DISTRIBUTION OF DIGESTIVE TUBULES AND FINE STRUCTURE
OF DIGESTIVE CELLS OF APLYSIA PUNCTATA (CUVIER, 1803)
NADIRA TAÏEB
Centre d’Etude des Ressources Animales Marines, Faculté des Sciences de St Jérôme, case 341, 13397 Marseille Cedex 20, France
(Received 16 November 1999; accepted 1 October 2000)
ABSTRACT
The distribution of digestive tubules of Aplysia punctata has been studied in animals under experimental
feeding conditions. Histological analysis of the digestive gland has revealed two types of tubules, called
tubules A and B. Tubules of type A were composed of basiphilic cells (calcium, excretory and thin cells)
and tubules of type B were lined by large digestive cells and basiphilic cells. The latter occur in small
groups, usually in the corners of the tubules. Type A tubules are involved in ion metabolism and show a
diphasic cycle (absorptive and reconstitutive) according to the height and the stage of calcium cells.
Type B tubules are involved in digestive processes and display a tetraphasic cycle (holding, absorption,
fragmentative and reconstitutive) depending upon the height and the stage of the digestive cells. The
tetraphasic cycle was compared with the four categories of tubules in bivalves. It is proposed that digestive processes may be continuous in digestive cells of A. punctata.
INTRODUCTION
MATERIAL AND METHODS
For molluscs, digestive processes have been mainly described in various species of bivalves (Platt, 1971; Langton, 1975; Mathers, 1976; Robinson & Langton, 1980;
Morton, 1983; Henry, 1987). While in gastropods, the
digestive gland has been the subject of numerous cytological studies (Taylor, 1968; Greene, 1969; Trench,
1969, Taylor, 1971; Runham, 1975; Graves, Gibson &
Bleakney, 1979; Greenwood & Mariscal, 1984; Griebel,
1993; Kress, Schmekel & Noo, 1994; Coehlo, Prince &
Nolen, 1998), the intracellular digestive processes, as reflected by morphological changes within the digestive
tubules, have not been investigated. In bivalves, digestive patterns are related to either the tidal cycle or food
availability. The digestive patterns have been differentiated by morphological features of the digestive cells,
which make up the tubules, as food is received and
digested intracellularly (Yonge, 1926; Morton, 1956;
McQuiston, 1969; Owen, 1970; Mathers, 1972). Digestive tubules have been classified into four types according to their intracellular digestive processes:absorptive,
disintegrative, reconstitutive and holding, which are
indicative of four phases in the dynamic processes of
intracellular digestion (Morton, 1973; Langton, 1975;
Robinson & Langton, 1980; Morton, 1983). Finally, it is
very likely that in bivalves, the morphological changes of
the digestive gland are correlated with food availability
(Langton & Gabott, 1974; Wilson & La Touche, 1978).
The aim of the present study was to investigate, using
light and electron microscopy techniques, if such foodrelated morphological changes also occur in the digestive gland of the herbivorous opisthobranch, Aplysia
punctata (Cuvier, 1803).
Aplysia punctata and Plocamium cartilagineum (red alga)
were collected from the Mediterranean littoral (Marseille,
France). Animals were fed ad libitum in the laboratory on P.
cartilagineum. Every 5 or 12 days, digestive glands were removed and the alimentary canal (gizzard, stomach, caecum
and intestine) of each animal checked for food material.
For routine light microscopy, sections of digestive glands
were fixed, cut and stained either with Heidenhain’s azan or
Masson’s trichrome, McManus periodic-schiff acid (PAS),
Mowry’s alcian blue, Perls Prussian blue; Von Kossa and
Schmorl methods were also used. For electron microscopy,
pieces of tissue were fixed for 1 h at 4°C in 2% glutaraldehyde,
and buffered at pH 7.4 with Sorensen’s buffer. Post-fixation
took place in 1% OsO4 in 0.1 M sodium phosphate buffer for
1–2 h at 4°C. The tissue was dehydrated through a series of
alcohols and embedded in Epon 812. Thin sections were cut
on an LKB ultra-microtome and mounted on copper grids.
They were stained with uranyl acetate and lead citrate, and
examined with a Philips 400T electron microscope.
The enzymatic profile was evaluated in the digestive glands
from five animals fed for 12 days on P. cartilagineum, using
the Apizym test. Measurements of glycerol hydrolase were
performed at 37°C in a pH-Stat apparatus at pH 6, 7 and 8,
using tributyrin as substrate. Hydrolase activity is expressed
in mole of released lipids per minute.
J. Moll. Stud. (2001), 67, 169–182
RESULTS
Histology
Whatever the period of feeding (5 or 12 days), the
digestive gland showed the same histological features.
It was composed of digestive tubules and highly devel© The Malacological Society of London 2001
NADIRA TAÏEB
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APLYSIA PUNCTATA DIGESTIVE TUBULES
Figure 1. A–E. Aplysia punctata digestive gland. A. The digestive gland consists of many digestive tubules (DT) and digestive ducts (DD),
bound together by connective tissue infiltrated by haemocytes (H). Digestive tubules are lined by calcium cells (arrow); digestive cell (double
arrows), excretory (Ec) and thin cells; Heidenhain’s azan. B. Tubule of type A1 (TA1) is lined by pyramidal basiphilic calcium cells of stage 1
(Cc1). These cells show brush borders (arrow) and delimited a reduced lumen. Haemocyte (H), calcium spherule (s); PAS. C. Tubule A1.
Reduced lumen (L); brush border (arrow); lipofuscin concretion (C); Masson’s trichrom. D. Semi-thin section of tubule A1 (TA1) stained with
azur blue. Tubule A1 is lined by basiphilic pyramidal cells and particularly by excretory cells represented by calcium cells of stage 4 (Cc4).
Granulofibrillar vacuole (GV); reduced lumen (L); brush border (arrow). E. Semi-thin section of tubule A2 (TA2) stained with azur blue. Short
basiphilic cells delimiting a large lumen (L) filled of spherules of fragmentation (SF), debris of cell membrane; calcium cell of stage 1 (Cc1);
calcium cell of stage 2 (Cc2); calcium cell of stage 2 (Cc2) showing apical granules (arrow); calcium cell of stage 3 (Cc3); calcium cell of stage 4
(Cc4). Scale bar 10 m for all figure parts.
oped collector ducts. Digestive tubules and ducts were
surrounded by a connective tissue infiltrated by muscle
fibres and haemocytes (Fig. 1A). Two categories of
tubules, called type A and type B were observed,
according to the characteristics of their cells. Type A
tubules (Fig. 1A–D) were characterized by the presence
of basiphilic cells (calcium, excretory and thin) (Taïeb
& Vicente, 1999), and type B tubules (Fig. 2A–C) by
the four cell types (calcium, excretory, thin, and large
digestive cells). In type B tubules, digestive cells were
numerous and close to each other, and basiphilic cells
formed a crypt localized in the tubule corners.
In all the animals studied, type A and B tubules displayed morphological variations. In type A (Fig. 1B–D),
we have distinguished four stages according to the features of the calcium cell. Stage 1 is characterized by the
presence of spherules, iron granules, crystals and concretions of calcium phosphate and lipofuscin. Some
spherules contained granulofibrillar material reacting
positively to the acid muco-polysaccharides tests; others
show internal concentric rings which stain black by the
Von Kossa method. The iron granules were seen at
the base of the cell but also extended to the apical part,
where they are accumulated and secreted into the lumen
by a merocrine process. Stage 2 is represented only by
the presence of concretions and granulofibrillar vacuoles containing amorphous material. Stage 3 is characterized exclusively by the presence of the voluminous
concretions. Stage 4 is represented by a voluminous
granulofibrillar vacuole, resulting from the fusion of
small vacuoles. Concerning the thin cells, the presence of
a small nucleus topped by vesicles with a fine granular
content that reacted negatively to iron and calcium tests,
was observed.
Type A tubules were classified into two groups
according to the height and the stage of their calcium
cells. Tubules A1 (Fig. 1B–D) contain three to 10 basiphilic cells. Some tubules contain exclusively calcium
cells at stage 1 (Fig. 1B and C), while others were lined
by calcium cells at various stages, and particularly at
stage 4 (Fig. 1D). All the cells of tubule A1 extended
into the lumen, and their surfaces delimited a reduced
lumen and possessed a well developed brush border. In
an advanced phase of ion absorption, the lumen was
often entirely occluded. Tubules A2 (Fig. 1E) were
characterized by short basiphilic cells with apical surfaces delimiting a large lumen. The lumen contained
numerous, fine, granular material that reacted positively to the iron test, and also fragmentation spheres
of cytoplasm with granulofibrillar material and/or
concretions of lipofuscin.
Type B tubules (Fig. 2A–C) were classified into four
groups, depending on the height and the stage of their
digestive cells. Tubules B1 were composed of high, columnar, digestive cells and pyramidal basiphilic cells,
which delimited a restricted lumen. Digestive cells contained cyanophilic vesicles, which reacted positively to
the AMPS (acid mucopolysaccharides) test and weakly
to iron tests. Tubule B2 (Fig. 2A and B) were characterized by highly vacuolated digestive cells containing
cyanophilic and/or erytrophilic vacuoles and crystals.
Vacuoles reacted positively to APMS, iron, calcium
and lipofuscin tests, and crystals to calcium and iron
tests. The apical cytoplasm of some digestive cells
appeared to be breaking off from the rest of the cell.
The size of the digestive cells varies, depending upon
the degree of fragmentation. Tubules B3 (Fig. 2C) were
characterized by a low epithelium and a large lumen
containing membrane debris, and spherules of fragmentation originating from digestive and crypt cells.
Pyramidal calcium and excretory cells could be easily
identified, while digestive cells lost their structure and
form. All the epithelial cells were at the same level and
their apical surfaces bear a distinct brush border.
Tubule B0 (Fig. 2C) had the appearance of tubule B3,
but the lumen was optically empty.
Cytochemistry
The extracts of digestive gland of A. punctata have
revealed various enzymatic activities. Apizym test reactions were positive except for -galactosidase, -glucosidase, -mannosidase and esterase (Fig. 3). In the case
of esterase, the lipolytic activity towards tributyrin is
shown in Fig. 4.
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NADIRA TAÏEB
Figure 2. A–C. Aplysia punctata digestive tubules of type B. A–B. Semi-thin section of a tubule B of type 2 (TB2) stained with azur blue. A. TB2
shows digestive cells (Dc) and crypt basiphilic cells (arrow) which delimit an irregular lumen (L); this tubule is characterized by digestive cells
whose apical cytoplasm appears to be broken off from the rest of the cell (short arrow); digestive vacuoles (v) weakly stained by azur blue
correspond to cyanophilic vacuoles; granules (g) stained highly with azur blue correspond to erytrophilic vacuoles containing iron, calcium and
lipofuscins; calcium cell (Cc); excretory cells (Ec). B. Digestive cell showing intracytoplasmic crystal (cr). The lumen (L) contains spherules of
fragmentation (SF), material, membrane debris and fine granulated material (arrow). C. Tubules B of type 3 (TB3) and of type 0 (TB0) are
characterized by short epithelial cells; TB3 shows a large lumen containing membrane debris (arrow), granules (double arrows) and spherules of
fragmentation (SF) stained with Masson’s trichrome; TB0 shows a lumen (L) optically empty of waste material. Scale bar 10m for all figure
parts.
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APLYSIA PUNCTATA DIGESTIVE TUBULES
Studied enzyme
Substrat
pH
Temperature
37c 20c
10c
Control
8
Alkaline phosphatase
2-naphtyl phosphate
Esterase (C 4)
2-naphtyl butyrate
–
–
–
Esterase (C 8)
2-naphtyl caprylate
–
–
–
Esterase (C 14)
2-naphtyl myristate
–
–
–
Leucine arylamidase
L-leucyl-2-naphtylamide
–
–
Valine arylamidase
L-valyl-2-naphtylamidase
–
–
Cystine arylamidase
L-cystyl-2-naphtylamide
–
–
Trypsine
N-benzoyl-DL-arginine-2-naphtylamide
–
Chymotrypsine
N-glutaryl-phénylalanine-2-naphtylamide
Acid phosphatase
2-naphty phosphate
Naphtol-AS-Bl-phosphohydrolase
Naphtol-AS-Bl-phosphate
–
galactosidase
6-Br-2-naphtyl- D-galactopyranoside
–
–
–
Galactosidase
2-naphtyl- D-galactopyranoside
Glucoronidase
Naphtol-AS-Bl- D- Glucoronide
Glucosidase
2-naphtyl- D- Glucopyranoside
Glucosidase
6-Br-2-naphtyl- D-Glucospyranoside
–
–
–
N-acétyl- b-Glucosaminidase
1-naphtyl-N-acétyl- D-mannopyranoside
–
–
–
mannosidase
6-Br-2-naphtyl- D-mannopyranoside
–
–
–
fucosidase
2-naphtyl- L-fucopyranoside
5
–
–
Figure 3. Detection of enzymatic activities in the A. punctata digestive gland (Apizym test).
Digestive cell ultrastructure
Digestive cells (Figs 5, 6A–E, 7A–F and 8A–C) were
linked apically to basiphilic cells (Fig. 6A) or to each
other (Fig. 6B) by desmosomes followed by a long septate junction. The free surface usually bore microvilli;
occasionally, microvilli were scarce or absent giving rise
to a straight or bulbous surface to the cells. The plasma
membrane at the base of the cell (Fig. 6C) showed little
infolding and interdigitation. The basal region contained numerous electron-transparent vacuoles which
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NADIRA TAÏEB
Activity
pH
(mmole/mn/g)
6
5
7
10
8
20
Figure 4. Tributyrylglycerol activity in the A. punctata digestive
gland (pH Stat).
extended toward the apical part of the cell. Mitochondria, free ribosomes, and elements of rough endoplasmic
reticulum (RER) were scattered throughout the cytoplasm. The nucleus (Fig. 6D), ellipsoid or round in shape,
had a round nucleolus surrounded by heterochromatin.
Numerous pores were observed on the nuclear envelope.
Two active Golgi complexes (Fig. 6E) were localized in
the supranuclear region of the cell and arranged more
or less concentrically to enclose an extensive cupshaped Golgi region. The content of both the saccules
and the peripheral vesicles was grey or dense. Secretory
vesicles might be fused and formed large dense granules. Coated vesicles were frequently seen. Numerous
small vesicles, dense tubules and multivesicular bodies
occured in the apical cytoplasm (Fig. 7A–B) and some
of these tubules surrounded areas of the cytoplasm.
Pinocytotic material was observed along the base of the
microvilli. The most characteristic feature of the digestive
cell was the presence of numerous membrane-bound
vacuoles. For descriptive purposes, it was convenient
to divide the vacuoles into three major categories. The
vacuoles of type Va (see above, Fig. 6C) were electron
transparent, round in shape and of various sizes. Vacuoles of type Vbn which corresponded to cyanophilic
vacuoles, occured in the subapical region of the cell;
they were relatively large and contained granulofibrillar material, dense crystallized needles and/or lipid-like
droplets and microfibrilles (Fig. 7B–C). Occasionally,
in the Golgi region, a smooth membrane-bound microvesicle appeared to be in the process of fusing with the
delimiting membrane vacuoles of type Vb (Fig. 7D). The
vacuoles of type Vc were found throughout the cytoplasm; they were dense and corresponded to erytrophilic vacuoles. Type Vc vacuoles could be divided into
three subgroups: type 1 (Vc1) (Fig. 5) contained moderately dense and amorphous material separated from
the limiting membrane by an electron-transparent space;
type 2 (Vc2) (Fig. 7D) were small and compact and represented by a dense granule which sometimes had been
torn out during sectioning; and type 3 (Vc3), were large
and composed of aggregates of dense granules generated
from the fusion of Vc2 vacuoles. Large spherules of
fragmentation containing vacuoles Vb and Vc were liberated in the lumen by an apocrine process (Fig. 8A and
B). Occasionally, some entire digestive cells were eliminated in the tubule lumen. In the old digestive cells, full
with residual bodies (Vc), the Golgi region occasionally
showed aggregations of straight tubules that were lined
by a finely granulated material of low electron density
(Fig. 8C). The granules of each tubule were much smaller than numerous ribosomes in close contact with them.
RER, Golgi apparatus and numerous vacuoles containing fine granular material of opaque density, were
present in the vicinity of the aggregated tubules. These
vacuoles of opaque density seemed to be secretory
granules, since small secretory vesicles, originating from
Golgi saccules, exhibited the same ultrastructure.
DISCUSSION
Cytochemistry
Aplysia enzymatic systems have been studied by Stone
(1957), Duffy & Duffy (1968), Elyakova, Shevcenco &
Avaeva (1981), Cho, Pyeum, Byum & Kim (1983), and
Onishi, Suzuki & Kikuchi (1985). According to Carefoot (1987), sea hares appear to digest starches and
simple sugars and show a lipase activity. Howells (1943)
reported that amylases are active in secretions from
salivary and digestive glands, and that the latter organ
secretes a number of enzymes that hydrolyse sucrose,
lactose, and maltose. The activities of acid and alkaline
phosphatases and proteases detected by the Apizym
test, and the esterase activity measured by the pH Stat
method, suggest that the Aplysia digestive gland is
involved in the process of the intracellular digestion.
Variability of tubules
Four major cell types (digestive, calcium, excretory
and thin) were described by Sumner (1965, 1966) in
molluscs. Similar types of cells were also presently
observed in A. punctata. Within gastropods, one type
of digestive tubule composed of the four cell types has
been described. Whereas in A. punctata we observed
two types of tubules (A and B) according to their cell
types, curiously, the topography of tubules A and B
was similar to that of some bivalve species. In the digestive gland of the protobranch Nucula (Owen, 1956), the
tubules are made up exclusively of secretory cells, and
in A. punctata tubules A were exclusively composed of
basiphilic cells at different stages (calcium, excretory
and thin). It has been suggested in a previous study
(Taïeb & Vicente, 1999) that calcium cells involved in
various functions (secretion, osmoregulation, ion de-
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APLYSIA PUNCTATA DIGESTIVE TUBULES
toxification and excretion) originated from young thin
cells, while excretory cells (called early, mature and postexcretory cells) represent different stages of degenerated
calcium cells. Early, mature and post-excretory cells
correspond to stages 2, 3 and 4 of calcium cells identified in this study, respectively. The digestive tubules of
many species of bivalve contain crypt cells (secreting
cells, undifferentiated and flagellated cells, and stem
cells) separating digestive cells. The type B tubules of
Aplysia showed the same topography with basiphilic
crypt cells. Digestive tubules of animals fed ad libitum
during 5 or 12 day periods showed a polyphasic cycle
Food material
Microvilli
Pinocytosis of food
material
Pinosome
Desmosome
Pinocytotic tubule
Vacuole “a”
Septate
junction
Vacuoloes “b”
Secretory vesicle
Golgi
apparatus
Gap junction
Golgi saccule
Secretory granule
Invagination
Vacuole “c1”
Vacuole “c2”
Vacuole “c3”
Rough endoplasmic
reticulum
Perinucleolar
heterochromatin
Euchromatin
Mitochondria
Ribosome
Intercellular space
Basal lamina
Muscle fibre
Connective
tissue
Figure 5. Diagram illustrating the fine structure of the A. punctata digestive cell.
175
NADIRA TAÏEB
Figure 6. Electron micrographs. A–E. A. punctata Digestive cell. A. Linkage between digestive and thin cells (DC, TC); the free surface of the
digestive cell is bulbous (short arrow); flagellum (fl); lumen (L); nucleus (N). B. Apical junctions between two digestive cells (DC); desmosome
(D); septate junction (sj). C. Basal region of a digestive cell; little infoldings (double short arrows); intercellular space (arrow); mitochondria
(m); rough endoplasmic reticulum (RER); basal lamina (BL); vacuole of type a (Va). D. Nucleus (N); nucleolus (n); numerous pores present in
the nuclear envelope (arrow); heterochromatin (He). E. Supranuclear Golgi apparatus (G); secretory vesicles (arrow); a coated vesicle (double
arrows); dense granule (DG) probably originating from the fusion of the secretory vesicles of different densities; nucleus (N). Scale bar 1 m
in all figure parts.
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APLYSIA PUNCTATA DIGESTIVE TUBULES
Figure 7. Electron micrographs. A–F. Aplysia punctata digestive cell. A–B. Apical region of a digestive cell. A. Vesicles (v) and concentric
dense tubules (dt); pinocytosis of food material (double arrows) at the base of microvilli (MV); lumen (L). B. Multivesicular bodies (mvb);
vacuole of type b (Vb) showing granulofibrillar material and highly dense needles of crystal (cr). C. Vacuole of type b (Vb) showing granulofibrillar material, microfibrils (FI) and droplet-like lipid (arrow). D. Fusion between a vacuole b (Vb) and a secretory vesicle (v); Golgi apparatus (G). E. Vacuole c of type 2 (Vc2) is represented by a highly dense and compact granule that was torn out during sectioning. F. A large
vacuole c of type 3 (Vc3), probably resulting from the fusion of vacuoles (Vc2), is composed by aggregates of dense granules (arrow). Scale
bar 1 m in all figure parts.
177
NADIRA TAÏEB
due to the morphological variations of calcium and
digestive cells. These variations result from changes
within the digestive and calcium cells of the digestive
gland as food was continuously received and digested
intracellularly.
Figure 8. Electron micrographs of an A. punctata digestive cell. A. A
spherule of fragmentation (SF) originated from apical part of a
digestive cell (Dc); lumen (L). B. Elimination of digestive cell in the
lumen; nucleus (N); vacuole b (Vb); vacuole c (Vc). C. Aggregates of
tubules (AT) in the vicinity of rough endoplasmic reticulum (RER);
mitochondria (m); Golgi apparatus (G); large vacuoles (V), which
probably result from the fusion of the small secretory vesicles (arrows);
a Golgi tubule (GT). Scale bar 1 m in all figure parts.
The results presented her indicate that tubules of
type A were not involved in intracellular digestive processes but were involved in ion metabolism. Tubules A
were classified into categories (tubules A1 and A2),
which might be compared to two of the four phases
described in digestive tubules of Bivalvia (absorptive,
disintegrating, reconstituting and holding phases) (Platt,
1971; Langton, 1975; Robinson & Langton, 1980).
Tubules A1 (absorptive phase) were represented by
basiphilic cells in the absorptive condition; they were
characterized by a reduced lumen, suggesting pinocytosis and mineral bioaccumulation phases. The bioaccumulation of calcium occurs in granulofibrillar
vacuoles, which evolve into calcium spherules or into
lipofuscin concretions (Taïeb & Vicente, 1999). Granulofibrillar vacuoles are also present in early, postexcretory and thin cells, suggesting that these cells
participate in ion absorption and that their vacuoles
constitute a reserve for ion storage. Tubules A2 (reconstituting phase) contained basiphilic short cells and
particularly post-excretory cells (stage 4 of calcium
cells). Calcium cells that accumulate proteinic and iron
granules in their apical cytoplasm (Taïeb & Vicente,
1999) secrete the fine granular material observed in the
lumen by a merocrine process. This secretory product
probably participates in ion regulation and extracellular digestion in different areas of the digestive tract.
Although the process of apical fragmentation of cells
was never observed, the presence in the lumen of spheres
containing granulofibrillar material and concretions
suggests that these spheres are excreted from the excretory cells (stage 2, 3 and 4 of calcium cells) through an
apocrine process. Tubules in a disintegrating phase
were not observed, probably because the secretion processes occur simultaneously for all basiphilic cells in a
short and a well defined period of the digestive cycle.
Tubules in the reconstituting phase reflected both a
massive cell elimination and an intense process of cell
regeneration. As long as animals are fed, the elimination of waste material from the tubule lumen occurs
immediately, and consequently, tubules in a holding
phase are never seen. When food appears in the individual clusters of tubules, the short basiphilic cells of
reconstituting tubules immediately increased in height
and absorbed ions (tubules A1). Later these cells fragmented and reconstituted (tubule A2) and another
cycle was reinitiated.
Whatever the duration of feeding, four aspects of type
B digestive tubules were observed in each digestive gland
of Aplysia, according to morphological variations, indicative of the state of intracellular digestion. Tubules
B1 showed pinocytosis and digestion-assimilation processes within the digestive cell. Tubules B2 contained
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APLYSIA PUNCTATA DIGESTIVE TUBULES
digestive cells that were characterized by the apocrine
secretion process. Fragmentation spherules, originating
from the nipped-off tips of digestive cells, may release
extracellular enzymes once they arrive in the stomach
(Owen, 1955, 1956; Palmer, 1979; Henry, 1987). In
addition to the process observed in tubules B2, tubule
B3 reflected the culmination of the breakdown process
and a massive regeneration of cells. Tubules B0 constituted the next stage of tubules B3 when their lumen had
been emptied by the muscular action of the walls of the
digestive tubule. At this stage, the lumen was in phase
to receive the predigested food material. Tubules of
type B showed a polyphasic digestive cycle depending
on the stage and height of digestive cells. The digestive
tubules displayed morphological variations, which reflect differences in the state of food material absorption
and intracellular digestion within the digestive cells
that make up the tubules. Thus, the height of digestive
cells of tubules B1 increased during the apocrine secretion (tubules B2) then decreased after the elimination
of spherules of fragmentation in the lumen (tubules
B3). Digestive cells reinitiated their growth at the end
of the type B0. There may exist different physiological
phases of a particular cell type. Pugh (1963) and Walker
(1970) attribute the apparent structural differences of
the cells to their various functional phases. Within
gastropods, phasic activity of digestive gland cells has
been described by some authors (Millot, 1937; Boghen
& Farley, 1974), and Morton (1955) identified successive phases of absorption, digestion and fragmentation
in the cycle of the digestive cell in the tubules of the pulmonate Leucophytia. The four aspects of tubules B we
found in Aplysia correspond to the four phases of the
digestive tubules described in bivalves, respectively:
holding (or normal), absorptive, disintegrating and
reconstituting. The presence of the four aspects of type
B tubules in the digestive gland of all animals studied
indicates that intracellular digestion occurs all the time.
The presence of food material in the alimentary canal
of Aplysia indicates that digestion processes were permanent. Robinson and Langton (1980) found the same
correlation between the heterogeneity of the digestive
gland of Mercenaria mercenaria and the presence of
food in the stomach and intestine.
The fine structure of the digestive cell
The A. punctata digestive tubules of type B were mainly
composed of digestive cells showing a typical vacuolar
system, composed of three groups of digestive vacuoles
(Va, Vb and Vc). The fine structure of these cells resembles that of cells with similar absorptive, aposecretion
and excretion functions in all gastropods and bivalves.
The presence of the membrane invaginations at the
apical surface of the digestive cell suggests a process of
pinocytosis of food particles. The small vesicles and the
short tubules occurring in the apical cytoplasm are
pinosomes, which may fuse and empty their content
into the vacuoles Va. These vacuoles correspond to the
P1 stage according to Owen (1970), who reported that
this stage constitutes a permanent reserve of exogenous
food material before digestive processes. Vacuoles Vb
and Vc correspond, respectively, to cyanophilic vacuoles and erytrophilic vacuoles observed using light
microscopy. Vacuoles Vb resemble the P2 stage (Owen,
1970), the heterolysosomes (Boucaud-Camou & Yin,
1980; Porteres & Tardy, 1995; Donval-Hilly, 1984), the
heterogenous vacuoles (Henry, 1987) and the green
granules (Sumner, 1966) described in other invertebrates. The needles of crystals present in the vacuoles
Vb contain a dense material, which is probably responsible for the weakly iron-positive reaction detected in
cyanophilic vacuoles. Kress et al. (1994) observed similar
crystalloid structures in cells containing microtubules
and suggested that the microtubule content, liberated
into the gland lumen, acts as a sort of glue to make the
faecal products. The presence of microtubules and
lipid-like droplets within digestive vacuoles (Vb) suggests the involvement of the processes of synthesis and
hydrolysis of proteins and lipids, as was reported by
Owen (1970) and Pal (1972), who observed similar
structures within bivalve digestive spheres. Fusion between secretory vesicles and digestive vacuoles (Vb)
clearly shows a transfer of enzymes from the cytoplasm
to the digestive vacuoles. The Golgi vesicles are primary
lysosomes containing hydrolytic enzymes (De Duve &
Wattiaux, 1966; Henry, 1987). Opisthobranchs have
been mainly studied due to their ability to retain chloroplasts within their digestive cells (Taylor, 1968; Greene,
1969; Trench, Boyle & Smith, 1973; Graves et al., 1979;
Griebel, 1993). Red algal chloroplasts (rhodoplasts)
have been found within large digestive vacuoles of the
rhodoplast digestive cells of Aplysia californica (Coehlo
et al., 1998). The digestive cells of A. punctata are characterized by an extensive digestive vacuole system, where
chloroplasts are not found. The process of phagocytosis
occuring in sacoglossans (McLean, 1976) is never seen
at the level of the apical membrane of A. punctata
digestive cells. Vacuoles Vc, which appear in three forms
(Vc1, Vc2 and Vc3), react strongly to iron and calcium
tests and contain dense granular material identified as
lipofuscin. They are comparable to the yellow granules
of gastropods, stage P3 (Owen, 1970) and to the residual
bodies of many bivalves. It is well known that in many
invertebrates, lysosomes are the site of bioaccumulation of heavy metals such as iron, zinc or copper in a
non-toxic form. According to Viarengo & Nott (1993),
179
NADIRA TAÏEB
this accumulating process represents a pathway of detoxification. Lysosomial catabolism occurs through different types of vacuoles, giving rise to a great variety of
structures (Kress et al., 1994). During lysosomial catabolism, the dense material accumulated in vacuoles Vb
undergoes a process of dehydratation, leading to the
formation of vacuoles Vc1, Vc2 and Vc3 (Fig. 5); the
two latter forms, compact in structure, correspond to
the final stage of catabolism. Vacuoles Vc may also
arise from cytoplasmic areas isolated by tubules of
pinocytosis and from multivesicular bodies that may
be involved during the process of endocytosis (Robbins
et al., 1964). The aggregations of straight tubules
observed in digestive cells filled with residual bodies
(vacuoles Vc) resemble the structures associated with
RER described in various molluscs (McLean, 1978;
Kessel & Beams, 1984; Roland-Cornejo, 1987). In most
cases, the presence of the tubules represents a pathological phenomenon. Abolins-Krogis (1970) observed
in the digestive and calcium cells of Helix pomatia an
arrangement of tubules that exhibit a peculiar hexagonal pattern. She suggested that this may be engaged in
the transport of lipids and calcium ions. The junction
between the straight tubules, Golgi apparatus and RER
suggests that the aggegation of tubules in Aplysia are
involved in protein secretion.
The present study of the digestive gland of A. punctata provides evidence that digestion in Aplysia is a
permanent process. This gastropod appears as an
opportunistic herbivore, which feeds and digests whenever it is exposed to food. Digestive cells of Aplysia
show characteristics of both gastropods (pulmonates)
and bivalves. During the digestive cycle, digestive and
calcium cells show morphological changes in relation
to ion metabolism and intracellular digestion, respectively. Finally, the digestive gland of Aplysia appears as
a major absorptive and secretory organ which may also
possess excretory and detoxifying functions.
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