Aminopeptidase reactivity in the digestive tract of adult abalone Haliotis asinina linnaeus.

ABSTRACT Histologically, the epithelium of the digestive tract is pseudostratified columnar type, which is composed of 3 kinds of cells (i.e., granulated cells, nongranulated cells, and mucus cells). The subsets of these three types of cells vary from region to region. By an enzyme-histochemical method, leucine aminopeptidase reactivity was localized principally within the cytoplasm of granulated cells in the buccal cavity, esophagus, esophageal pouch, hepatopancreas, and the third intestinal region, which implies that these sites are responsible for the synthesis and secretion of this peptidase for the digestion of proteinaceous nutrients.

KEY WORDS: Haliotis asinina, abalone, digestive tract, aminopeptidase

INTRODUCTION

The abalone Haliotis asinina is considered the most economically important abalone among the three species found along the coastal water of Thailand (Nateewathana & Bussawarit 1988) because of its good taste, high proportion of flesh, and the fastest growth rate. Due to increased demand, the collection of these abalone from natural habitat could not keep pace. To increase the abalone stocks and yields, the animals have been cultured and fed artificial diets for enhancing the growth rate (Serviere-Zaragoza et al. 1997). The rate of growth and nutritional composition of abalone were significantly affected by the level of proteins in the diet (Britz & Hecht 1997), which could be increased up to 30% (Fallu 1991). This high protein content could be broken down by proteolytic enzymes in the digestive tract of abalone, even though abalone are considered to be herbivorous animals that feed on mainly macroalgae, which are richer in carbohydrates. One possible protease inducible in the abalone is aminopeptidase, which is a family of zinc-dependent enzyme that catalyzes the hydrolysis of amino acid residues at the amino terminus of peptide substrates (Acosta et al. 1998). The source of this enzyme is most likely the epithelium of the digestive tract. The epithelial cells of the digestive tract of gastropods have been classified into 3 cell types and termed by different names; for example, columnar storage cell, secretory cell and mucus-producing cell (Tiebskorn & Kunast 1990), nonciliated, ciliated, and glandular cell (Leal-Zanchet 1998, Roldan & Garcia-Corrales 1988). Campbell (1965) and Chitramvong et al. (2000) have studied the abalone and also classified these epithelial cells into 3 types, which include pigmented cell, secretory cell, and mucus cell. The aim of this study is to investigate the presence of aminopeptidase in epithelial cells of some parts of the digestive tract of H. asinina by an enzyme-histochemical method.

MATERIALS AND METHODS

Collection of Abalone Specimens

Adult abalone H. asinina (age >16 mo old) of both sexes were obtained from the Coastal Aquaculture Development Center, Department of Fisheries, Prachuabkirikan province, Thailand. The animals were reared in the raceways of concrete tanks, which were well flushed with mechanically circulated seawater and air delivery system to maintain controlled environment. Abalone were fed with a diet of macroalgae (Gracilaria spp. and Laminaria spp.) supplemented with artificial food, and kept under normal daylight cycle.

Burstone and Folk Method for Detecting Aminopeptidase

Abalone with body weights of 36.5 [+ or -] 2 g were anesthetized with 5% magnesium chloride for about 30 min. The shells (average size of 6.5 cm x 2.8 cm) were removed and digestive organs were dissected out and diced into small pieces before being embedded in the cryo-supporting medium. Frozen specimens were sectioned at 5-[micro]m thick. The cryosections were fixed in two changes of chloroform for 4 min each, then hydrated in graded series of acetone, and finally in distilled water. The sections were incubated in a substrate solution containing the mixture of L-leucyl-bnaphthylamide, distilled water, tris buffer and garnet GBC for 1h at 37[degrees]C, then in tap water for 5 min and counterstained by Hematoxylin. After the final run through tap water for 10 min, the sections were mounted on glass slides in buffered glycerol (Burstone & Folk 1956).

Histology of the Digestive Tract

To identify the cell types and characterize the cellular details for the enzyme localization, the histology of the abalone digestive tract was also studied from paraffin and semi thin plastic sections by preparing the specimens as follows.

For paraffin sections, various parts of the digestive tract were cut and fixed in the fixative of Bouin at 4[degrees]C overnight. Specimens were washed in 70% ethyl alcohol for removal of the fixative. Then, they were dehydrated through a graded series of ethyl alcohol (70% to 100%) for 1-2 h each, cleared with dioxane, infiltrated and embedded in paraffin wax, sectioned at 5 [micro]m thick, and finally stained with Hematoxylin-Eosin (H&E) or Hematoxylin-PAS (H&PAS). The specimens were observed and evaluated for the characteristics of each cell type under microscope equipped with a digital camera.

For semithin plastic sections, the corresponding parts of the digestive tract were fixed in a solution of 4% glutaraldehyde and 2% paraformaldehyde in 0.1 M Millonig buffer at 4[degrees]C overnight, After washing with buffer, the samples were postfixed in 1% osmium tetroxide in 0.1 M Millonig buffer at 4[degrees]C for 1 h, and then dehydrated in graded series of ethanol and pure propylene oxide. After that the specimens were infiltrated and embedded in Araldite 502 resin, and finally polymerized at 60[degrees]C for 24 h. Blocks of plastic embedded specimens were sectioned at 1 [micro]m. These semithin sections were stained with 1% methylene blue, and then observed under microscope equipped with a digital camera.

RESULTS

Histology and Aminopeptidase Reactivity

The Buccal Cavity

The epithelium of buccal cavity consists of 3 cell types, the tall granulated columnar cells bearing microvilli; which appear as a "brush border" under the light microscope, the nongranulated cells, and the mucus cells having a "goblet" appearance; which are widely scattered in the epithelium (Fig. 1a). The granulated cells show red orange product in the apical cytoplasm overlapping on the granulated area (Fig. 1c), indicating the presence of aminopeptidase in this area of the cytoplasm, and at the luminal surface, whereas nongranulated and mucus cells are not stained. The control section does not show the presence of aminopeptidase (Fig. 1b).

[FIGURE 1 OMITTED]

The Esophagus

The epithelium of the esophagus contains 4 cell types: 2 types of granulated columnar cells, nongranulated columnar cells, and mucus cells (Fig. 1d to f). The two types of granulated columnar cells are distinguished by the presence of different size granules and color (i.e., in H&E stain type 1 contains small brownish granules whereas type 2 contains large reddish eosinophilic granules, whereas in semithin section all the granules appear bluish with different intensities (Fig. 1d to f)). The granulated cells with large reddish granules, nongranulated cells, and the mucus cells do not show aminopeptidase reactivity, whereas the granulated cells with small brownish granules show intense aminopeptidase reactivity in the granules (Fig. 1h). The control section does not show red product (Fig. 1g).

[FIGURE 1 OMITTED]

The Crop

The epithelium of crop appears to be pseudostratified columnar epithelium that is composed of only one-cell type (i.e., the tall columnar cells, bearing a brush border). These cells also contain small clear mucin granules that are tightly packed together in both the apical and basal cytoplasm (Fig. 1i). This cell type shows only weak reactivity at its luminal surface but not in the cytoplasm (Fig. 1k), hence probably does not contain aminopeptidase enzyme.

The Stomach

The stomach epithelium comprises 3 cell types: mucus cell, tall granulated columnar cells with fine granules in the apical cytoplasm, and nongranulated columnar cells. The epithelium is covered by a thick layer of PAS-positive gastric shield (Fig. 1l). The intense aminopeptidase reactivity is present in the gastric shield overlying the epithelium but absent in the epithelial cells' cytoplasm. (Fig. 1n). The control section shows negative reaction (Fig. 1m).

The Intestine

The intestinal epithelium contains four types of columnar epithelial cells. The former group comprises two types of granulated cells with different characteristics of granules (i.e., the first type of granulated cell appears to be similar to those found in the stomach whereas the second type possesses larger granules with varying density (Fig. 2a, b, c)). There is one type of nongranulated cell (Fig. 2b), and one type of mucus cell appearing similar to those in the crop (Fig. 2a). All two types of granulated cells show intense aminopeptidase reactivity in their cytoplasm (Fig. 2d, g). These four cell types occupy almost the entire epithelium except at the two prominent longitudinal folds, which contain only nongranulated cells that are devoid of aminopeptidase reactivity (Fig. 2d, e). The mucus cells, which are dispersed in the epithelium do not show any aminopeptidase reactivity either. The control section does not show any reaction product (Fig. 2f).

[FIGURE 2 OMITTED]

Hepatopancreas

The hepatopancreas comprises a large number of acini (Fig. 2h). The apical part of the acinar cells near the lumen exhibits intense aminopeptidase reactivity (Fig. 2j). The control section shows no reaction of aminopeptidase (Fig. 2i).

DISCUSSION

In this study epithelial cells of the abalone digestive tract is classified into 3 types. The granulated cells (GC) are so called because they contain dense granules (serous type) with different sizes. This cell type could correspond to secretory cells as reported by Campbell (1965) and Chitramvong et al. (2000). In contrast to these earlier works we could identify at least four subtypes of these cells in various parts of the digestive tract because of their well preserved characteristics in semithin plastic sections. All GC except those in the stomach exhibit the aminopeptidase activity, which overlaps on the granulated area of the cytoplasm. There is only one type of nongranulated cell (NC), which does not exhibit aminopeptidase activity. There are at least 2 types of mucus cells (MC), in contrast to only one type of mucin producing cell reported by Campbell (1965) and Chitramvong et al. (2000). The first type of MC appears like goblet cells containing large mucin granules in the apical part, which tend to be dissolved away as shown in buccal cavity, esophagus, and stomach: whereas the second type is usually loaded with small tightly packed mucin granules as shown in the crop and intestine. Again the identification of these two subtypes of mucus cells is possible because of the use of plastic embedment. The presence and distribution of proteolytic enzyme was studied by detecting aminopeptidase (AP) reaction in different regions of the alimentary tract, from the buccal region to the anus. Almost all parts of the digestive tract show positive AP activities restricted at the brush border of the epithelial cells or in the lumen. The distinct appearance of AP reactivity was only observed in the cytoplasm of the granulated cells of buccal cavity, esophagus, esophageal pouch, as well as intestine. In addition, the intense enzyme reactivity was also detected in the acinar cells of the hepatopancreas. Hence these cells are producing zymogen granules that may contain aminopeptidase. The results agree with the work of Serviere-Zaragoza et al. (1997) who detected the proteolytic activity in the hepatopancreas, crop and stomach content, and intestine and rectal fluid of the blue abalone, H. fulgens. The proteolytic activity observed in the intestinal and rectal fluid has optimal pH at alkaline range, whereas those from the hepatopancreas, crop, and stomach content were at acid pH. In the hepatopancreas, many proteases (three types of hydrolases and a carboxypeptidase) were detected by enzyme assays in H. midae (Erasmus et al. 1997, Hernandez-Santoyo et al. 1998). Moreover, trypsin and chymotrypsin activities were also detected in the intestine and rectum by using specific-synthetic substrates and inhibitors in kinetic and electrophoresis assays (Groppe & Morse 1993, Serviere-Zaragoza et al. 1997). The AP reactivity recognized in other parts of the digestive tract observed in the present study could possibly be the result of the released enzyme being mixed with the nutrients passing through these regions of the digestive tract rather than the enzyme being synthesized by epithelial cells of these regions. This could explain the weak AP activity at the luminal surface of the crop, the first and second intestinal parts, and in the gastric shield of the stomach.

The absence of AP activity on the secondary foldings in the crop epithelium indicates that this organ may be the main part for food absorption rather than secreting the enzymes. Our observation is in agreement with Mclean (1970) who found, by using autoradiography with [sup.14]C-labeled algae, that in H. rufescens the crop is the major absorptive area. This author also demonstrated that the crop epithelium is permeable to phenylalanine and glucose. However, enzymes other than protease may be present in crop, because high lipase activity was detected in the crop (Mclean 1970). Interestingly, in the intestine there is an area called the longitudinal fold (typhlosole), which does not exhibit any AP activity as most cells are nongranulated, and this structure probably is involved mainly in the absorption, whereas the rest of intestine synthesizes and secretes the protease enzyme.

ACKNOWLEDGMENT

This research was supported financially by the Thailand Research Fund (Senior Research Scholar Fellowship to Prasert Sobhon)

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PORNCHARN SAITONGDEE, (1) PORNRUT RABINTOSSAPORN, (1) PRAPEE SRETARUGSA, (1) TANES POOMTONG, (2) AND PRASERT SOBHON (1), *

(1) Anatomy Department, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand; (2) The Coastal Aquaculture Development Center, Department of Fisheries, Ministry of Agriculture and Cooperatives, Klong Wan, Prachaubkirikhun 77000, Thailand

* Corresponding author. Fax: [phone]; E-mail: [e-mail]