber are diverse and contain paramyosin (Chantler 1983).

Types of muscle fiber composing the foot muscle of mollusc are categorized based on the arrangement of dense body, distribution of thick filament, ratio of thick microfilament and thin microfilament, invagination of sarcolemma, composition of sarcoplasmic reticula, and mitochondrial arrangement (Nicaise & Amsellem 1983). Foot muscle of Batillus rhodostoma of Turbinidae illustrates intermediate type of muscle forms found in some reported molluscs (Lowy & Vibert 1967, Sobieszek 1973).

The foot muscle ofNassarius kraussianus, a gastropod, is composed mostly of smooth muscle fibers and collagen fibers. Although there were some muscle fibers with 4-9 [micro]m in diameter, the majority had 3-4 [micro]m in diameter, and has numerous membrane interdigitations between each cell. Muscle fiber is composed of thick filament with density of 131 [+ or -] 9.1 [micro][m.sup.-1], 40.6 [+ or -] 4.1 nm in diameter, and length of 7-10 [micro]m. Well-developed weakly acidic glycogen granules of 2 [micro]m in length and 0.5 [micro]m in diameter are found in the sarcoplasm (Trueman & Hodgson 1990).

In the foot muscle of the whelk, Bullia rhodostoma of Nassariidae of Gastropoda, two types of muscle fibers, namely A and B types were distinguished. Among these, A-type is striated muscle fiber observed mainly in propodium, which enables fast shortening of the foot when crawling or burrowing. Dense body (94.70 [+ or -] 7.50 nm in diameter) of the A-type muscle fiber is arranged vertically along the long axis of the muscle fiber. Ratio of thin filament to the thick filament is more than 1:10 and the mitochondrial area is approximately 25%. The B-type is of smooth muscle fiber type that is mostly found in the metapodium, and functions to maintain greater tension. Dense body is rarely found in the B-type muscle fiber. The diameter of the dense body is 84.40 [+ o r-] 2.47 nm with irregular arrangement. Ratio of thin filament to the thick filament is approximately 1:12-1:30 with mitochondrial area at 35%. The mitochondrial distribution in the A-type muscle fiber is a dispersed pattern, whereas that in the B-type muscle fiber is clustered (da Silva & Hodgson 1987).

Frescura & Hodgson (1992) categorized the muscle fibers of columellar muscles of six species (Bullia rhodostoma, Burnupena cincta, Haliotis spadicea, Siphonaria capensis, S. concinna, Turbo sarmaticus) belonging to Prosobranchia into Type I and Type II. Muscle fiber of Type I has small number of dense body, mitochondria and sarcoplasmic reticula. Thin and thick filaments are irregularly mixed in these muscle fibers. The diameter of thick filament measured in the preparation was 26 nm for Turbo sarmaticus and 69 nm for Burnupena cincta, illustrating differences depending on the species. In comparison, muscle fibers in the Type II category are composed of thin filament and have a striated appearance. These muscle fibers are connected with collagen fibers and exhibit difference in the development of cell organelles.

In this study, transverse striations were not clearly observed in the muscle fiber of the foot of the spiny top shell. Types of muscle fiber were distinguished into a type in which thin and thick filaments were present in mixture and the other type composed only of thin filament. It was determined that these two types corresponds to the "Type I" and "Type II" muscle fiber reported by Frescura & Hodgson (1990a, 1990b, 1992). Such result of this study and distribution of thick filament, invagination of sarcolemma and arrangement of dense body, and sarcoplasmic reticula and mitochondria illustrated the same features as previous reports. However, the finding that muscle fiber composed only of thin filament is categorized into two types is deemed to be a characteristic of muscle fiber of the spiny top shell, Bartillus cornutus.

LITERATURE CITED

Bullock, T. H. 1965. Mollusca, Gastropoda. In: T. H. Bullock & G. A. Horridge, editors. Structure and function of the nervous systems of invertebrates Vol. 2. San Francisco: Freeman. pp. 1283-1386.

Chantler, P. D. 1983. Biochemical and structural aspects of molluscan muscle. In: A. S. M. Saleuddin & K. M. Wilbur, editors. The Mol lusca Vol. 4, Physiology, Part 1, New York: Academic Press. pp. 77-154.

da Silva, F. M. & A. N. Hodgson. 1987. Fine structure of the pedal muscle of the whelk Bullia rhodostoma Reeve: Correlation with function. Comp. Biochem. Physiol. A 87:143-149.

Eble, A. F. 2001. Anatomy and histology of Mercenaria mercenaria. In: J. N. Kraeuter & M. Castagna, editors. Biology of the hard clam. New York: Elsevier. pp. 117-220.

Frescura, M. 1987. An ultrastructural study of limpet shell muscle. Proc. Electron Microsc. Soc. Southern Afr. 17:43-44.

Frescura, M. & A. N. Hodgson. 1990a. A comparative study of gastropod colnmellar muscle with special reference to limpets. Proc. Electron Microse. Soc. Southern Afr. 20:145-146.

Frescura, M. & A. N. Hodgson. 1990b. The fine structure of the shell muscle of patellid prosobranch limpets. J. Moll. Stud. 56:435-447.

Frescura, M. & A. N. Hodgson. 1992. The fine structure of the columellar muscle of some gastropod mollusks. Veliger 35:308-325.

Gainey, L. F. 1976. Locomotion in the Gastropoda: Functional morphology of the foot in Neritina reclivata and Thais rustica. Malacologia 15: 411-431.

Grenon, J.-F. & G. Walker. 1978. The histology and histochemistry of the pedal glandular system of two limpets, Patella vulgata and Acmaea tessulata (Gastropoda: Prosobranchia). J. Mar. Biol. Assoc. UK 58: 803-816.

Jones, H. D. & E. R. Trueman. 1970. Locomotion of the limpet, Patella vulgata (L.). J. Exp. Biol. 52:201-216.

Lowy, J. & P. G. Vibert. 1967. Structure and organization of actin in a molluscan smooth muscle. Nature 215:1254-1255.

Millman, B. M. & P. M. Bennett. 1976. Structure of the cross-striated adductor muscle of the scallop. J. Mol. Biol. 103:439-468.

Nicaise, G. & J. Amsellem. 1983. Cytology of muscle and neuromuscular junction. In: A. S. M. Saleuddin & K. M. Wilbur, editors. The Mollusca, Vol. 4, Physiology, Part 1. New York: Academic Press. pp. 1-33.

Sobieszek, A. 1973. The fine structure of the contractile apparatus of the anterior byssus retractor muscle of Mytilus edulis. J. Ultrastruct. Res. 43:313-343.

Trueman, E. R. & A. C. Brown. 1976. Locomotion, pedal retraction and extension, and the hydraulic systems of Bullia (Gastropoda: Nassariidae). J. Zool. 178:365-384.

Trueman, E. R. & A. C. Brown. 1987. Locomotory function of the pedal musculature of the nassariid whelk, Bullia. J. Moll Stud. 53:278-288.

Trueman, E. R. & A. N. Hodgson. 1990. The fine structure and function of the foot of Nassarius kraussianus, a gastropod moving by ciliary locomotion. J. Moll. Stud. 56:221-228.

Voltzow, J. 1990. The functional morphology of the pedal musculature of the marine gastropods Busycon contrarium and Haliotis kamtschatkana. Veliger 33:1-19.

Voltzow, J. 1994. Gastropoda: Prosobranchia. In: F. W. Harrison & A. J. Kohn, editors. Microscopic anatomy of invertebrates Vol. 5, Mollusca I. New York: Wiley-Liss, Inc. pp. 111-252.

Yoo, J. S. 1988. Korean shells in color. Seoul: Iljisa Publishing Co. 196 pp.

GUI KWON JUNG, JUNG JUN PARK, JAE WOO LEE, AND JUNG SICK LEE * Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Korea

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