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Histology of hypobranchial gland and gill of Haliotis asinina Linnaeus.

ABSTRACT The hypobranchial gland of Haliotis asinina exhibits major and minor folds that are called leaves and leaflets. When viewed by scanning electron microscope, numerous tufts of rod-shaped cilia, paddle-like cilia, and clusters of granules being exocytosed from the pores were observed on the surface of the leaves and the leaflets. In transverse sections, each of the leaf and the leaflet can be divided into two areas: the basal area consists mainly of two types of large goblet cells, and the apical area that contains a mixture of supporting cells, sensory cells and four types of mucus-secreting cells. H. asinina has bipectinate gills. Gills are observed in abalone at the age of 1 mo; and the number and length of their filaments increase with age. In mature abalone, there are about 17 filaments per each gill with equal numbers on both sides. The length of the longest filament is approximately 2.48 mm. Each filament is supported axially by a thin collagenous connective tissue. On the efferent side, there is a V-shaped chitinous skeleton rod. Epithelium lining the filament is composed of tall columnar cells bearing microvilli mixed with ciliated columnar cells and mucus cells.

KEY WORDS: H. asinina, gill, hypobranchial gland, histology, SEM

INTRODUCTION

The hypobranchial gland is a single or paired, highly glandular area of the epidermal lining the roof of the mantle cavity (Hyman 1967). Two such glandular areas, one on each side of the rectum, occur in Haliotis, and the left one is larger than the right one (Crofts 1929). The hypobranchial gland consists of regular folds or lamellae oriented at right angles to the mantle wall (Hyman 1967). The histology of the hypobranchial gland has been investigated (Crofts 1929, Bevelander 1988). Crofts (1929) described three types of cells in the hypobranchial gland of Haliotis tuberculata: mucus cells with spindle shaped secretion, mucus cells with granules, and ciliated cells. Bevelander (1988) described 3 types of cells in the hypobranchial gland of H. rufescens: mucus cells with rod-like elements, mucus cells with granular cytoplasm, and supporting cells.

Gills are the principal organs for respiratory gas exchange in mollusks. They are positioned in the mantle cavity (Crofts 1929, Eertman 1996), so they are affected by many substances that flow through the mantle cavity (Schulte-Oehlmann et al. 2000). The studies on gastropod gill morphology is very limited; a few papers have been published on the structure of gills of pulmonate Siphonaria capensis (De Villiers & Hodgson 1987) and of some caenogastropod species. The gill structure of the investigated gastropods shows basic uniformity because the gill filaments are composed of a ridge and an extended sheet of nonciliated cells. However, the gill filaments of these various species of gastropods differ in the shape of the filaments (corrugated, triangular, or rounded). Each gill filament is covered with a single layered epithelium of either cuboidal (Schulte-Oehlmann et al. 2000) or columnar cells (Crofts 1929). However, there seems to be a difference in the thickness of the epithelial cells. A hemocoelic space occupies the center of each filament (Eertman 1996, De Villiers & Hodgson 1987). Crofts (1929) found that in H. tuberculata, the V-shaped chitinous skeletal rod, attached to one side of the gill epithelium was similar to that in cephalopoda (Haszprunar 1987). The epithelium of the gill of S. capensis and H. tuberculata consists of three types of cell: nonciliated cell, ciliated cell, and secretory cell (De Villiers & Hodgson 1987).

To the best of our knowledge, there is still no information on the histology of hypobranchial glands and gills in H. asinina, a common abalone species found along the coastal water of Thailand, which is considered to be one of the economic aquatic animals that has been cultured for commercial exploitation. Hence, this study reports on the histology of hypobranchial glands and gills of this species.

MATERIALS AND METHODS

Collection of Abalone Specimens

Abalones were obtained from a land-based culture system at Coastal Aquaculture Development Center, Department of Fisheries, Prachaubkirikun Province, Thailand. They were reared in concrete tanks housed in the shade, and well flushed with mechanically circulated sand-filtered seawater, and provided with an air delivery system to maintain the stable controlled environment. The optimum level of salinity is ~22.5-32.5 ppt, and the temperature ~22[degrees]C to 26[degrees]C (Singhagraiwan & Doi 1993). They were fed with macroalgae (usually Gracilaria spp. and Laminaria spp.), supplemented with artificial food.

They were anesthetized in 5% Mg[Cl.sub.2], after which their shells were removed. The gills and hypobranchial glands were dissected out and processed for light microscope (LM) and scanning electron microscope (SEM) studies.

Specimen Preparation for LM

Specimens were fixed in Bouin fluid in 0.14 M NaCl for 24 h and washed with 70% ethyl alcohol. Then, they were dehydrated through a graded series of ethanol, cleared in dioxane, infiltrated and embedded in paraffin. Five-micron-thick sections were cut and stained with hematoxylin and eosin.

For semithin sections, specimens were fixed in Karnovsky fixative (2% paraformaldehyde and 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.8) at 4[degrees]C for overnight, and washed with 0.1 M sodium cacodylate buffer. They were postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer at 4[degrees]C for 1 h. Then, they were dehydrated in graded series of ethanol and embedded in Araldite 502 resin. Sections were cut at 1-[micro]m thickness with Porter Blum MT-2 [micro]Ltramicrotome, and stained with methylene blue or PAS-methylene blue (Hayat 1993). Examination of the tissues sections was done under an Olympus Vanox light microscope.

Specimen Preparation for SEM

Hypobranchial glands were cut and fixed in a Karnovsky fixative (4% glutaraldehyde 2% paraformaldehyde in 0.1 M sodium cacodylate buffer), pH 7.8 at 4[degrees]C, for overnight, and washed in 0.1 M sodium cacodylate buffer. They were postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at 4[degrees]C. Then, they were dehydrated in graded series of ethanol, and dried in a Hitachi HCP-2 critical point drying machine, using liquid C[O.sub.2] as a transitional medium. They were then mounted on aluminum stubs and coated with platinum and palladium in an ion sputtering apparatus, Hitachi E 5000. The specimens were examined in a Hitachi S-2500 scanning electron microscope with an accelerating voltage of 15 kV.

RESULTS

Hypobranchial Gland

The hypobranchial gland of H. asinina is located on the dorsal surface of the mantle cavity and runs parallel to the gill. It appears as a large yellow pectinated ridge whose lateral sides are connected to the gills by a thin mantle membrane (Figs. 1A and B, 2A). Each gland consists of many axes, from each of which 8 to 11 leaves arise (Fig. 1B). Under SEM, the major folds or leaves decline from the axis to the lower level and branch into two to three terminals. Many minor folds or leaflets branch off from both sides of each leaf (Fig. 1C). The hypobranchial gland exhibits many tufts of rod-shaped cilia, paddle-liked cilia, and granules that are exocytosed from pores on the surface of leaves and leaflets (Figs. 1E and F). In contrast, the junction between the axis and leaf has fewer ciliary tufts, round granules that are being exocytosed from pores on the fold, and there are deep grooves between the folds (Fig. 1D).

[FIGURES 1-2 OMITTED]

In transverse section, each leaf and leaflet can be divided into 2 areas: the basal area, which consists mainly of large mucus cells and the apical area that contains a mixture of supporting cells, sensory cells, and small mucus-secreting cells having goblet appearance (Figs. 2B and C, 3A). These cells have the following characteristics.

[FIGURE 3 OMITTED]

1. Supporting cells. These cells vary in shape; they can be triangular, oval, or spindle (Figs. 3B and C). The nucleus is oval and contains mostly pale-stained euchromatin. The upper part of this cell type is wide and shows many long microvilli.

2. Sensory cells. These cells are elongated in shape, and they contain euchromatic oval nuclei with many nucleoli (Figs. 3B and C). The upper part of this cell type is narrow and reaches the surface of the epithelium where it bears some cilia.

3. Mucus cells. These cells are filled with many mucin granules and have oval shape. Based on the staining characteristics with PAS-methylene blue on semithin sections, there are 4 types of mucus cells (Figs. 3B and C). Type-1 is a mucus cell with a round nucleus and PAS and methylene blue negative granules in the cytoplasm. It is located near the basement membrane (Figs. 3B and C). Type-2 is a mucus cell with flattened euchromatic nucleus and numerous blue granules packed tightly together in the cytoplasm (Figs. 3B and C). Type-3 is a small mucus cell whose cytoplasm is filled with PAS-positive round granules (Figs. 3B and C). Type-4 is the largest mucus cell containing large heterogenous granules with either pinkish or bluish (methylene blue positive) hues (Fig. 3C).

In the basal area, there are 2 types of mucin ceils lying in alternation. Type-1 is the mucus cell, which contain homogenous purple (methylene blue positive) material in the cytoplasm (Fig. 3D). Type-2 is the mucus cell containing homogeneous pale pink (PAS positive) material with streaks of deep pink rod-liked materials embedded in the former (Fig. 3D).

Gills

H. asinina has two bipectinate gills, positioned slightly to the left of the center in the mantle cavity and pointing anteriorly (Figs. 1A and B). The gills are attached to the mantle by a thin membrane. There are equal numbers of filaments on both sides (i.e., about 17 filaments per each gill). All filaments lie parallel to each other. Each is a delicate pleat with blunt free tip and is corrugated in the middle (Fig. 4A).

[FIGURE 4 OMITTED]

Transverse sections through the gill filaments reveal that they are covered with a single layered epithelium (Figs. 4B to E). Each filament is supported axially by a thin collagenous connective tissue, enclosing the hemocoelic space, which contains hemocytes (Fig. 4E). On the efferent side, there is a V-shaped opaque chitinous skeletal rod (Fig. 4B), serving as the attachment site for muscles that could bring about considerable movement of the gill filament. The gill filament is lined by a columnar epithelium that varies much in thickness in different parts (Figs. 4B to E). Most of the epithelial cells on the proximal and distal ends of a filament are tall columnar bearing microvilli, which appears as brush border under LM, with ciliated epithelial cells (Figs. 4B, C, and E). There are numerous cilia on the region of the apical ciliary band (Fig. 4B) and the lateral ciliary band (Fig. 4C). However, in most of the filaments, the cells are cuboidal and have no cilia (Fig. 4D). Four types of cells can be identified in the epithelium covering the efferent side of the filament (Fig. 4B): (1) the cuboidal cells with round nuclei on the lateral side of the skeletal rod; (2) the ciliated tall columnar cells in the distal end of the filament; (3) the small columnar cells with dense granules and microvilli; (4) the mucus cells with large basophilic or small metachromatic granules in the efferent filament.

The afferent epithelium of the gill filament comprises of 4 types of cells (Fig. 4E). These are: (1) the cuboidal cells with round nuclei with euchromatin and distinct nucleoli on the lateral side; (2) the tall columnar cells with round or oval nuclei located in the terminal epithelium; (3) the ciliated columnar ceils in the distal end of the filament; (4) the mucus cell with numerous dense granules packed tightly together in the apical cytoplasm (Fig. 4E).

DISCUSSION

Hypobranchial Gland

There are some controversies on the types of cells found in the hypobranchial gland. Crofts (1929) and Bevelander (1988) reported 3 cell types in the hypobranchial gland of Haliotis tuberculata and H. rufescens, respectively. Both authors described 2 types of mucus cells that contain different secretion. Type-1 mucus cell contains rodlike or spindle-shaped secretion while type-2 mucus cell contains granular secretion (Crofts 1929, Bevelander 1988). The third cell type is the supporting cell (Bevelander 1988), which is ciliated (Crofts 1929). These two types of mucus cells correspond to those found in the basal area of hypobranchial gland of H. asinina. The first type of mucus cell contains large, rodlike mucin granules that gave an intense positive reaction to PAS, indicating the presence of neutral mucopolysaccharide. In H. rufescens, the mucus cell also contains rodlike granules that were identified to be acid mucopolysaccharide in nature (Bevelander 1988). The second type of mucus cell in the basal area of H. asinina hypobranchial gland appears homogenous and stains purple with PAS-methylene blue. This may be equivalent to the second type of mucus cell of H. rufescens, but the content appears more homogenous in H. asinina. Bevelander (1988) suggested that the granules in these cells were glycoprotein.

In H. asinina, the apical areas of hypobranchial gland leaves and leaflets represent specialized zones where at least 6 types of cells are formed, (i.e., supporting cell, ciliated sensory cell, and 4 types of mucus cells) whose classification is based on the appearance and staining characteristics of the granules. These mucus cells appear very different from those found in the basal area, both in cell shape and secretion. Judging from the staining pattern to PAS-methylene blue, type-1 and type-3 mucus cells contain neutral mucopolysaccharide granules, whereas type-2 cells contain basophilic granules suggesting acidic protein in mature (Humason 1972). Type-4 mucus cell contains both types of granules, neutral mucopolysaccharide and basophilic granules.

Because of the vast number and considerable variety of mucus cells, the principal function of hypobranchial gland should be the secretion of mucin (Hyman 1967). The surface area of the gland is increased by the folding of the glandular epithelium into large pleats to increase the mucus-secreting area. The quantity of mucus discharging into the respiratory chamber increases suddenly if the animal is irritated. Mucus is thus produced and secreted for the protection and for clearing away debris from the anus and renal organs to keep the gills and mantle cavity clean. Furthermore, when foreign irritating particles from turbid water are attached to the surface of the gland, the mucus cells may release mucus to bind particles that will be brushed away by the ciliary action of the epithelial cells. The mucus cells that perform this function may be type-2 mucus cells because they have similar characteristics to those observed in the gill epithelium that may perform similar function. Crofts (1929) found that irritating oils introduced into the entrance of the respiratory chamber seemed to be perceived at once by the hypobranchial gland, and the shell closed down abruptly, and at once a large amount of mucus was released from the mucus cells. In a similar experiment, Alexander (1970) introduced milk into the respiratory chamber, and obtained similar responses. Furthermore, some mucus cells may release mucin into the seawater, perhaps to clear away the offending substance, as well as to adjust pH of seawater to be suitable for respiration. The mucus cells participating in this protective action may belong to 2 types (i.e., the acidic and basic mucus cells types-1 and 3). The remaining mucus cell (type-4) may act as general mucus cells that produce mucin to lubricate the organ and protect the gills. Furthermore, the heterogeneity of the mucus cells of hypobranchial gland implies that the secretion of mucin for clearing irritants and debris may not be the only function. The complexity of the mucin released could be involved in other process, such as acting as the inducers for the larval settlement, because it has been shown that mucus trial from adult animals is one of the most important factors for settlement.

Gill

Light microscopic observation shows that the internal structure of the gills and the gill filaments of H. asinina have an internal architecture similar to those of other mollusks. The gills are bipectinate, with individual filaments showing basic similarities. All filaments are positioned parallel to each other and are linked by a common base through which the hemolymph is directed and distributed to the individual filaments (Eertman 1996). Each filament is corrugated in the middle part, which encloses the hemocoelic space, thus it may be a modification to enlarge the total gill surface area and improve respiratory gas exchange (Crofts 1929, Eertman 1996).

The epithelium of the gills in H. asinina is a simple columnar or cuboidal type, which presumably helps to enhance the rapid gas exchange. This feature has been found in most species of mollusks studied so far (Crofts 1929, Eertman 1996, De Villiers & Hodgson 1987). The gill filaments possess areas of ciliated cells alternating with areas of nonciliated cells. In our study, the efferent margin of the filament and both sides of the hemocoelic space in the efferent side consist of ciliated cells (Crofts 1929). Ciliary movement may take part in sweeping mucus secretions from the mucus cells that serve to capture foreign particles and remove them from the gills (Nuwayhid et al. 1978). Hence, the ciliated ceils lining the gills observed in the present study may play both roles in making water current and removing irritating particles.

Nuwayhid et al. (1978) did not find any mucus cells in the gills of Patella vulgata, whereas in Siphonaria capensis there were a larger number of these cells (De Villiers & Hodgson 1987), and in Austrocochlea constricta there were two types of mucus goblet cells (Eertman 1996). The present study revealed two types of mucus cells on the gills of H. asinina. All of them are grouped at the efferent and afferent sides of the filament. It was suggested that these mucus cells function primarily in the cleaning of gills by removing dirt in coordination with muscle contraction and ciliary movement (Yonge 1952). The chitinous skeletal rods found in the efferent side may serve for attachment of muscles that bring about considerable movement of the gill filament (Crofts 1929, Haszprunar 1987). In S. capensis, it was found that the muscle fibers were located at intervals on the hemocoelic surface (De Villiers & Hodgson 1987). On the contrary, the present study reveals that the muscle fibers are attached to the inner surface of the rod. The function of this muscle may be the same as described earlier.

ACKNOWLEDGMENTS

This research was supported financially by the Thailand Research Fund to Prasert Sobhon for a Senior Research Scholar Fellowship.

LITERATURE CITED

Alexander, C. G. 1970. The osphradium of Conusflavidus. Marine Biol. 6:236-240.

Bevelander, G. 1988. Abalone gross and fine structure. Pacific Grove California: The Boxwood Press. pp. 1-80.

Crofts, D. R. 1929. Haliotis. Liverpool Mar. Biol. Comm. Mere. 29:1-174.

De Villiers, C. J. & A. N. Hodgson. 1987. The structure of the secondary gills of Siphonaria capensis (Gastropoda: Pulmonata). J. Moll. Stud. 53:129-138.

Eertman, R. H. M. 1996. Comparative study on gill morphology of gastropods from Moreton Bay, Queensland. Moll. Res. 17:3-20.

Haszprunar, G. 1987. The fine structure of the ctenidial sense organs (bursicles) of vetigastropoda (Zeugobranchia, Trochoidea) and their functional and phylogenetic significance. J. Moll. Stud. 53:46-51.

Hayat, M. A. 1993. Stains and cytochemical methods. New York: Plenum Press. pp. 1-455.

Humason, G. L. 1972. Animal tissue techniques. 3rd ed. USA: W.H. Freeman and Company. pp. 1-641.

Hyman, L. H. 1967. The invertebrates, Mollusca 1, vol. 6. New York: McGraw-Hill. pp. 1-792.

Nuwayhid, M. A., D. P. Spencer & H. Y. Eider. 1978. Gill structure in the common limpet Patella vulgata. J. Mar. Biol. Assoc. UK. 58:817-823.

Schulte-Oehlmann, U., M. Tilmann, B. Markert, J. Oehlmann, B. Watermann & S. Scherf. 2000. Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part II: Triphenyltin as a xeno-androgen. Ecotoxicology 9:399-412.

Singhagraiwan, T. & M. Doi. 1993. Seed production and culture of a tropical abalone, Haliotis asinina Linne. Bull. Tokai Reg. Fish. Res. Lab 5:29-102.

Yonge, C. M. 1952. The mantle cavity of Siphonaria alternata. Proc. Malacol. Soc. London. 29:190-199.

CHAITIP WANICHANON, (1) * PRAPHAPORN LAIMEK, (1) VICHAI LINTHONG, (1) PRAPEE SRETARUGSA, (1) MALEEYA KRUATRACHUE, (2) EDWARD SUCHART UPATHAM, (3) TANES POOMTONG, (4) AND PRASERT SOBHON (1)

(1) Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand; (2) Department of Biology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand; (3) Department of Medical Science, Faculty of Science, Burapha University, Chonburi 20130, Thailand; (4) The Coastal Aquaculture Development Center, Department of Fisheries, Klongwan, Prachuabkirikun Province 77000, Thailand

* Corresponding author. E-mail: sccwn@mahidol.ac.th
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Author:Sobhon, Prasert
Publication:Journal of Shellfish Research
Date:Dec 15, 2004
Words:3426
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