Sensory receptors on cephalic and epipodial tentacles of Haliotis asinina Linnaeus.

ABSTRACT Haliotis asinina, a tropical abalone, has a pair each of cephalic, optic, appendage tentacles at the anterior end of the head, and numerous epipodial tentacles distributed on the periphery of its body. The cephalic and epipodial tentacles are essentially sensory organs with similar general structure. In a mature adult (about 16 mo-old), the cephalic tentacle measures about 3.77 cm in length and 0.14 cm in diameter, whereas the epipodial tentacle measures about 9.27 mm in length and 0.59 mm in diameter. In cross sections, each tentacle has a bundle of nerve fiber forming the core structure, surrounded by a thick layer of circular muscle, which also branches into radially oriented fibers. These fibers are interlaced with thick paraxially orientated fibers; together they account for the size and mobility of the tentacles. The surface of both types of tentacles can be divided into three parts: the basal part exhibits slight corrugation consisting of small folds alternated with grooves, the middle part has numerous short hillock-shaped papillae, and the top part has a very high concentration of cone-shaped papillae. Each papilla comprises a group of densely stained ciliated neuroepithelial cells, surrounded by lightly stained supporting epithelial cells bearing microvilli. Gamma amino butyric acid (GABA) was found, by immunohistochemistry, to be highly concentrated in the neuroepitbelial cells. The rest of epithelium of both kinds of tentacles is of a columnar type, consisting of clear cells bearing microvilli and goblet cells. The GABA-containing neuroepithelial cells are scattered widely among them.

KEY WORDS: H. asinina, tentacle, histology, sensory receptor, GABA

INTRODUCTION

Tentacles are important sense organs of gastropods, which contain tactile and chemoreceptor cells in abundance (Chase 1981, Chase & Croll 1981). The cephalic tentacles are among the most important prosobranch sense organs, although little attention has been paid to their histologic structure and function. They are richly endowed with sensory cells, which may be significantly different in each of the major groups of gastropods (Haszprunar 1988, Beesley et al. 1998). Ito et al. (2000) stained the neurons in the tentacles in a terrestrial slug, Limax marginatus, by backfilling of the tentacular nerves with Lucifer yellow. Four types of stained neurons comprising sensory neurons, gamma cells, ganglion cells, and lateral cells, were identified in the superior and inferior tentacles. In Haliotis tuberculata, a temperate species of abalone, the cephalic tentacle has a mixed sensory and motor nerve, which are centrally located and extends along the length of the tentacle. On the periphery of the nerve, there are sinuses located between muscle fibers, and the latter are arranged longitudinally, obliquely and transversely. Covering the muscle is the surface epithelium that is shown to have folds or papillae (Crofts 1929). The epithelium is of cuboidal type, which consists of 3 cell types: sensory cells, supportive epithelial cells, and widely scattered mucus cells. The sensory cells are spindle-shaped with darkly stained nuclei and supporting cells that are oval and more transparent (Crofts 1929, Bevelander 1988). The epipodium is a series of small tentacles arising from the dorsal part of the foot. They often have the same shape and structure as the cephalic tentacles, although they are much smaller (Crofts 1929, Haszprunar 1988). Crofts (1929) indicated that the epipodial tentacles of H. tuberculata had 2 types of epithelial cells: supporting cells and sensory cells.

The present study reports on the structure of both the cephalic and epipodial tentacles of Haliotis asinina Linnaeus, a tropical abalone commonly distributed along the Thai coastal water, as observed by light and scanning electron microscopes. In addition, immunohistochemistry showed that GABA, a major neurotransmitter of gastropods, is highly concentrated in the sensory epithelial cells.

MATERIALS AND METHODS

Collection of Abalone Specimens

Adult abalones over 12-mo-old 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 a stable controlled environment. The optimum level of salinity used was ~22.5-32.5 ppt, and the temperature about 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 tentacles (cephalic, epipodial and appendage) were measured, dissected out and processed for light microscope (LM) and scanning electron microscope (SEM) studies.

Specimen Preparation for SEM

Tentacles were cut and fixed in a Karnovsky fixative (4% glutaraldehyde 2% paraformaldehyde in 0.1M sodium cacodylate buffer), pH 7.8, at 4[degrees]C 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.

Specimen Preparation for LM

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

For semithin section, specimens were fixed in Karnovsky fixative, pH 7.8, at 4[degrees]C for overnight, and washed with 0.1M sodium cacodylate buffer. They were postfixed in 1% osmium tetroxide in 0.1M sodium cacodylate buffer, at 4[degrees]C, for 1 h. Then, they were dehydrated in a graded series of ethanol and embedded in Araldite 502 resin. Sections were cut at 1-[micro]m thickness with Porter Blum MT-2 [micro]L tramicrotome, and stained with methylene blue or PAS-methylene blue. Examination and photographing of the tissue sections were done under a Nikon eclipse E600 microscope and Nikon digital camera DXM1200.

Immunoblotting for GABA

The neural and tentacular tissues were tested for the presence of GABA by homogenizing the cerebral ganglia, pleuropedal ganglia, tentacular, and epipodial tentacles collected from adult H. asinina in 0.1 M sodium phosphate buffer saline (PBS) containing 1 mM pheuyl-methyl-sulfunyl-fluoride (PMSF). The homogenate was centrifuged at 12000g for minutes, after which the supernatant was collected, and the protein contents determined by Lowry method (Lowry et al. 1951). Proteins were adjusted to be about 1 mg/mL by adding the extraction buffer. A 1-[micro]L of each suspension at the dilutions at 1:1, 1:10, and 1:100 were spotted onto nitocellulose (NC) sheets. The NC sheets were then incubated in 5% skimmed milk for blocking of nonspecific binding before being incubated in the primary antibody (mouse monoclonal antibody against GABA, Sigma Chemical Co, USA). Peroxidase-conjugated goat antimouse IgG (Zymed Laboratories) was used as the secondary antibody. The antigen-antibody complexes were then visualized by enhanced chemiluminescence using a LumiGlo kit (KPL, Gaithersburg, MA), with detection made on X-ray film. For positive control neural tissues, the mouse brain was collected and treated similarly.

Immunohistochemistry

Additional 5-[micro]m sections of paraffin-embedded tentacular and epipodial tentacles, as prepared for LM study, were placed on glass slides, deparaffinized, and rehydrated in ethanol and distilled water. They were then stained by an immunoperoxidase method, by first immersing in 0.1M PBS, pH 7.4, containing 0.3% [H.sub.2][O.sub.2] to block endogenous peroxidase, followed by 0.1% glycine in buffer to block free aldehyde groups from the fixative, then in 10% normal goat serum in the buffer for 1 h, in monocloual antibody (MoAb) (Sigma Chemical Co, USA) at 1:300 dilution, at 20[degrees]C overnight. The sections were thoroughly washed with PBS, followed by incubation in HRP-conjugated secondary antibody (Goat antimouse IgG-HRP, Zymed Laboratories) at 1:200 dilution, for 2 h, and labeled with an aminoethyl carbazole substrate kit (AEC, Zymed Laboratories) for 30 min. Finally, the sections were counter-stained with Harris hematoxylin before being mounted in buffered glycerol, and photographed in Nikon eclipse E600 microscope and Nikon digital camera DXM1200.

RESULTS

Cephalic Tentacle

Cephalic tentacles of mature female abalone (age about 16 mo) were 3.77 [+ or -] 0.59 cm in length and 0.14 [+ or -] 0.03cm in diameter (Table 1): whereas the length of cephalic tentacles and the diameters of mature male abalone of similar age was 3.38 [+ or -] 0.57 cm and 0.12 [+ or -] 0.02 cm, respectively. Noticeably, at the same age the female shell was longer than that of male, and its weight was also heavier than that of male (Table 1).

A cephalic tentacle is round and tapered from the base to the tip (Figs. 1A to C). Based on the surface features as observed under SEM, the tentacle could be divided into 3 parts (i.e., 1/10 basal, 1/10 middle and 8/10 top). There are gradual changes between adjacent parts. The basal part has a smooth surface. It consists of many folds, alternated with grooves (Fig. 1E). Many sensory papillae are located on the folds. On the middle part of the tentacle, there are more papillae on each fold (see Fig. 3C). These papillae are distributed separately and appear taller than those at the base. Each appears as a hillock covered with microvilli and encircling cilia at the tip (Figs. 2C, E). On the top part of the tentacle, the papillae are longer and more numerous than those on the middle part (Fig. 1F). Each appears as a slender truncated cone, projecting perpendicularly to the tentacle's surface (Figs. 2A, F). On top of each papilla, there is a tuft of long cilia, whereas the rest of its surface is covered with microvilli (Figs. 2D,F).

[FIGURES 1-3 OMITTED]

In paraffin sections, the longitudinal section of a cephalic tentacle also exhibits the three parts, based on the degree of surface folding that show gradual changes between adjacent parts (Fig. 3A). Along the axis of each tentacle, there is a central tentacular nerve running along its whole length, and gives rise to the radial branches that innervate the surrounding muscle (Figs. 3B, C). Covering the tentacle is a simple columnar epithelium lying on a thick basement membrane (Figs. 3D, E). The cells in the epithelium covering the papilla can be classified into three types, based on their histologic characteristics and staining affinities. They are: (1) a sensory cell that is spindle shaped with a round or oval shape nucleus, and contains darkly-stained chromatin (Figs. 3D, E). The cytoplasm is dense and stained blue with methylene blue and pinkish purple with PAS-methylene blue. There are long cilia at the tip of each sensory cell, which give rise to the ciliary tuft at the top of each papilla as observed under SEM; (2) a supporting cell which has a columnar or pyramidal shape, and it is generally larger than the sensory cells. It has a round or oval nucleus that contains mostly euchromatin with a few blocks of heterochromatin (Figs. 3D, E). The cytoplasm is clearer than sensory cell, and appears light blue after staining with methylene blue, and light pinkish with PAS-methylene blue stain. The supporting cell has a brush border that is made of microvilli; and (3) a goblet cell, which is a type of mucin-producing cell with a small nucleus and oval shape that is mostly euchromatin (Fig. 3D). The cytoplasm is large, clearer, and stained light blue with methylene blue and pinkish with PAS-methylene blue. At the point where sensory cells aggregate to form a papilla, fibers from the tentacular nerve terminate at a group of these cells that have cilia on top (Fig. 3E).

Epipodial Tentacle

The general structure of an epipodial tentacle is similar to that already described for a cephalic tentacle, but each tentacle is about two times narrower and four times shorter than the cephalic tentacle (Fig. 1A, Table 1). Based on the surface features observed under SEM, each tentacle could also be divided into 3 parts (i.e., 1/5 basal, 1/5 middle, and 3/5 top) (Fig. 2A). Each part has characteristic structures (Figs. 2B, C, D), but there are gradual changes between adjacent parts. The surface of the basal part has folds alternated with grooves (Fig. 2B). On the folds are many short bulbous papillae, each has a circle of cilia on the top (Fig. 2B). In contrast, at the middle part the grooves and folds become less obvious, whereas the papillae are wider and longer, and each appears as a hillock with a circle of cilia on top (Figs. 2C, E). On the top part of an epipodial tentacle there are numerous papillae that are more slender than those on the lower parts, and each appears as a truncated cone with a circle of cilia at the top (Figs. 2D, F).

In spite of its much shorter length, the epipodial tentacle histologically resembles the cephalic tentacle. There are a bundle of epipodial tentacle nerves in the central axis (Figs. 4A, B). Most of surface epithelium, particularly at the basal part of the tentacle, is covered by columnar cells bearing a brush border (Figs. 4C, D). The papilla comprises of three types of epithelial cells with the same characteristics and staining affinities as those of papillae located on a cephalic tentacle (Figs. 4C, D).

[FIGURE 4 OMITTED]

Appendage Tentacle

In addition to the cephalic, optic, and epipodial tentacles, H. asinina has a pair of vestigial appendage tentacles. Each is short with a half circle shape, and is covered by numerous irregular folds, alternated with grooves (Fig. 1D). The folds are covered by epithelial cells bearing numerous microvilli.

Transverse sections of the appendage tentacle reveal similar interior structure (i.e., nerve and muscle) to that of the cephalic and epipodial tentacles. The columnar epithelium, which covers the tentacle, also comprises three types of cells that are evenly spread over the surface without being organized into sensory papillae.

Immunoblots of GABA

The extracts of H. asinina neural tissues (i.e., cerebral and pleuropedal ganglion) are strongly stained for GABA almost equal to that of mouse brain extract (Fig. 5). However, both tentacular and epipodial tentacular tissues are only slightly stained for GABA.

[FIGURE 5 OMITTED]

Sensory Cells Containing GABA

Both of the cephalic and epipodial tentacles have similar patterns of immunostaining for GABA in the cells of epithlium and in the muscle (Fig. 6B). The most prominent immunoreactive cells were sensory cells aggregated at the center of each papilla, which could be readily identified by the presence of cilia on their apical surface (Fig. 6C). Of the remaining surface epithelial cells, the immunoreactive cells are sensory cells, which are widely scattered among the supporting cells (Figs. 6D to F). The nerve bundle and its radiating nerve fibers in both types of tentacles exhibited GABA immunoreactivity (Fig. 6B). In the control the immunostaining is negative (Fig. 6A)

[FIGURE 6 OMITTED]

DISCUSSION

Cephalic and Epipodial Tentacles

As in other prosobranch mollusks, the cephalic tentacles of H. asinina are the major sensory organs |or perceiving the environment conditions because they are the outgrowths from the anterior end of the head (Zaitseva 1997, Beesley et al. 1998). In cross sections the basic structure of the cephalic tentacle is similar to that of other species of Haliotis because the tentacular nerve is in the middle of longitudinally orientated muscle fibers, and it is covered by a simple columnar epithelium (Croft 1929, Bevelander 1988, Beesley et al. 1998). In this study, it was found that the epithelium of cephalic tentacles of H. asinina has 3 types of cells: sensory ceils, supporting cells; which are the majority of cell type, and mucus cells. This is in agreement with the study by Croft (1929) who reported similar finding in the cephalic tentacle of H. tuberculata. The sensory cells of the tentacles of Haliotis are not subepithlial as in pulmonate mollusks (Wright 1974), but are part of the epithelium similar to that in P. elegans (Zaitseva 1997). The sensory cells could be easily identified as having oval shaped nucleus with densely stained chromatin and cytoplasm in contrast to the supporting cells that have much clearer nuclei and cytoplasm. In the basal part of the cephalic tentacle, they are widely scattered in the epithelium and are located in the middle and top parts and concentrated in the center of each papilla. Underneath the papilla, there is a nerve branch from the central tentacular nerve bundle that may supply the sensory cells. This situation is similar to the nerve bundles of P. elegans, which connect their sensory dendrites to the epithelium of the tentacle tip (Zaitseva 1997). On the apical surface of each papilla, SEM showed that there was a bundle of long twisted cilia that in cross section were demonstrated to belong to the sensory cells. In contrast, the supporting cells bear only microvilli that appear in LM as a brush border. The goblet cells are also widely scattered in the epithelium, and few are located in the papilla epithelium. Unlike the epithelium lining the mantle and the hypobranchial gland, the tentacular epithelium is probably not the major site for mucin production because it specializes mostly in the sensory perception.

The epipodial tentacles are smaller and shorter than the cephalic tentacles. They are numerous and located around the foot muscle. They have been shown in this study, as well as in the study by Crofts (1929), to have a basic structure similar to that of the cephalic tentacles. However, the muscle bundles in the epipodial tentacles are smaller and do not appear as regularly arranged as those in the cephalic tentacles. The cephalic tentacles are used for exploring the environment and for seeking out food, while avoiding danger; therefore, they need longer and stronger muscle bundles to stretch and retract them over a long distance. Alternatively the epipodial tentacles function more in support of the cephalic tentacles in finding food and receiving chemical stimuli around the abalone's body. Hence, epipodial tentacles are smaller and shorter than the cephalic tentacles and only project out for a short distance around the body, therefore these tentacles do not need as strong a muscle as in the former.

The group of ciliated sensory cells in the papillae of cephalic and epipodial tentacles are structurally very much like those in the olfactory epithelium or taste buds of vertebrates (Kierszenbaum 2002). Thus, the papillae may function mostly as chemoreceptors similar to the olfactory epithelium and taste buds of the vertebrates. This is supported by many investigators who have shown that, in addition to tactile reception, the cephalic tentacles also act as chemical receptors because they can respond to the chemical stimuli, odor, and food (Preston & Lee 1973, Farkas & Shurey 1976, Chase 1981, Bell & Tubin 1982, Voss & Schmidt 2001). Shimozono et al. (2001) reported that the metacerebro-procerebral neuron (MPN) of Limax marginatus is an output-neuron from the procerebrum. The MPN receives monosynaptic inputs from the superior and inferior tentacle nerves. The MPN, thus, may receive olfactory information via two pathways (i.e., one directly from the tentacle and the others by way of the procerebrum) and possibly functions by integrating both sources of inputs. Similar sensory inputs may be gathered by epipodial tentacles because their papillae have identical structural organization.

Immunohistochemistry

This study has demonstrated the presence of GABA cells of both cephalic and epipodial tentacles. Identification of the immunostained cells is based on the characteristics and the position of cells. These cells are mostly the sensory cells aggregated at the center of each papilla, or single sensory cells widely scattered in the rest of the tentacular epithelium. GABA is the major inhibitory neurotransmitter in the central nervous system and a neuromodulator in certain peripheral tissues vertebrates (Sattelle 1992). In marine invertebrates, GABA is a neurotransmitter that stimulates growth and metamorphosis of the larvae into more mature stage (Aminur & Veharai 2001), and in abalone it specifically stimulates settlement and metamorphosis (Morse et al. 1988) and feeding of larvae (Arshavsky et al. 1993). Therefore, it is highly possible that the GABAergic sensory cells in the papillae of both cephalic and epipodial tentacle play important parts in sensing the availability of food and perhaps also controlling the feeding behavior of the postmetamorphosed abalone up to and during the adult stage.

Appendage Tentacle

The pulmonate mollusks have 2 pairs of tentacles: the superior and inferior tentacles, and the anterior and posterior tentacles, which are called the cephalic tentacle and rhinophore (Beesley et al. 1998). Rhinophores have shapes ranging from simple tapering rods to elaborate lamellae or tubular organs (Beesley et al. 1998). In the prosobranch mollusks, there is one pair of cephalic tentacles, and they do not have a rhinophore (Beesley et al. 1998). In this study, we found a pair of short and small tentacles that had a dome shape and were located dorso-medial to the base of the cephalic tentacle. They were called appendage tentacles. It is possible that these tentacles are homologous to the pulmonates rhinophores, but they remain as vestigial structures. The appendage tentacle has a simple columnar epithelium containing supporting and mucus cells but much fewer sensory cells; there were no papilla like those on the cephalic and epipodial tentacles, and there are no well organized muscles and nerve fibers. Thus, they may just be rudimentary structures.

TABLE 1.

Sizes of cephalic tentacles and epipodial tentacles of adult H.
asinina.

                    Shell                     Cephalic
          Weight    Length                    Tentacle
Sex        (g)       (cm)     [+ or -] SD    Length (cm)    [+ or -] SD

Male        29       3.77        0.62           3.38           0.57
Female      33       4.11        0.64           3.77           0.59

           Diameter of                     Epipodial
            Cephalic                       Tentacle
Sex       Tentacle (cm)    [+ or -] SD    Length (cm)    [+ or -] SD

Male          0.12            0.02           0.80           0.24
Female        0.14            0.03           0.93           0.32

           Diameter of
            Epipodial
Sex       Tentacle (cm)    [+ or -] SD

Male          0.06            0.01
Female        0.06            0.02

All data are based on 16-mo-old fresh abalone of each sex (n = 15). SD,
standard deviation.

ACKNOWLEDGMENTS

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

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

Departments of (1) Anatomy and (2) Biology, Faculty of Science, Mahidol University, Rama VI 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: [e-mail].