Thalassas, 27 (2): 61-75 An International Journal of Marine Sciences

HISTOLOGICAL AND ULTRASTRUCTURAL CHARACTERISATION OF THE STOMACH AND INTESTINE OF THE OPISTHOBRANCH BULLA STRIATA (HETEROBRANCHIA: CEPHALASPIDEA)

ALEXANDRE LOBO-DA-CUNHA(1,2,3), ANA RITA MALHEIRO(4), ÂNGELA ALVES(1), ELSA OLIVEIRA(1), RITA COELHO(3) & GONÇALO CALADO(3,5,6)

Key words: Digestive tract, microscopy, histochemistry, cytochemistry, Mollusca, Gastropoda

ABSTRACT the intestine. Internally, the stomach is characterized by the presence of a typhlosole and many mucus- In order to obtain more data for a comparative secreting cells that are strongly stained by PAS analysis of the digestive system in opisthobranchs, the reaction and alcian blue. Significant amounts of stomach and intestine of Bulla striata were studied proteins were not detected in the mucus-secreting with light and electron microscopy. A 3D-model of cells of the stomach, but protein-rich secretory the stomach and its connections with the posterior material was found in the apical region of another oesophagus, digestive gland ducts and intestine type of secretory cells present in both stomach was created from a series of histological sections. and intestine. The end of the typhlosole can be The U-shaped stomach is just a segment of the considered the transition point between the stomach digestive tube without any external distinction from and intestine. Mucus-secreting cells are also abundant in the intestine and all of them stain with alcian blue. However, most mucus-secreting cells of the intestine (1) Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4099-003 Porto, are not significantly stained by PAS reaction, but Portugal. E-mail: [email protected] contain more proteins than the mucus-secreting cells (2) Centre of Marine and Environmental Research (CIIMAR), of the stomach. The granular cells with a large number 4050-123 Porto, Portugal (3) Portuguese Institute of Malacology (IPM), 8201-864 Guia, of small electron-dense secretory vesicles containing Portugal proteins and neutral polysaccharides were found only (4) Vale do Sousa Higher School of Health - CESPU, Paredes - Portugal. in the intestine. The available data show that despite (5) Lusophone University of Humanities and Technologies, some anatomical and histological differences several 1749-024 Lisbon, Portugal (6) Institute for Marine Research (IMAR), FCT/UNL, 2829-516 cell types are identical in the digestive systems of Caparica, Portugal Aplysia depilans and B. striata.

61 ALEXANDRE LOBO-DA-CUNHA, ANA RITA MALHEIRO, ÂNGELA ALVES, ELSA OLIVEIRA, RITA COELHO & GONÇALO CALADO

Figure 1: Anatomy of the stomach and intestine of B. striata. A. Tip of the digestive gland (dg) including the stomach (st) and initial part of the intestine (in). Terminal portions of the posterior oesophagus (po) and long digestive gland duct (ld) are also visible. B-D. Frontal, lateral and rear views of a 3D-model of the U-shaped stomach (st) and initial part of the intestine (in). A short digestive gland duct (sd) can be seen behind the terminal portion of the posterior oesophagus (po). The arrowhead mark, approximately, the zone where the stomach typhlosole ends. INTRODUCTION comprising the Umbraculoidea, Cephalaspidea, Runcinacea, Anaspidea (or Aplysiomorpha) and In recent years, molecular approaches have been Pteropoda has received a good support (Klussmann- used to investigate opisthobranch phylogeny (Wägele Kolb et al., 2008; Dinapoli & Klussmann-Kolb, et al., 2003; Grande et al., 2004; Vonnemann et al., 2010; Jörger et al., 2010). In this clade diets are 2005; Malaquias et al. 2009a; Dinapoli & Klussmann- very diversified: umbraculoideans feed on sponges, Kolb, 2010; Jörger et al., 2010). Due to these efforts, a anaspideans and runcinaceans are herbivores, new vision of the relationships among opisthobranch pteropods can be carnivores (Gymnosomata) or clades, and between these and other heterobranch omnivores (Thecosomata), and cephalaspideans gastropods is now emerging. The molecular data include both herbivorous and carnivorous species do not support the monophyly of the traditional (Kohn, 1983; Wägele & Klussmann-Kolb, 2005; opisthobranchs, but a clade (Euopisthobranchia) Malaquias et al., 2009b).

62 CHARACTERISATION OF THE STOMACH AND INTESTINE OF THE OPISTHOBRANCH Bulla Striata (HETEROBRANCHIA: CEPHALASPIDEA)

Dietary specialization and the related modifications Morphology of the digestive system were considered fundamental aspects in opisthobranch evolution (Thompson, For light microscopy, tissue pieces containing the 1976; Mikkelsen, 2002; Malaquias et al., 2009b), stomach, initial portion of the intestine and part of the but in spite of that the digestive systems of these digestive gland were fixed for 24 h in Bouin solution, animals is not fully investigated yet. Some light dehydrated in increasing concentrations of ethanol and microscopy studies dedicated to the digestive system embedded in paraffin. Middle and terminal segments of cephalaspideans were published many years ago of the intestine were processed in the same way. Tissue (Fretter, 1939; Rudman, 1971, 1972b, 1972c). The sections were stained with haematoxylin and eosin. radular teeth and gizzard plates of some species were For transmission electron microscopy (TEM), samples studied using SEM (Gosliner, 1994; Malaquias & Reid, of stomach and intestine were fixed for about 2 h at 2008), whereas histochemistry and TEM were used to 4°C in 2.5% glutaraldehyde and 4% formaldehyde investigate the digestive system only in a very limited (obtained from hydrolysis of para-formaldehyde), number of species. The digestive system of Aplysia diluted with 0.4 M cacodylate buffer pH 7.4 (final is fairly well studied, with several articles reporting buffer concentration 0.28 M). After washing in buffer, histochemical and ultrastructural aspects of its organs samples were postfixed with 2% OsO4 buffered with (Taïeb & Vicente, 1999; Lobo-da-Cunha, 1999, 2000; cacodylate, dehydrated in increasing concentrations of Taïeb, 2001; Lobo-da-Cunha, 2001, 2002; Lobo- ethanol and embedded in Epon. Semithin sections (2 da-Cunha & Batista-Pinto, 2003, 2005, 2007). The µm) for light microscopy were stained with methylene salivary glands and the oesophagus of the herbivorous blue and azure II. Ultrathin sections were stained with cephalaspidean Bulla striata have already been studied uranyl acetate and lead citrate, before being observed with histochemical and ultrastructural methods (Lobo- in a JEOL 100CXII transmission electron microscope da-Cunha & Calado, 2008; Lobo-da-Cunha et al., operated at 60 kV. 2010a, 2010b), but the digestive system of carnivorous cephalaspideans has just started to be investigated Histochemistry with the same level of detail (Lobo-da-Cunha et al., 2009). To our best knowledge, the digestive systems of The tetrazonium coupling reaction for protein umbraculoideans and pteropods were never studied by detection was applied to 2 µm sections of Epon TEM, and in runcinaceans only the digestive gland was embedded stomach and intestine fragments. These studied at the ultrastructural level (Kress et al., 1994). semithin sections were treated with a 0.6% solution

of H2O2 for 10 min., to remove the osmium tetroxide In order to obtain more data for a comparative fixative from the tissue. After washing in water, sections analysis of the digestive system in opisthobranchs, the were treated for 10 min with a freshly-prepared 0.2% stomach and intestine of B. striata were studied with solution of fast blue salt B in veronal-acetate buffer light and electron microscopy methods. pH 9.2, washed in water and treated for 15 min with a saturated solution of β-naphthol in veronal-acetate MATERIALS AND METHODS buffer pH 9.2 (Ganter & Jollès, 1970). The PAS reaction for polysaccharide detection was applied to sections of Specimens of Bulla striata Bruguière, 1792 about paraffin embedded pieces and to semithin sections of 2-3 cm in length were collected in Ria de Alvor Epon embedded fragments. After oxidation with 1% and Ria de Faro, two estuaries in the South coast of periodic acid for 10 min, tissue sections were washed Portugal. Stomach and intestine samples collected with water and stained with Schiff reagent for about 15 from 5 animals were processed for light and electron min (Ganter & Jollès, 1970). Alcian blue staining was microscopy as reported below. applied to sections of paraffin embedded material. For

63 ALEXANDRE LOBO-DA-CUNHA, ANA RITA MALHEIRO, ÂNGELA ALVES, ELSA OLIVEIRA, RITA COELHO & GONÇALO CALADO

Figure 2: Histology and histochemistry of the stomach of B. striata. A. Transverse section through the middle of the U-shaped stomach (st) showing the typhlosole (arrows). Numerous PAS positive secretory cells (arrowheads) can be seen in the stomach epithelium, but not in the long digestive gland duct (ld). Digestive gland tissue (dg) surround the stomach. B. A few PAS positive secretory cells (arrowheads) can be seen in the epithelium of the long digestive gland duct (ld) at the connection point with the stomach (st). C. Detail of the stomach epithelium showing lining epithelial cells (asterisk) and PAS positive bottle-shape secretory cells (arrows). The unstained zone (arrowhead) in the basal region of these mucus-secreting cells corresponds to the nucleus. D. Alcian blue stains stomach mucus-secreting cells (arrows) that are also present in the typhlosole (ts). E. Stomach mucus-secreting cells (arrowheads) are not significantly stained by the tetrazonium coupling reaction, but this technique revealed protein-rich secretory material at the apex of another type of secretory cells (arrow). nu - nuclei. F. Apical secretory material is not stained by PAS reaction (arrow), but glycogen deposits in neighbour cells are strongly PAS positive (arrowhead). detection of carboxylated polysaccharides, sections Cytochemistry were stained for 20 min. with 0.5% alcian blue in 3% acetic acid (pH 2.5). To demonstrate the presence For polysaccharide detection by TEM, ultrathin of sulphated polysaccharides, sections were stained sections collected on copper grids were treated with a for 20 min. with 0.5% alcian blue diluted in a HCl 5% solution of tannic acid for 10 min, briefly washed solution with pH 1.0 (Ganter & Jollès, 1970). Sections in water, stained with 2% uranyl acetate for 10 min of paraffin embedded tissues were subsequently and finally washed in water (Sannes et al., 1978). stained with haematoxylin, washed, dehydrated and mounted with DPX. Epon sections were washed, air For localization of acidic polysaccharides by dried and mounted with DPX. TEM, the colloidal iron method described by Knight

64 CHARACTERISATION OF THE STOMACH AND INTESTINE OF THE OPISTHOBRANCH Bulla Striata (HETEROBRANCHIA: CEPHALASPIDEA)

Figure 3: Histology and histochemistry of the intestine of B. striata. A. Transverse section of the intestine (in) on the edge of the digestive gland (dg). Haematoxylin and eosin stain. B. Semithin section of the intestine showing a granular cell (arrow) and epithelial cells with cilia (arrowhead) and without cilia (asterisk). mu - muscular layer; nu - nuclei. C. The secretory material of granular cells is PAS positive (arrows), and positive reaction is also detected in the basal region of a mucus-secreting cell (asterisk). nu - nucleus. D. The tetrazonium coupling reaction reveals protein-rich secretion in the granular cells (arrows) and in mucus-secreting cells (asterisk). E. Semithin section of the intestine showing a PAS positive mucus- secreting cell (arrowhead) and unstained ones (arrows). F. Intestine section stained with alcian blue and PAS reaction. Some mucus-secreting cells are stained only by alcian blue (arrows), but the dark blue ones are stained by both procedures (arrowheads). and Lewis (1992) was used. A stock solution of RESULTS colloidal iron was prepared adding 1 ml of a 25%

FeCl3 solution drop-by-drop to 50 ml of boiling water. Anatomy, histology and histochemistry The dark red stock solution was filtered and dialysed. Just before use, 1.5 ml of stock solution were diluted The stomach of B. striata is embedded in a with 7.5 ml of an acetic acid solution with a pH about narrow tip of the digestive gland (Fig. 1A). On the 1.8. Ultrathin sections were collected on gold grids surface of the digestive gland it is possible to see one and stained for 5 min with the diluted colloidal iron long straight duct that opens into the stomach near solution. The grids were washed in 10% acetic acid the connection point between the stomach and the and finally in distilled water, before being observed posterior oesophagus (Fig. 1A). In order to disclose with the transmission electron microscope. what lies underneath the surface of the digestive

65 ALEXANDRE LOBO-DA-CUNHA, ANA RITA MALHEIRO, ÂNGELA ALVES, ELSA OLIVEIRA, RITA COELHO & GONÇALO CALADO

Figure 4: Ultrastructural aspects of stomach and intestine lining epithelium. A. Stomach epithelial cells with a boarder of cilia (arrowheads) and microvilli (asterisk). Mitochondria (arrows) are abundant in the apical region of these cells. B. Multivesicular body (arrow) close to the base of the microvilli (asterisk) in an intestinal epithelial cell. C. Electron-dense lysosome-like bodies (ly) in the supranuclear region of an intestinal epithelial cell. A Golgi stack can be seen in one of the cells (arrow). D. Basal region of stomach epithelium showing large and small patches of cytoplasm filled with glycogen granules stained by the tannic acid-uranyl acetate method (arrows). The secretory vesicles of a mucous cell are also stained (asterisks). ct - connective tissue. gland, a 3D model was created from a series of around the tip of the digestive gland, being visible at 200 histological sections stained with haematoxylin its margin (Fig 1A-D). In the largest animals used in and eosin. To obtain an unobstructed view of the this study, the intestine was about 2 cm long. stomach and its connections with the posterior oesophagus, digestive gland ducts and intestine, Two sections of the U-shaped stomach can be seen the digestive gland tissue was excluded from the when this organ is transversely cut (Fig. 2A). The model (Fig. 1B-D). In the model it is possible to see stomach wall forms a typhlosole along the inner curve a short digestive gland duct that opens into the top of this U-shaped organ, and a very large number of of the stomach, behind the terminal portion of the secretory cells are present in the stomach epithelium posterior oesophagus (Fig, 1 C-D). In the animal, (Fig. 2A). On the other hand, secretory cells are rare this short digestive gland duct is completely covered in the epithelium of digestive gland ducts (Fig. 2A-B). by digestive gland tissue being invisible on the The lining epithelium of the stomach is formed surface. The U-shaped stomach is just a segment of by thin ciliated and non-ciliated columnar cells the digestive tube without any external distinction interspersed with two types of secretory cells. Bottle- from the anterior portion of the intestine that circles shaped mucus-secreting cells are very abundant, with

66 CHARACTERISATION OF THE STOMACH AND INTESTINE OF THE OPISTHOBRANCH Bulla Striata (HETEROBRANCHIA: CEPHALASPIDEA)

a thin apical neck and a larger basal region containing 1). A thin layer of muscular cells surrounds the the nucleus (Fig. 2C-E). Much less frequent are the epithelium of the intestine (Fig. 3B). cells with an apical mass of secretion (Fig. 2E-F). The histochemical properties of both cell types can be Ultrastructure and cytochemistry seen in Table 1. A thin muscular layer surrounds the stomach epithelium. The lining epithelium is identical in both the stomach and the intestine. It is formed by thin elongated In the intestine the typhlosole is absent (Fig. 3A). cells with a border of microvilli between 2 and 3 µm This structure ends not far after the middle of the long, and most of them also have cilia (Fig. 4A). digestive tube segment that goes from the curve of the Vesicles, multivesicular bodies, some lysosome-like U-shaped stomach to the first curve of the intestine bodies and several mitochondria can be seen in the around the tip of the digestive gland (Fig. 1C). Most supranuclear region (Fig. 4A-C). A few lipid droplets intestine epithelial cells possess cilia, but some are can be found in these epithelial cells, but glycogen is non-ciliated (Fig. 3B). The most abundant secretory the main reserve substance filling a substantial part of cells present in the intestine are the granular cells the cytoplasm in some cells (Fig. 4D). and mucus-secreting cells. The granular cells are thinner and contain a large number of small secretory The mucus-secreting cells of the stomach are granules most of them in the cytoplasm above the filled with a large number of secretory vesicles of nucleus, which is positioned approximately in the variable dimensions with low or median electron- centre of the cell (Fig. 3B-D). The histochemical density. They can be oval or irregular in shape properties of this cell type are included in Table 1. (Fig. 5A) and some of them may be fused forming Mucus-secreting cells of the intestine are bottle- larger compartments with secretory material. The shaped (Fig. 3C-E), but the histochemical techniques vesicles contain very fine filaments forming a show that two kinds of mucus secreting cells are reticulate pattern that can be denser in some vesicles present in the intestine (Table 1). In sections stained than in others (Fig. 5B). The secretory vesicles are by both PAS reaction and alcian blue, it can be seen moderately stained by tannic acid-uranyl acetate that the larger mucus-secreting cells (mucous cells II, method for polysaccharide detection (Fig. 4D), and in Table 1) are stained by alcian blue showing a light with the colloidal iron method for acid polysaccharide blue colour. The thinner ones (mucous cells I, in Table detection iron particles are distributed over the 1) are stained by both procedures appearing with a secretory material contained in the vesicles (Fig. dark blue coloration in result of the superposition 5C). These cells possess several Golgi stacks formed of light blue and the magenta colour of PAS positive by a large number of flat cisternae, mainly located cells, but none are stained by PAS reaction alone around the nucleus (Fig. 5A-B). A few mitochondria (Fig. 3F). However, the change in the staining pattern and rough endoplasmic reticulum cisternae are also of mucus-secreting cells occurs gradually at the present. Very frequently, an intraepithelial nerve transition zone between the stomach and the intestine. terminal was seen in direct contact with the basal After the end of the typhlosole the number of PAS region of a mucus-secreting cell. In ultrathin sections positive cells gradually diminishes in the digestive of these zones the basal lamina has a reticulated tube, and after the first curve of the intestine only appearance, and some perforations are clearly visible about one third of the mucus-secreting cells are PAS in the basal lamina (Fig. 5D). positive (Fig. 3E). In addition to these cell types, the cells with an apical mass of protein-rich secretory In the intestine, the mucous cells I are similar material found in the stomach epithelium are also to the mucous cells of the stomach. However, the present along the intestine in small amounts (Table majority of intestinal mucous cells (type II) possess

67 ALEXANDRE LOBO-DA-CUNHA, ANA RITA MALHEIRO, ÂNGELA ALVES, ELSA OLIVEIRA, RITA COELHO & GONÇALO CALADO

Figure 5: Ultrastructure of stomach mucous cells. A. General view of the dilated basal region showing the nucleus (nu), a large number of secretory vesicles (asterisks) and some Golgi stacks (arrows). B. The Golgi stacks (Gs) are formed by many flat cisternae, and the secretory material creates a fine reticular pattern within the vesicles (asterisk). C. Colloidal iron particles attach to the secretory material within the vesicles (asterisks). D. Intraepithelial nerve terminal (nt) attached to the base of a mucous cell (asterisk). In this region the basal lamina (bl) has a reticulated appearance and is perforated (arrow). ct - connective tissue. large electron-lucent vesicles in which the secretory secretory material is strongly marked by colloidal iron material forms a web of fine filaments linking small particles. Very fine strings of iron particles cover the cores with low electron-density (Fig. 6A). These thin web of filaments and a high concentration of iron vesicles also fuse with each other, but the substances particles is seen around the cores of secretory material they enclose are not significantly stained by the tannic (Fig. 6B). The concentration of iron particles on these acid-uranyl acetate method. On the other hand, the cores creates a pattern with coarse spots, which is

68 CHARACTERISATION OF THE STOMACH AND INTESTINE OF THE OPISTHOBRANCH Bulla Striata (HETEROBRANCHIA: CEPHALASPIDEA)

Figure 6: Ultrastructure of intestinal secretory cells A. Intestinal mucous cell containing secretory vesicles with a web of very fine filaments (arrowheads) connecting small cores of secretory material (arrows). nu - nucleus. B. In the secretory vesicles, colloidal iron particles attach to the thin web of filaments (arrowheads) and around the cores of secretory material (arrows). C. Cell containing an apical mass of secretory material (asterisks) showing some microvilli at the apex (arrowhead). The secretory material of the granular cells has a higher electron density (arrows). D. Apical region of a granular cell showing microvilli (asterisk), multivesicular bodies (arrows), and many electron-dense secretory vesicles (arrowheads). very different from the more uniform distribution of flattened cisternae resemble the ones observed in the iron particles in the secretory vesicles of the stomach stomach mucus-secreting cells. type of mucous cells. The secretory vesicles fill the cytoplasm almost completely and usually just some Cells with an apical mass of secretory material Golgi stacks can be seen in peripheral zones of the were observed in ultrathin sections of both stomach cytoplasm. These Golgi stacks formed by many and intestine epithelia. These cells possess some

69 ALEXANDRE LOBO-DA-CUNHA, ANA RITA MALHEIRO, ÂNGELA ALVES, ELSA OLIVEIRA, RITA COELHO & GONÇALO CALADO

Table 1: Table 1. HistochemicalHistochemical properties properties of stomach of stomach and intestine and intestine secretory secretory cells cells in Bula in Bula striata striata and and Aplysia Aplysia depilans depilans..

PAS Alcian blue Alcian blue Tetrazonium PAS Alcian blue Alcian blue Tetrazonium reaction pH 1.0 pH 2.5 reaction reaction pH 1 pH 2.5 reaction

Cell types Bulla striata - Stomach Aplysia depilans - Stomach

Mucous cells ++ ++ ++ - ++ ++ ++ ++ Cells with apical - - - ++ Cell type not observed mass of secretion Bulla striata - Intestine Aplysia depilans - Intestine

Mucous cells I + ++ ++ ±/+ ++ ++ ++ ++ Mucous cells II -/± ++ ++ ±/+ Cells with apical - - - ++ Cell type not observed mass of secretion Granular cells ++ - - ++ ++ - - ++

- negative reaction; -/± negative or weak reaction; ±/+ weak or moderate reaction; + moderate reaction; ++ strong reaction

microvilli and their apical region is typically filled by As in the stomach, intraepithelial nerve terminals a membrane bound mass of secretory material with attached to the basal region of secretory cells and a

a very irregular shape and median electron-density, reticulated basal lamina with perforations were also that seems to result from the fusion of smaller vesicles observed in the intestine of B. striata. (Fig. 6C). Material with identical electron-density and texture is also present in round vesicles and Golgi stack DISCUSSION cisternae. Several small Golgi stacks and many rough endoplasmic reticulum cisternae are present in these Cephalaspideans and Anaspideans are two cells that also contain a few lysosome-like bodies. opisthobranch clades with a close phylogenetic relationship (Dinapoli & Klussmann-Kolb, 2010; The intestinal granular cells are characterised Jörger et al., 2010), including the herbivorous by a large number of spherical secretory vesicles species B. striata and Aplysia depilans, respectively. containing electron-dense material. These vesicles With the existing anatomical, histochemical and are very abundant in the cytoplasm above the nucleus, ultrastructural data, it is now possible to make a reach a diameter about 0.8 µm and never fuse with each more detailed comparative analysis of the digestive other (Fig. 6D). The cell apex is covered by microvilli system of these two species. Both possess long about 2 µm long. Some multivesicular bodies and ribbon-shaped salivary glands, starting from the small electron-lucent vesicles are usually present in posterior part of the buccal mass and ending the apical region, but endoplasmic reticulum cisternae near the gizzard. Moreover, histochemical and are not abundant (Fig. 6D). The nucleus is located in ultrastructural studies revealed that the salivary the central part of the cell. Below the nucleus only glands of these species have in common two a small number of secretory vesicles are present, types of mucus-secreting cells, named granular because the basal region of these cells is filled with mucocytes and vacuolated mucocytes. Nevertheless, very deep cell membrane invaginations associated the ciliated cells present in the salivary glands with a large number of mitochondria. of these species are different, being secretory

70 CHARACTERISATION OF THE STOMACH AND INTESTINE OF THE OPISTHOBRANCH Bulla Striata (HETEROBRANCHIA: CEPHALASPIDEA)

in A. depilans and non-secretory in B. striata stomach of the carnivorous cephalaspideans Philine (Lobo-da-Cunha, 2001, 2002; Lobo-da-Cunha & aperta and Scaphander lignarius just one type of Calado, 2008). Few mucous cells were found in mucus-secreting cells has been reported (Fretter, the oesophagus and crop of A. depilans while in 1939). However, the carnivorous cephalaspidean B. striata mucous cells are very abundant in the Melanochlamys cylindrica and the herbivore Bulla anterior and posterior oesophagus (Lobo-da-Cunha quoyi seem to be devoid of secretory cells in the & Batista-Pinto, 2005; Lobo-da-Cunha et al. 2010a, stomach (Rudman, 1971, 1972c). On the other 2010b). Both species have a gizzard, although with hand, the stomach epithelium of Haminoea is more a different number of hard plates. In A. depilans, complex, containing additional types of secretory a filter chamber is positioned between the gizzard cells (Fretter, 1939; Rudman, 1971). The stomach and the stomach, containing many acicular teeth epithelium of the herbivore Haminoea hydatis to prevent the entrance of larger algal fragments includes a cell type characterized by an apical into the stomach (Howells, 1942; Fretter & Ko, accumulation of secretory material (Fretter, 1939), 1979). In B. striata, the gizzard is followed by the that could be related to the cells found in the stomach posterior oesophagus, but some acicular teeth are and intestine of B. striata also containing a mass of present in the most anterior region of the posterior secretory material in the apical region. Among the oesophagus (Lobo-da-Cunha et al. 2010b). Acteonidae, secretory cells of the mucous type were reported in the stomach of some species, but not in In both species the stomach is embedded in the others (Fretter, 1939; Rudman, 1972a). digestive gland and linked to it by ducts, and the intestine is attached to the digestive gland for most Mucus-secreting cells are allegedly responsible for of its length. Nevertheless, substantial anatomical the lubrication of the luminal surface of the digestive differences can be found in the stomach, which in tube and were also found in the stomach of many Aplysiidae ends in a caecum that does not exist in B. other gastropods (Bolognani Fantin et al., 1982; striata (Howells, 1942; Fretter & Ko, 1979). A caecum Roldan & Garcia-Corrales, 1988; Soto et al., 1990; is also present in the stomach of the Thecosomata, Leal-Zanchet, 1998). However, only a few papers which are closely related with the Anaspideans reported ultrastructure aspects of the stomach cells of (Klussmann-Kolb & Dinapoli, 2006). gastropods (Pipe, 1986; Triebskorn, 1989; Boer & Kits, 1990; Leal-Zanchet, 2002; Lobo-da-Cunha & Batista Mucus-secreting cells are very abundant in Pinto, 2003; Martin et al., 2010). the stomach epithelium of these opisthobranchs. However, the secretory material of these cells is rich In the intestine, A. depilans and B. striata contain in proteins in A. depilans (Lobo-da-Cunha & Batista- granular cells and mucus-secreting cells (Table 1). Pinto, 2003) and that is not true for the stomach of The former are characterized by the presence of B. striata (Table 1). Despite that, in each of these electron-dense secretory vesicles containing proteins species stomach mucous cells form secretory vesicles and neutral polysaccharides, and could be classified with low or medium electron-density containing as serous cells (Lobo-da-Cunha & Batista-Pinto, thin filaments of secretory material (Lobo-da- 2007). In the intestine of B. striata, the extensive Cunha & Batista-Pinto, 2003). In addition to these system of cell membrane invaginations associated to mucus-secreting cells, the stomach epithelium a large number of mitochondria in the basal region of of B. striata contains cells specialised in protein the granular cells suggests an intense active transport secretion, whereas in the stomach of Aplysia just the in these cells. In the intestine of A. depilans just one mucous cells have been reported so far (Howells, kind of mucous cells was detected (Lobo-da-Cunha & 1942; Lobo-da-Cunha & Batista-Pinto, 2003). In the Batista-Pinto, 2007), while in B. striata some are PAS

71 ALEXANDRE LOBO-DA-CUNHA, ANA RITA MALHEIRO, ÂNGELA ALVES, ELSA OLIVEIRA, RITA COELHO & GONÇALO CALADO

positive and others PAS negative (Table 1). In addition layer, preventing them from disintegrating (Bush, to this difference in PAS reactivity, the mucous cells 1988). These cells are also similar to the granular of B. striata intestine can be distinguished by the cells of B. striata and A. depilans, which may have content of the secretory vesicles and by the pattern similar functions, but the secretory granules in P. of colloidal iron staining. They may represent two vulgata club-shaped cells are not stained by PAS sub-types of mucous cells or different stages of cell reaction. However, considering the data that support maturation. The secretory vesicles containing a web an absorptive function for the intestine, secretion of of thin filaments linking small cores of secretory digestive enzymes cannot be ruled out for intestinal material in the intestinal type of B. striata mucous cell cells containing proteins in their secretory vesicles. resemble the vesicles described in mucus-secreting Two or more secretory cell types were encountered cells of the anterior oesophagus of this species (Lobo- in the intestine of pulmonate gastropods, including da-Cunha et al., 2010a). mucous cells and other secretory cells (Triebskorn, 1989; Boer & Kits, 1990; Franchini & Ottaviani, The cells with an apical mass of secretion 1992; Leal-Zanchet, 1998, 2002). observed in the stomach and intestine of B. striata were not reported in Aplysia. These cells belong to a The presence of many intraepithelial nerve different type, and can easily be distinguished from terminals in close association with the base of the granular cells by the difference in electron- secretory cells in both stomach and intestine of B. density of the secretory material, absence of PAS striata put in evidence the importance of nervous staining and fusion of secretory vesicles creating control over the activity of these parts of the digestive the large mass of secretory material in the cell tract, as previously reported in other species of apex, an aspect never observed in the granular cells gastropods (Bush, 1988; Boer & Kits, 1990; Lobo-da- of both species (Lobo-da-Cunha & Batista-Pinto, Cunha & Batista-Pinto, 2003). 2007). The columnar cells of the stomach and intestine Two types of secretory cells were also reported lining epithelium are also identical in B. striata and in the intestine of the opisthobranchs A. punctata A. depilans. Lysosomes with arilsulphatase activity (Howells, 1942), B. quoyi (Rudman, 1971), S. are very conspicuous in the supra-nuclear region lignarius (Fretter, 1939) and in some species of Philine of stomach and intestine columnar epithelial cells (Fretter, 1939; Rudman, 1972b), but although their of A. depilans and probably are responsible for characterization was based only on light microscopic the intracellular digestion of substances collected observations, those cells seem to correspond to the from the lumen by endocytosis (Lobo-da-Cunha & intestinal mucous and granular cells of B. striata Batista-Pinto, 2003, 2007). The digestive gland is and A. depilans (Lobo-da-Cunha & Batista-Pinto, recognized as the main sites of digestion and nutrient 2007). However, in Haminoea zelandiae just one absorption in gastropods, but it was demonstrated type of secretory cell was reported in the intestine that absorption of nutrients occurs along the digestive (Rudman, 1971). Ultrastructural observations of tract (Walker, 1972; Orive et al., 1979). According to the intestinal epithelium of the marine snail Nerita some authors, the function of the gastropod intestine picea revealed secretory cells very similar to the is the consolidation of fecal pellets (Mikkelsen, granular cells of B. striata and A. depilans (Pfeiffer, 1996). However, the presence of a microvillous 1992). In the limpet Patella vulgata, the intestinal border, lysosomes and the accumulation of lipids and epithelium contains club-shaped protein secreting glycogen in epithelial cells of the digestive tract of cells rich in rough endoplasmic reticulum cisternae gastropods were considered as signs of an absorptive and its secretion coats the fecal rods with a protein function (Roldan & Garcia-Corrales, 1988; Boer &

72 CHARACTERISATION OF THE STOMACH AND INTESTINE OF THE OPISTHOBRANCH Bulla Striata (HETEROBRANCHIA: CEPHALASPIDEA)

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