THE FUNCTIONAL ORGANIZATION OF THE SALIVARY GLAND OF
BIOMPHALARIA STRAMINEA (GASTROPODA: PLANORBIDAE):
SECRETORY MECHANISMS AND ENZYMATIC DETERMINATIONS
KIEIV R. S. MOURA 1 , WALTER R. TERRA 2 AND ALBERTO F. RIBEIRO 1
1
Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo C.P. 11461, 05422-970 São Paulo, SP Brazil; and
2
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo C.P. 26077, 05513-970 São Paulo, SP Brazil
(Received 17 September 2002; accepted 13 May 2003)
ABSTRACT
A detailed morphological analysis of the salivary gland of adults of the freshwater snail Biomphalaria
straminea was carried out by light and electron microscopy, complemented by the biochemical assay of
key digestive enzymes in gland homogenates. The salivary gland is a paired tubular organ with the anterior portion (the tubular ducts) inserted into the buccal mass and the posterior region (the secretory
portion) joined by their tips, forming a loop. The entire gland is made up of a simple columnar epithelium, resting on a thin connective tissue, and three regions can be recognized: the duct, the intermediary
and the secretory regions. The duct is formed by a single cell type, the duct epithelial cell, covered
apically by cilia and microvilli. The intermediary region presents two cell types: one similar to the duct
cells and the other to the secretory cells found in the secretory region. Two cell types are also observed in
the secretory region: the intercalary (non-secretory cells) and the secretory cells. The intercalary cells
have many cilia and microvilli on their apical surface which, along with the cilia present in the duct
epithelial cells, should act in mixing and propelling the salivary secretion in the gland lumen from the
secretory region into the buccal cavity. According to the general cell morphology, size and secretory
activity, five phases of differentiation (I–V) can be identified in secretory cells. Based on the ultrastructural aspect of the secretion vesicles and the rough endoplasmic reticulum, two cell sub-types can be
further distinguished in secretory cells: L-cells and H-cells. The mechanism of elimination of secretion
in secretory cells is initially apocrine (phases III–IV), ending as a holocrine mechanism in the final
(phase V) steps of the secretory process. To detect the presence of digestive hydrolases in the salivary
gland, five enzymes (amylase, cellulase, maltase, aminopeptidase and trypsin) were tested in gland
homogenates. Except for trypsin, the other enzymes were found to be significantly active, indicating an
important role of the salivary secretion in the initial digestion of food ingested by these animals.
INTRODUCTION
The secretion of the salivary glands of gastropods has been related
to several physiological functions including the lubrication and
ingestion of food particles, the initial phases of the digestive process and even the capture of prey, due to the presence of pharmacologically active substances that act as venom (Andrews, 1991;
Bhanu, Shyamasundari & Rao, 1981; Charrier & Rouland, 1992;
Serrano, Gómez & Angulo, 1996).
Morphological studies have revealed a great anatomical and
histological variation among the salivary glands of several molluscan groups, which should be related to phylogeny and the
diverse dietary habits exhibited by these organisms. Although
herbivorous species of Pulmonata possess one pair of salivary
glands (Marcuzzi, 1950; Boer, Bonga & Rooyen, 1967; Moreno,
Piñero, Hidalgo, Navas, Aijon & Lopez-Campos, 1982), some
carnivorous species of Prosobranchia may have two pairs of
glands, one acinar and the other tubular, the latter known as
accessory glands (Andrews, 1991; Voltzow, 1994). In fact, a great
variation in gland morphology is found among prosobranchs.
Most of the species in this group typically possess one pair of
acinous salivary glands which may be very complex, as in patellogastropods and mesogastropods, reduced, as in many ciliary
feeders, or even absent in some species (Fretter, 1965; for
reviews, see Andrews, 1991; Voltzow, 1994).
The salivary glands of Pulmonata generally show a complex
histological organization revealing a remarkably diverse range
Correspondence: A. F. Ribeiro; e.mail: [email protected]
J. Moll. Stud. (2004) 70: 21–29
of cell types, varying from about five to 17 types or conditions
of the cells in two main categories, mucous and serous (for
review, see Luchtel, Martin, Deyrup-Olsen & Boer, 1997). Such
complexity results in controversy related to the morphological
identification and function of the various cell types found in the
salivary glands. Sometimes, discrepant results for the same
species have been published, as in Helix aspersa, where three
(Moreno et al., 1982), four (Serrano et al., 1996) or six (Charrier,
1988) different secretory cells types are identified. Certainly,
further and more detailed studies are necessary to clarify this
important aspect of the salivary gland organization.
In Basommatophora, the salivary glands have been studied
in a few species, mainly in Biomphalaria glabrata (Marcuzzi, 1950;
Pan, 1958) and Lymnaea stagnalis (Carriker & Bistald, 1946;
Boer, Bonga & Rooyen, 1967). Apart from the works of Marcuzzi
(1950) and Pan (1958) there is no information concerning the
detailed morphological organization of the salivary glands or
the role of their secretion in Biomphalaria.
The species B. straminea (Dunker, 1848) (Planorbidae) is an
important vector of schistosomiasis in the tropics, mainly in
the north and northeast areas of Brazil (Abdel-Malek, 1985;
Carvalho, 1992; Barbosa, 1995). Increased knowledge of the
morphology and physiology of the internal organs of this and
other vector species of Biomphalaria may contribute to the development of new strategies of biological control of their natural
populations in endemic areas.
The aim of this study is to examine the morphological and
functional organization of the salivary glands of B. straminea,
© The Malacological Society of London 2004
K. R. S. MOURA ET AL .
its posterior region, forming a loop (Fig. 1A). The anterior
region is formed by the glandular ducts, which open into the
buccal cavity on either side of the oesophagus; the posterior
region constitutes the secretory portion. The ducts have a
smooth external surface, whereas the secretory portion shows a
rough surface with many bulges and invaginations constituting
the so-called glandular lobes (Pan, 1958).
Histologically and ultrastructurally, the glandular epithelium
can be divided into three main regions: the duct, the intermediary and the secretory regions.
The salivary gland lumen is lined with a simple columnar
epithelium, resting on a thin basal lamina with a slender connective tissue sheath underneath (Fig. 1C–E), where elongated
cells, rich in cytoplasmic microfilaments, are occasionally noted
(Fig. 3A). Spaces between cells, filled with cell debris, are sporadically observed in the glandular epithelium (Figs 1D, 4D).
Along the entire epithelium, adjacent cell boundaries display
interdigitations, with desmosomes near the lumen and septate
junctions along the boundary (Fig. 2E). In the intermediary and
secretory regions, dilated and complex basal and intercellular
spaces, formed by the undulating basolateral plasma membrane, are frequently observed (Fig. 2B).
The duct is lined with an epithelium formed by a single cell
type, the duct epithelial cell (Figs 1C, 2A). This cell is apparently
non-secretory and covered by many cilia and microvilli on its
apical surface. The cytoplasm is rich in mitochondria and shows
small vesicles, some cisternae of the rough endoplasmic reticulum and few Golgi areas. The nucleus is centrally located and a
large number of free ribosomes and glycogen inclusions are
present in the cytoplasm. The intermediary region, located
between the duct and the secretory region, exhibits two cell
types: one similar to the duct cells and the other to the secretory
cells found in the secretory region. The secretory region has two
well-defined cell types: the intercalary cell and the secretory cell
(Figs 1E, 2B, C; see also the general diagrammatic representation of the secretory epithelium in Fig. 5). The intercalary
cell, with no detectable secretory activity, is very slender in the
basal region of the epithelium, enlarging toward the apical
region (Figs 1E, 2B, C). The nucleus is usually round and apically
located. The cytoplasm contains numerous mitochondria
mainly concentrated at the cell apex. Some cisternae of the
rough endoplasmic reticulum and rare Golgi areas can also be
observed. This cell type pocesses many cilia (Fig. 1B) and
microvilli at its apical surface.
The secretory cells exhibit distinctive morphological aspects
and secretory activity, which represent different phases of differentiation (Figs 3, 5). Five phases (I–V) can be identified.
Secretory cells in phase I, present characteristics of undifferentiated cells, showing a large basal nucleus and a typical triangular
shape (Fig. 3A). They are located in the basal portion of the
epithelium with no direct contact with the gland lumen. The
cytoplasm is rich in cisternae of rough endoplasmic reticulum
and mitochondria, with few Golgi areas. Secretory vesicles are
including hydrolase assays of five key digestive enzymes in gland
homogenates, to determine the possible role of the gland secretion in food digestion.
MATERIAL AND METHODS
All investigations were performed on sexually mature specimens of the snail B. straminea, maintained in the laboratory at
25–27°C on plastic trays with aerated filtered dechlorinated tap
water and fed with fresh lettuce leaves. The founder population
of B. straminea was originally collected in Mucambeiro (State of
Minas Gerais, Brazil), and kept in the laboratory as decribed
elsewhere (Tomé & Ribeiro, 1998).
For light microscopy, the snails were dissected in the fixative
under the stereomicroscope and the salivary glands carefully
pulled apart. The samples were kept in Bouin’s fixative for 3 h or
Zamboni solution (Stefanini, Martino & Zamboni, 1967) for
18 h, at room temperature, upgraded in ethanol and embedded
in paraffin wax (m.p. 58°C) or historesin (Leica, Heidelberg).
Serial sections (2–5 m thickness) were cut and stained with
Hematoxylin–Eosin–Phloxyne, Periodic Acid–Schiff (PAS) or
Toluidine Blue (Kiernan, 1981).
For transmission electron microscopy (TEM), the salivary
glands were fixed in an ice-cold solution of 2% glutaraldehyde
plus 1% osmium tetroxide in 0.05 M sodium cacodylate buffer
pH 7.4 for 1 h at 4°C. The tissues were rinsed in cacodylate
buffer, dehydrated in graded ethanol and embedded in Spurr
resin. Ultra-thin sections were cut using a Leica Ultracut UCT
ultramicrotome, stained with uranyl acetate and lead citrate,
and examined in a Zeiss EM 900 electron microscope.
For scanning electron microscopy (SEM), the tissues were
fixed as for TEM and, after rinsing in distilled water, the samples
were transferred to an aqueous solution of 1% tannic acid for
10 min, followed by aqueous solution of 1% osmium tetroxide
for 30 min at 4°C. After rinsing in distilled water, the glands were
dehydrated in graded ethanol at room temperature, criticalpoint dried and gold-coated according to standard procedures.
The preparations were examined in a Zeiss DSM 940 electron
microscope.
For hydrolase assays the salivary glands were removed, quickly
washed in a cold solution of 0.06% NaCl and homogenized in
cold double-distilled water using a Potter-Elvehjem homogenizer.
Protein was determined according to Bradford (1976), using
Comassie Blue G as a stain and ovalbumin as a standard. Enzyme
assays were accomplished as described in Table 1, using tissue
processed from freshly dissected animals.
RESULTS
Morphology
The salivary gland of B. straminea is a paired tubular organ, with
the anterior region inserted into the buccal mass and joined in
Table 1. Assay conditions and methods used in the determination of enzymatic activities.
Enzyme
Substrate
Concentration
pH
Substance or group determined
Reference
Amylase
Starch
0.5%
4.8–10
Reducing groups
Noelting & Bernfeld (1948)
Aminopeptidase
LpNa
1 mM
5–9
Nitroaniline
Erlanger, Kokowsky & Cohen (1961)
Maltase
Maltose
7 mM
4.5–10
Glucose
Dahlqvist (1968)
Celulase
CMC
1%
5–10
Reducing groups
Hoffman (1937)
Trypsin
BAPA
0.8 mM
8.5
Nitroaniline
Erlanger et al. (1961)
Assays were performed at 30°C at the indicated pH values. The buffers (50 mM) used were: citrate–phosphate (pH 4.5–7.0), phosphate (pH 7.0–8.0), glycine–NaOH
(pH 8.0–10.0). The reaction medium with starch contained besides buffer, 10 mM NaCl. Incubations have been carried out for at least four differents periods of time and
the initial rates of hydrolysis calculated. All assays were performed under conditions such that activity was proportional to protein concentration and to time. A unit of
enzyme is defined as the amount that catalyses the cleavage of 1 mol of substrate (or bond) per minute. Abbreviations: BAPA, -N-benzoyl-DL-arginine;
CMC, carboxymethyl cellulose; LpNA, L-leucine-p-nitroanilide.
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THE SALIVARY GLAND OF BIOMPHALARIA STRAMINEA
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9
10
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9
20
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6
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8
9
30
1
2
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6
7
8
9
40
1
2
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4
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9
50
1
2
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5
6
7
8
9
60
1
2
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7
8
lumen and display well-developed microvilli and no cilia (Figs
2B, 3C). In general, these cells have a centrally located nucleus
with a large nucleolus. The cytoplasm is rich in elements of the
rough endoplasmic reticulum and shows many well-developed
Golgi areas (Fig. 3E). Numerous PAS-positive secretory vesicles,
larger then the ones seen in the previous phase, can also be
noted. Some vesicles are seen in the apical cytoplasm in process
not evident at this phase. Secretory cells in phase II show almost
the same morphological characteristics as the cells in the previous phase (Fig. 3B). Nevertheless, they are larger, with more
Golgi areas, showing small, PAS-positive secretory vesicles, scattered or clustered in certain regions of the cytoplasm. Cells in
phase III are still larger and more elongated than cells in phase
II. They have the apical surface in direct contact with the gland
Figure 1. A, B. Scanning electron micrographs of the salivary gland. A. External view showing the ducts inserted into the buccal mass and the secretory regions.
B. Internal view of the secretory region exhibiting abundant cilia (arrows). C–E. Histological sections of historesin-embedded tissues, fixed in Bouin’s (C, D) or
Zamboni (E) and stained with Hematoxylin-Eosin-Phloxyne (C, E) or Toluidine Blue (D). C. Duct, with abundant cilia projecting into the lumen.
D. Secretory region showing cells rich in secretory vesicles; note the presence of apical bulges (arrows) and a space between cells filled with cell debris (asterisk).
E. Detail of the secretory region showing the intercalary and the secretory cells and the underlying connective tissue (arrows). Abbreviations: BM, buccal mass;
Dt, duct; IC, intercalary cell; L, lumen; N, nucleus; SC, secretory cell; SR, secretory region. Scale bars: A 0.5 mm; B 10 m; C, D, E 25 m.
23
K. R. S. MOURA ET AL .
of elimination to the gland lumen, along with the plasma membrane and their limiting membrane. Free double membrane
vesicles, similar to the secretory vesicles, are frequently present
in the gland lumen (Fig. 4C). The cytoplasm is also rich in mitochondria, glycogen particles and free ribosomes. Occasionally,
organelles similar to lysosomes, with membranous electrondense contents, are observed. Gland cells in phase IV display the
cytoplasm filled with secretory vesicles (Fig. 3D), which are eliminated in clusters into the glandular lumen along with parts of
the apical cytoplasm, forming typical apical bulges (Fig. 4A). In
most secretory cells at this phase, a fusion of the secretory vesicles
is observed, particularly in the apical region of the cytoplasm.
The final stages of secretion occur during phase V, when the
entire cell contents are eliminated into the gland lumen (Fig.
4B). Epithelial spaces, filled with cell debris, are frequently
observed between intercalary cells (Fig. 4D).
Secretory cells in phases III and IV can be further subdivided
into L- and H-secretory cells, according to the ultrastructural
aspect of their secretory vesicles and rough endoplasmic reticulum. L-secretory cells possess secretion vesicles of types A and B
(Figs 3C–E, 4A, 5). The most common type-A vesicles are round,
with a granular content of low electron density; sometimes one
or more internal electron-dense spots are also noted. Type-B
vesicles are round, sometimes elongated, with homogeneous,
Figure 2. Transmission electron micrographs of the salivary gland. A. Duct cells; note the presence of both apical cilia (small arrows) and microvilli (large
arrows). B. H-secretory cells in phase III and intercalary cells in the gland secretory region; note the complex basal and intercellular spaces, formed by the undulating basolateral plasma membrane (asterisk). C. Apical portion of an intercalary cell showing the nucleus, the cilia (small arrows) and microvilli (large
arrows). D. Detail of the apical surface of an intercalary cell showing a cilium (small arrow) with a basal body and microvilli (large arrows). E. Detail of the intercellular junctions between intercalary cells exhibiting a desmosome and a septate junction. Abbreviations: BB, basal body; D, desmosome; HSC, H-secretory
cell; IC, intercalary cell; L, lumen; N, nucleus; SV, secretory vesicles; SJ, septate junction. Scale bars: A, C 1 m; B 10 m; D, E 0.5 m.
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low electron-dense contents. In L-secretory cells the cisternae of
the rough endoplasmic reticulum are dilated exhibiting a fine
granular content.
H-secretory cells show secretion vesicles of types C and D (Figs
2B, 3C, 4B, 5). The most frequently observed type-C vesicles
possess a large, usually round, electron-dense internal area surrounded by a circular, low density, homogeneous area, which
sometimes has a granular aspect. Type D vesicles have a high
electron density and homogeneous contents which occasionally
show a granular aspect. The rough endoplasmic reticulum
Figure 3. Transmission electron microscopic images of the secretory region. A. Secretory cell in phase I. Note a myoepithelial-like cell (arrows) under the basal
lamina. B. Secretory cell in phase II. C. H- and L-secretory cells in phase III. D. L-secretory cell in early phase IV. E. Detail of a Golgi complex in an L-secretory
cell in phase III; note the associated secretory vesicle. Abbreviations: BL, basal lamina; G, Golgi complex; HSC, H-secretory cell; L, lumen; LSC, L-secretory cell;
N, nucleus; SV, secretory vesicles. Scale bars: A, B, E 1 m; C, D 10 m.
25
K. R. S. MOURA ET AL .
cisternae of these cells are generally narrower than the ones
observed in L-cells.
Table 2. Enzymatic activities.
Enzyme
Biochemical assays
Each pair of salivary gland has 46 4 g of protein and displays
several hydrolases (Table 2). The carbohydrases (amylase, cellulase and maltase) are very active, aminopeptidase is less active
and trypsin is beyond detection by the methods used. Amylase
showed the highest activity among all tested enzymes. This
enzyme loses activity during storage in frozen state, a characteristic not observed with the other enzymes.
pH optimum
Activity: mU/an ± SEM (mU/mg ± SEM)
Amylase
7.0
16 ± 1 (350 ± 30)
Celulase
6.5
3.0 ± 0.3 (70 ± 9)
Maltase
5.0
0.179 ± 0.006 (3.9 ± 0.1)
Aminopeptidase
7.0
0.135 ± 0.005 (2.9 ± 0.1)
Trypsin
nd
Activities are expressed in milliunits per animal (mU/an) and specific activities
(in parenthesis) in milliunits per mg protein (mU/mg). Abbreviations: nd, not
detected; SEM, standard error of mean.
Figure 4. Transmission electron micrographs of secretory cells. A. Detail of the apical cytoplasm of an L-secretory cell in phase IV; note the secretory vesicles in
process of elimination into the gland lumen. B. Bulging of the apical cytoplasm with secretory vesicles of an H-secretory cell in early phase V. C. Free secretory
vesicles in gland lumen. D. An epithelial space (asterisk) in the secretory region limited by an intercalary cell, filled with cellular debris; note the presence of a
neighbouring secretory cell. Abbreviations: IC, intercalary cell; L, lumen; SC, secretory cell; SV, secretory vesicles. Scale bars 1 m.
26
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Figure 5. A. General diagrammatic representation of the secretory epithelium showing the two main cell types, the intercalary (IC) and the secretory cells in different phases of differentiation (SC-I to SC-V). The two secretory cell sub-types, L and H (LSC and HSC), are shown. B. The four main types (A–D) of secretory
vesicles present in secretory cells.
presence of muscle fibres surrounding the gland. Thus, in the
salivary glands of Helix aspersa (Moreno et al., 1982) and Eobania
vermiculata (Bani, Formigli & Cecchi, 1990), for instance, the
glandular epithelium is not ciliated but the gland is surrounded
by a conspicuous connective tissue rich in muscle fibres. In these
species, the propelling of saliva inside the gland lumen is probably
due to peristaltic contraction of the muscle fibres which surround the gland. In contrast, the salivary glands of Lymnaea stagnalis and Helisoma trivolvis show, as in B. straminea, a well-ciliated
epithelium along the gland with scarce muscle fibres in the
surrounding connective tissue. In these species, the main propulsive force should be synchronized beating of cilia.
An important ultrastructural feature in B. straminea is the
simultaneous presence of cilia and microvilli on the apical
surface of duct and intercalary cells, and the presence of welldeveloped microvilli in the secretory cells. The abundance of
microvilli along the entire gland suggests the existence of some
capacity of absorption by the epithelium (Fawcett, 1981; Weiss,
1983), possibly related to the final processing or concentration
of saliva before elimination from the gland. The presence of
dilated and complex intercellular spaces in the intermediary and
secretory gland regions, resembling the mammalian gall bladder
epithelium (Junqueira et al., 1992), may also be involved in water
absorption and concentration of secretion in the gland lumen.
The lateral membranes of adjacent gland cells display apical
belt desmosomes followed by septate junctions, similar to the
junctional specializations found in several tissues of other pulmonate species (Luchtel et al., 1997; Tomé & Ribeiro, 1998;
Bojat, Sauder & Haase, 2001). According to Willott, Balda,
Fanning, Jameson, Itallie & Anderson (1993) the septate junctions found in invertebrates should be involved in formation of
a selective permeability barrier across the epithelia resembling
DISCUSSION
The salivary gland of B. straminea is an important organ of the
digestive system, exhibiting an intense and complex secretory
activity which should perform a significant role in ingestion
and/or digestion of food. As compared to the salivary gland of
other related pulmonate species, it shows a relatively simple
morphological organization being very similar, as expected, to
the gland described in B. glabrata (Pan, 1958). In the two
Biomphalaria species, the glandular lobes do not form an acinar
structure with a complex ramified duct system as observed, for
instance, in salivary glands of Lymnaea stagnalis (Carriker &
Bistald, 1946; Boer et al., 1967), Helix aspersa (Moreno et al., 1982)
and Agriliomax reticulatus (Walker, 1970).
A remarkable morphological characteristic of the salivary
gland is the presence of abundant cilia along the entire gland,
associated with the apical surface of duct and intercalary cells.
Ciliated cells, similar to the intercalary cells of B. straminea, were
also described in the principal and interlobular ducts of the salivary gland of Lymnaea stagnalis but not in the region of the
glandular acini (Boer et al., 1967). The rich ciliated epithelium
found in B. straminea should be involved in mixing and propelling the salivary secretion (saliva) from the secretory region
of the gland to the buccal cavity. Although ciliary currents are
probably the main motor force inside the lumen, the sparse
occurence of cells rich in cytoplasmic microfilaments, similar to
the myoepithelial cells of vertebrates (Weiss, 1983; Junqueira,
Carneiro & Kelley, 1992), in the subjacent connective tissue,
may help in generating a further propelling force for the secretion through their synchronized contraction. It is interesting
to note that there seems to be an indirect relationship between
the occurrence of a well-developed ciliary epithelium and the
27
K. R. S. MOURA ET AL .
the function attributed to tight junctions of vertebrates (Ribeiro
& David-Ferreira, 1996).
The morphological data suggest that the salivary gland
secretion of B. straminea is mainly produced by the secretory
cells found in intermediary and secretory regions of the gland.
In fact, this cell type is very rich in cytoplasmic organelles related
to secretory activity, including a well-developed rough endoplasmic reticulum, many conspicuous Golgi areas and abundance
of different types of secretion vesicles (Kelly, 1985; Rothman &
Orci, 1992). These cells show a complex secretory activity along
their differentiation path, which can be followed from cells
showing undifferentiated characteristics and absence of visible
secretory vesicles (phase I) to cells exhibiting an intense production and elimination of secretion (phases III–V). According
to the morphology of the rough endoplasmic reticulum cisternae and the secretory vesicles, two secretory cell subtypes can be
defined, the L- and H-secretory cells. The L-cells are similar to
the mucous cells found in the salivary gland of the pulmonate
Eobania vermiculata and called mucocyte I by Bani et al. (1990),
and to the mucous cells described in the salivary gland of six
species belonging to the superfamily Helicoidea, named granular mucocytes by Serrano et al. (1996). On the other hand, the
H-cells of B. straminea are similar to the serous cells (granular
cell) found in the salivary gland of Eobania vermiculata (Bani
et al., 1990) and to the D-type serous cells present in glands of
Helix aspersa, Hygromia limbata, Cernuella aginnica and Cepaea
nemoralis (Serrano et al.,1996). Thus, the L- and H-secretory cells
seem to be equivalent to the mucous and serous cells, respectively,
described in salivary glands of other pulmonate species.
There are three main cellular mechanisms of secretion depending on the way the secretory products are released from cells
(Weiss, 1983; Junqueira et al., 1992). In the merocrine mechanism, the secretory vesicles leave the cell by exocytosis, involving
membrane fusion between the vesicle-limiting membrane and
the plasma membrane. In the process of apocrine secretion, the
secretory product is discharged along with parts of the apical
cytoplasm, whereas, in the holocrine mechanism, the product of
secretion is eliminated with the whole cell, involving destruction
of the secretion-filled cells. In the salivary gland of B. straminea
two mechanisms of secretion were detected in the secretory
cells. During phases III and IV, individual secretory vesicles are
discharged into the gland lumen along with their limiting membranes (phase III) or in clusters, including their limiting
membranes and parts of the apical cytoplasm (phase IV), through
a typical apocrine mechanism. In the final stages of the secretion (phase V), the entire secretory cell, filled with vesicles, is
discharged into the lumen, characterizing a holocrine process
of secretion. The final, holocrine secretion, was also described by
Pan (1958) in his histological studies in B. glabrata. The epithelial spaces with cell debris, frequently noted between intercalary
cells, probably represent the result of holocrine secretion. Thus,
these spaces form and disappear in the salivary epithelium due
to the processes of holocrine secretion and cell renewal through
differentiation of secretory cells in phase I. Secretory cells in
phase I are similar to the basophilic cells observed by Boer et al.
(1967) and also considered as precursors of all secretory cells
present in the salivary gland of Lymnaea stagnalis.
Despite the morphological diversity and complexity of prosobranch salivary glands very few species seems to secrete digestive
enzymes in their saliva; it usually contains only mucus and occasionally protein (Fretter & Graham, 1962; Voltzow, 1994). In
neogastropods, the salivary glands may serve to lubricate and
bind food particles and they may have a pharmacological or,
rarely, an enzymatic activity (Andrews, 1991). In Pulmonata, a
major postulated role of the salivary secretion is also the lubrication of food particles with mucus-containing fluid (Luchtel et al.,
1997); the presence of digestive enzymes is only described for a
few species (see below).
Among the enzymes detected in the salivary gland of B.
straminea, amylase showed the highest activity, followed by cellulase, maltase and aminopeptidase. Nevertheless, trypsin was not
detected. Although information concerning the presence of
digestive enzymes in the salivary glands of other molluscan
species is scarce, the detected activities are considered significant when compared, for example, with the enzymes found in
the salivary gland of Helix lucorum (Flari & LazaridouDimitriadou, 1996), and insects (Ferreira, Ribeiro & Terra,
1993). The occurrence of digestive enzymes, mainly amylase, is
also described in the salivary gland of other pulmonate species
such as Lymnaea stagnalis (Carriker & Bistald, 1946; Boer et al.,
1967), Agriolimax reticulatus (Walker, 1970), Helix lucorum (Flari
& Lazaridou-Dimitriadou, 1996) and Helix aspersa (Charrier &
Rouland, 1992). Thus, besides the probable function of salivary
secretion as an auxiliary means in the capture and ingestion of
food, the presence of significant activities of digestive enzymes
in salivary glands of B. straminea strongly suggests, as pointed out
by Luchtel et al. (1997) for other molluscan species, an important role of this secretion in the initial stages of food digestion.
ACKNOWLEDGMENTS
The authors are indebted to M. V. Cruz and W. Caldeira for efficient technical assistance with the morphological techniques
and to T. Rezende and A. Batista for their help with the biochemical assays. This work was supported by Brazilian Agencies
CNPq, FINEP and FAPESP. K.R.S.M. is a graduate fellow from
CAPES. W.R.T. and A.F.R. are staff members of their respective
departments and research fellows from CNPq.
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