Camp.
Biochem.
Printedin Great
Physiol. Vol. 103A, Britain
No.
3,
pp. 45.5-460,
1992
030%9629/92 $5.00 + 0.00 0 1992 PcrgamonPressLtd
PHOSPHATASE ACTIVITY IN THE HEPATOPANCREAS HELIX A.
OF
ASPERSA
AL~NDROS
and D.
PORCEL*
Department of Cell Biology, University of Granada; *Microscopy Service, University of Granada, 18071 Granada, Spain. Tel.: (958) 22-8806. (Received 16 March 1992; accepted 15 April 1992)
Abstract-l. Phosphatase acid (PhA) activity in the digestive gland (hepatopancreas) of the common garden snail Helix aspersa has been investigated using cytochemical methods. 2. All the cells composing this gland show PhA activity, the distribution pattern differing according to
the cell type. 3. The digestive cells show the most widely dis~but~
reaction product (brush border, phagolysosomes, multivesicular bodies and autophagic vacuoles). 4. In the excretory cells this activity appears in large sacs, while in the calcium calls the reaction product is abundant in the calcium granules. 5. Cellular digestion processes performed by each of these cell types is discussed together with their role in the detoxification of heavy elements derived from the environment.
~RODU~ON
The digestive gland of pulmonate gastropods has been the subject of a number of investigations, which concentrate primarily on the study of cell types using both light microscopy and scanning electron microscopy. These studies deal mainly with the morphology and structure of these cells (Krigsman, 1925, 1928; McGee-Russell, 1955; Bani, 1962; David and Gotze, 1963; Sumner, 1965, 1966a,b, 1969; AbolinsKrogis, 1965, 1970; Gwen, 1970; Walker, 1970; Navascues et al., 1978, 1980; Abadla-Fen011 et ai., 1982; Almendros et al., 1988). Other studies have demonstrated the presence of diverse enzymes, principally acid hydrolases, using either biochemical methods (Reid, 1968; Jarrige and Henry, 1952; Billet, 1954; Leon et al., 1960; Orzel, 1967; Bielawski and Kesa, 1986; Assaka et af., 1987) or cytochemical methods (Ellet and McGee-Russell, 1955; Rosenbaum and Ditzion, 1963; Reid, 1966, 1968; Bowen, 1968a,b, 1970a,b, Bowen and Davies, 1971; Bowen and Lloyd, 1971; Rowen et al., 1988; Ireland, 1986). The digestive gland of pulmonate gastropods serves as a useful experimental model for the study of digestive processes since its cells take part in a wide range of extra- and intra-cellular digestive processes involving a great variety of lithic bodies. The majority of the enzymes used by these animals for digestion are secreted by the cells produced in the digestive gland. Some are used in the cells of the hepatopanc~atic adenomere itself for intracellular digestion, while others flow into the stomach via an acidic, viscous liquid (Yonge, 1926). A knowledge of the position and distribution of the enzymes of this gland is important in order to understand the physiology of the digestive process. The object of our research has been to determine the distribution of phosphatase acid (PhA), a lysosomic enzyme marker, in the different cell types of the hepatopancreas of the snail Helix asperm. To do this
we have studied its ~ochemica1 ~st~bution, which has enabled us to establish the degree to which this gland takes part in intracellular and/or extracellular digestion. We found phosphatase acid activity to be associated with several different structures in the cells that make up the hepatopancreas. Our distribution pattern for this activity coincides with the model given by Bowen and Davies (1971) for the slug Aritln hortensis. MATRRIAlS
AND METHODS
Six adult Helix aspersa snails were collected in the field and kept in a suitable environment in the laboratory where we controlled their diet, photoperiod and level of humidity. We anesthetized the snails with a 10% solution of magnesium chlorate (Runham et al., 1965), removed the hepatopancreas, and obtained thin slices with a vibratome. The fixer solution was ~utar~dehyde (0.5%) and parafo~aldehyde (2%) in PBS buffer, at pH 7.4. Fine slices were incubated for enxymate development according to Rarka and Anderson’s method (1962). They were then fixed with a 2% osmium tetroxide PBS buffer, which was dehydrated in increasing concentrations of acetone and embedded in low-density resin (Spurr, 1969). Material incubated without substrate was used as a control. Medium-fine cuts were stained with blue toluidine
to locate suitable sections for examination. Unstained ultr&ne slices were examined with a Zeiss 902 electron microscope. RESULTS Phosphatase acid activity is associated with lysosomes. Practically all of the cells in the digestive gland of pulmonate gastropods are involved in digestive processes, whether of extracellular material (heterophagia) or intracellular material (autophagia). A great variety of lithic bodies, widely distributed in the different cell types of the h~atopancre~, are involved in these digestive processes.
455
A. ALMENDROS and D. PORCEL
456 Digestive
ceils
PhA activity is quite marked in the brush border of these cells (Figs 1, 2 and 3). The reaction product appears as a fine precipitate extending unevenly the whole length of the brush border. The numerous apical vesicles are small (0.2545 pm) and lightcoloured (see arrows in Fig. 2) and show no PhA activity, as is also the case for the large, ovoid, light-coloured sacs. Below the apical zone the digestive cells have a great variety of different-sized sacs with varied contents. Small sacs of approximately 1 pm have a homogenous grey content, while the contents of medium-sized sacs and large ones of more than 5 pm are lumpy and uneven. All of these lithic sacs contain electrodense reaction products, which vary in size and distribution depending on the type of sac. In the small sacs, presumably primary lysosomes (Fig. l), the reaction product appears as a fine, irregularly distributed precipitate. In larger-sized sacs (secondary lysosomes) the electrodense reaction product is distributed in two ways: as a precipitate in a centralized cluster in medium-sized sacs and as various precipi-
tates of uniform size distributed throughout the interior in large sacs (Fig. 1). In summary, the location of FhA-activity reaction product within different parts of the cell appears to confirm that these cells participate in both intra- and extra-cellular digestive processes, although primarily the latter. We have also found small amounts of PhA activity in endoplasmic reticulum cisterns, but not in the Golgi apparatus.
The location of the reaction product in this ceil type is very specific. It appears in the form of large electrodense stains inside the central vacuole that occupies almost all of the cytoplasm. There is no PhA activity in the few remaining organellae nor in the apical brush border. Calcium cells
The dist~bution of the enzyme in these cells is completely restricted to the calcium granules. The reaction product is localized in the membranes consti-
Fig. 1. General view showing an electron-dense reaction product in some digestive vesicles (arrow), 2178x .
Phosphatase activity in Helix aspersa
Fig. 2. PM activity in the apical membrane and brush border of the digestive cell (arrow). Negative reaction in apical vesicles and clear sacs, 13,860 x
tutmg the ring growths and, quite evidently, in the centre of the calcium granules (Fig. 4). The calcium granules that appear to be empty show no ring growths because the salts in them resisted slicing and slipped out whole during the cutting process, leaving an empty-shell structure. Therefore they only show signs of reaction product in the external membrane. The small sacs, described above as primary lysosomes, also contain reaction product. The control snails show a negative cytochemical reaction for PhA in the cells of the digestive gland.
DISCUSSION
Our results show that phosphatase acid activity takes place in all the cell types that constitute the hepatopancreas of the snail Helix aspersa. It is known that this type of hydrolithic activity is characteristic of lysosomes (Novikoff et al., 1971). De Table 1. The distribution of PhA activity in the different organellae and cell types of the hepatopancreas of the snail Cell types Brush border Apical vesicles Lysosomes Lytic vesicles Endoplasmic reticulum Golgi Calcium granules
Digestive
Excretory
+++ ---
--
+++ +++
++ +++
+i-
-+I-
Calcium
+++ _ +++
Strong reaction (+ + +); medium reaction (+ +); moderate reaction (+/-); negative activity (-).
Duve and Wattiaux (1966) defined lysosomes cytochemically as being characterized by a surrounding membrane and showing a positive reaction to phosphatase acid. Fawcett (1986) further specify that, apart from being bounded by a membrane, lysosomes contain acid hydrolases and are extremely varied in appearance. From a functional point of view, it is known that digestion in molluscs is both extra- and intra-cellular, the latter having evolved more recently (Sumner, 1969). The distribution of PhA in the digestive cells revealed by our experimental technique coincides with the distribution described by Bowen and Davies (1971) for Arion hortensis. The activity we found that the brush border is similar to that reported by Ugolev (1960) for the intestine of vertebrates. This indicates that the enzyme is located in the plasmatic membrane of the brush border which, given the excretory tubules of this gland, leads us to suspect that the enzyme is involved in extracellular digestive processes (as Bielawski suggests for Helix pomatia). The majority of the vacuolar formations, which are very abundant in this type of cell, seem to play a role in lysosome activity. In our study we have found enzyme activity to be distributed mainly in these vacuolar structures. These results confirm that the digestive cell is responsible for the majority of both intra- and extra-cellular digestive processes in the hepatopancreas of these molluscs. Phosphatase acid activity would be quite important in hibernating snails since it must play a key role in
4.58
A. ALMENUROS and D. PORCEL
Fig. 3. Detail of electron-dense precipitate in brush border, 23,760 x
Fig. 4. Calcium granule, showing an intense PhA reaction in its core and growth rings, 39,600 x
Phosphatase activity in Helix aspersa autophagic processes. Thus, the images obtained in this study of active snails mainly depict heterophagosomes; we found no autophagosomes. The excretory cells show PhA activity in the large cytoplasmic vacuoles. The presence of the electrodense precipitate may be due to the aggregation of numerous digestive vesicles. If this is the case, it would point to the conclusion that this cell type is derived from a digestive cell which has undergone an extreme intensification of lithic digestive processes. This explanation is given by a number of authors who have carried out structural studies (Fretter, 1952; Billet and McGee-Russell, 1955; Sumner, 1965, 1966a,b, 1969; Navascues et al., 1978; and Almendros et d., 1988), and contrasts with the explanation given by Thiele (1953) who considers them to derive from the calcium cells. (Calcium cells have marked PhA activity, which is associated with the calcium granules. The precipitate apoears both in the growth rings and in the centre of thr cells. These results agree with those found for other molluscs such as Arion (Sumner, 1969). We have concluded that PhA is widely distributed in ,.he different cell types forming the hepatopancreas of the snail, but that it is found especially in the digestive cells. It is least evident in the excretory cells and is found only at one specific site in the calcium cells. All this leads us to the conclusion that the digestive cells play the major role in processes of storage and digestion. The excretory cells would thus be a final stage in the cycle of the digestive cell, responsible for expelling digestive residue by apocrima or holocrinia. Finally, the calcium cell would seem to be a different type of stem cell, involved primarily in processes related with the metabolism of calcium and cell detoxification (Howard et al., 1981).
REFERENCES Ab;idia Fenoll F., Rois A., Almendros A. and Navascues J. ( 1982) An electron microscopic study on the muscle cells
and nerve fibres in the digestive gland of the snail ~iyptotnphailus aspersa. 2. L&zig. 96, 873-884. Abolins-Krogis A. (1965) On the
Mikrosk,
Anat.
Forsch
protein stabilizing substance in the isolated g-granules and in the regenerating membranes on the shell of Helix pomatia. Arkiu. Zool. 15,475.
AbcJlins-Krogis A. (1970) Electron microscope observations on calcium cells in the hepatopancreas of the snail Helix pomatia 1. Arkiv. Fiir. Ziolo&
18, 85-91.
Almendros A., Rios A.. Bueno J. D.. Parcel D. and Abadia-Molina F. (1988) Ultrastructure of cells from the adenomera of the digestive gland of Helix aspersa. M. Inst. Phys. Conf Ser. 93, 155-156. Assaka L., Marchand C. R. and Strosser M. T. (1987) Detection of a somatostatin-14 peptide in the hepatopancress of the snail Helix aspersa. C. R. Seances Sot. Biol. Fil. 181, 187-193.
Bani G. (1962) Struttura e uhrastruttura dell’epatopancreas di Chginulus borellianus (colosi). Mod.
Zool. Ital. 69, 157.
Barka T. and Anderson P. J. (1962) Histochemical methods for acid phosphatase using hexazonium oararosanilin as coupler. 3. Histochem. Cyjochem. 10, 741-753. Bielawski J. and Kesa H. (1986) Seasonal variation of phosphatase activity in the hepatopancreas of the snail Helix pomatia. Comp. Biochem. Physiol. 83A, 105-108.
Billet F. (1954) The /I-glucuronidase
of the Roman snail
(Helix pomatia). Biochem. J. 51, 159.
459
Billet F. and McGee-Russell S. M. (1955) /I-Glucuronidase in the digestive gland of the Roman snail (Helix pomatia). Q. JI Microsc. Sci. %, 35-48.
Bowen I. D. (1968a) Electron cytochemical localization of acid phosphatase activity in the digestive caeca of the desert locus. J. R. Microsc. Sot. 88. 279-289. Bowen I. D. (1968b) Electron cytochemical studies on autophagy in the gut epithelial cells of the locust, Schistocerca gregaria. J. Histochem. 1, 141-151.
Bowen I. D. (1970a) The fine structural localization of acid phosphatase in the gut epithelial cells of the slug, Arion ater. Protoplasma 70, 247-260.
Bowen I. D. (1970b) Golgi associated acid phosphatase in muscle and nerve from Arion ater 1. Protoplasma 71, 409-417.
Bowen I. D. and Davies P. (1971) The fine structural distribution of acid phosphatase in the digestive gland of Arion hortensis. Protoplasma 73, 73-8 1.
Bowen I. D. and Lloyd D. C. (1971) Technique for electron cytochemical localization of acid hydrolases. J. Microsc. 94, 71.
Bowen I. D., Worril N. A., Winters C. A. and Mullarkey K. (1988) The use of back-scattered electron imaging, Xray microanalysis and X-ray microscopy in demonstrating physiological cell death. Scanning Microsc. 2, 1453-1462.
David H. and Gotze J. (1963) Elektronenmikroskopische befunde an der mitteldarmdriise von Schnecken. Z. Mikr. Anal. Forsch. 70, 252. de Duve C. and Wattiaux R. (1966) Functions of lysosomes. A. Rev. Physiol. 28, 435492.
Fawcett D. W. (1986) A textbook of Histology, Saunders, PA. Fretter V. (1952) Experiments with P32 and 1131 on species of Helix, Arion, and Agrolimax. Q. JI Microsc. Sci. 93, 135.
Howard B., Mitchell P. C. and Ritchie A. (1981) Composition of intra cellular granules from the metal accumulating cells of the common garden snail Helix aspersa. Biochem. J. 194, 507-512. Ireland M. P. (1986) Effects of normal healing on zinc distribution and alkaline phosphatase activity of Helix aspersa. J. Mall. Stud. 52, 169-173. Jarrige P. and Henry R. (1952) Etude de I’activite glucuronidasique du sue digestif d’Helix pomatia (L) et de sa phenolsulfatase. Bull. Sot. Chim. Biol. 34, 872. Krijgsman B. J. (1925) Arbeitsrhythmus der der verdaunngsdriisen bei Helix pomafia. 1. Die natiirlichen bedingungen. Zeit. Vergl. Physiol. 2, 264. Krijgsman B. J. (1928) Arbeitsrhythmus der verdaunngsdriisen bei Helix pomatia. 2. Sekretion, resorption und phagocytose. Zeit. Vergl. Physiol. 8, 187. Leon Y. A., Bulbrook R. D. and Corner E. D. S. (1960) Steroid sulphatase, arylsulfatase and /?-glucuronidase in the mollusca. J. Biochem. 75, 612617. McGee-Russell S. M. (1955) A cytological study of tissues concerned in the secretion of shell in the snail, Helix pomatia. Ph.D. thesis. Oxford. Navascues J., Almendros A., Aijon J. and Abadia-Fenoll F. (1978) Estudio a microscopia electronica de las formaciones granulares de1 hepatopancreas de Helix hortensis. Morfologia Normal y Parologica. A(2), 401412.
Navascues J., Almendros A., Abadia-Fen011 F. and Valverde F. (1980) Estudio de las mitocondrias de1 hepatopancreas de Helix hortesis. Bol. R. Sot. Espariola Hist. Nat. 78, 169-178.
Novikoff P. M., Novikoff A. B., Quintana N. and Haun J. (1971) Golgi apparatus, GERL and lysosomes of neurons in rat dorsal ganglia, studied by thick section and thin section cytochemistry. J. Cell Biol. 50, 859-886. Orzel R. (1967) Arylsulfatase and p-glucuronidase activity in the various species of tropical molluscs. Can. J. Zool. 45, 134135.
460
A. ALMENDROS and D. PORCEL
Owen G. (1970) The fine structure of the digestive tubules of the marine bivalve, Caridium edule. Phil. Trans. R. Sot. (B)258, 245. Reid R. G. B. (1966) Digestive tract enzymes in the bivalves Lima hians and Mya arenaria. Comp. Biochem. Physiol. 17, 417. Reid R. G. B. (1968) The distribution of digestive tract enzymes in lamellibranchiate bivalves. Comp. Biochem. Physiol. 24, 727-744. Rosenbaum R. M. and Ditzion B. (1963) Enzymatic histochemistry of granular components in digestive gland cells of the Roman snail Helix pomatia. Biol. Bull. 124, 211. Runham N. W., Isarankura K. and Smith B. J. (1965) Methods for narcotizing and anaesthetizing gastropods. Malacologia. 2, 23 l-238. Spurr A. R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 3143. Sumner A. T. (1965) The cytology and histochemistry of the digestive gland cells of Helix. J. Microsc. Sci. 106, 173-192.
Sumner A. T. (1966a) The fine structure of digestive-gland cells of Helix, Succinea and Testacelia. J. R. Microsc. Sot. 85, 181-192. Sumner A. T. (1966b) The fine structure of the digestive gland cells of Anodonta. J. R. Microsc. Sot. 417423. Sumner A. T. (1969) The distribution of some hydrolytic enzymes in the cells of the digestive gland of certain lamellibranchs and gastropods. J. Zool. Lond. 158, 277-291. Thiele G. (1953) Vergleichende untersuchungen iiber den feinbau und die frunktion der mitteldarmdrtise einheimischer gastropoden. Z. Zellforsch. 38, 87. Ugolev A. M. (1960) Parietal contact digestion. Bull. exp. Biol. Med. 49, 12-17. Walker G. (1970) The cytology, histochemistry and ultrastructure of the cell types found in the digestive gland of the slug, Agriolimax reticulatus. Protoplasma 71, 91-109. Yonge C. M. (1926) Structure and physiology of the organs of feeding and digestion in Ostrea edulis. J. mar. Biol. Ass. U.K. 14, 295.
Biochem.
Printedin Great
Physiol. Vol. 103A, Britain
No.
3,
pp. 45.5-460,
1992
030%9629/92 $5.00 + 0.00 0 1992 PcrgamonPressLtd
PHOSPHATASE ACTIVITY IN THE HEPATOPANCREAS HELIX A.
OF
ASPERSA
AL~NDROS
and D.
PORCEL*
Department of Cell Biology, University of Granada; *Microscopy Service, University of Granada, 18071 Granada, Spain. Tel.: (958) 22-8806. (Received 16 March 1992; accepted 15 April 1992)
Abstract-l. Phosphatase acid (PhA) activity in the digestive gland (hepatopancreas) of the common garden snail Helix aspersa has been investigated using cytochemical methods. 2. All the cells composing this gland show PhA activity, the distribution pattern differing according to
the cell type. 3. The digestive cells show the most widely dis~but~
reaction product (brush border, phagolysosomes, multivesicular bodies and autophagic vacuoles). 4. In the excretory cells this activity appears in large sacs, while in the calcium calls the reaction product is abundant in the calcium granules. 5. Cellular digestion processes performed by each of these cell types is discussed together with their role in the detoxification of heavy elements derived from the environment.
~RODU~ON
The digestive gland of pulmonate gastropods has been the subject of a number of investigations, which concentrate primarily on the study of cell types using both light microscopy and scanning electron microscopy. These studies deal mainly with the morphology and structure of these cells (Krigsman, 1925, 1928; McGee-Russell, 1955; Bani, 1962; David and Gotze, 1963; Sumner, 1965, 1966a,b, 1969; AbolinsKrogis, 1965, 1970; Gwen, 1970; Walker, 1970; Navascues et al., 1978, 1980; Abadla-Fen011 et ai., 1982; Almendros et al., 1988). Other studies have demonstrated the presence of diverse enzymes, principally acid hydrolases, using either biochemical methods (Reid, 1968; Jarrige and Henry, 1952; Billet, 1954; Leon et al., 1960; Orzel, 1967; Bielawski and Kesa, 1986; Assaka et af., 1987) or cytochemical methods (Ellet and McGee-Russell, 1955; Rosenbaum and Ditzion, 1963; Reid, 1966, 1968; Bowen, 1968a,b, 1970a,b, Bowen and Davies, 1971; Bowen and Lloyd, 1971; Rowen et al., 1988; Ireland, 1986). The digestive gland of pulmonate gastropods serves as a useful experimental model for the study of digestive processes since its cells take part in a wide range of extra- and intra-cellular digestive processes involving a great variety of lithic bodies. The majority of the enzymes used by these animals for digestion are secreted by the cells produced in the digestive gland. Some are used in the cells of the hepatopanc~atic adenomere itself for intracellular digestion, while others flow into the stomach via an acidic, viscous liquid (Yonge, 1926). A knowledge of the position and distribution of the enzymes of this gland is important in order to understand the physiology of the digestive process. The object of our research has been to determine the distribution of phosphatase acid (PhA), a lysosomic enzyme marker, in the different cell types of the hepatopancreas of the snail Helix asperm. To do this
we have studied its ~ochemica1 ~st~bution, which has enabled us to establish the degree to which this gland takes part in intracellular and/or extracellular digestion. We found phosphatase acid activity to be associated with several different structures in the cells that make up the hepatopancreas. Our distribution pattern for this activity coincides with the model given by Bowen and Davies (1971) for the slug Aritln hortensis. MATRRIAlS
AND METHODS
Six adult Helix aspersa snails were collected in the field and kept in a suitable environment in the laboratory where we controlled their diet, photoperiod and level of humidity. We anesthetized the snails with a 10% solution of magnesium chlorate (Runham et al., 1965), removed the hepatopancreas, and obtained thin slices with a vibratome. The fixer solution was ~utar~dehyde (0.5%) and parafo~aldehyde (2%) in PBS buffer, at pH 7.4. Fine slices were incubated for enxymate development according to Rarka and Anderson’s method (1962). They were then fixed with a 2% osmium tetroxide PBS buffer, which was dehydrated in increasing concentrations of acetone and embedded in low-density resin (Spurr, 1969). Material incubated without substrate was used as a control. Medium-fine cuts were stained with blue toluidine
to locate suitable sections for examination. Unstained ultr&ne slices were examined with a Zeiss 902 electron microscope. RESULTS Phosphatase acid activity is associated with lysosomes. Practically all of the cells in the digestive gland of pulmonate gastropods are involved in digestive processes, whether of extracellular material (heterophagia) or intracellular material (autophagia). A great variety of lithic bodies, widely distributed in the different cell types of the h~atopancre~, are involved in these digestive processes.
455
A. ALMENDROS and D. PORCEL
456 Digestive
ceils
PhA activity is quite marked in the brush border of these cells (Figs 1, 2 and 3). The reaction product appears as a fine precipitate extending unevenly the whole length of the brush border. The numerous apical vesicles are small (0.2545 pm) and lightcoloured (see arrows in Fig. 2) and show no PhA activity, as is also the case for the large, ovoid, light-coloured sacs. Below the apical zone the digestive cells have a great variety of different-sized sacs with varied contents. Small sacs of approximately 1 pm have a homogenous grey content, while the contents of medium-sized sacs and large ones of more than 5 pm are lumpy and uneven. All of these lithic sacs contain electrodense reaction products, which vary in size and distribution depending on the type of sac. In the small sacs, presumably primary lysosomes (Fig. l), the reaction product appears as a fine, irregularly distributed precipitate. In larger-sized sacs (secondary lysosomes) the electrodense reaction product is distributed in two ways: as a precipitate in a centralized cluster in medium-sized sacs and as various precipi-
tates of uniform size distributed throughout the interior in large sacs (Fig. 1). In summary, the location of FhA-activity reaction product within different parts of the cell appears to confirm that these cells participate in both intra- and extra-cellular digestive processes, although primarily the latter. We have also found small amounts of PhA activity in endoplasmic reticulum cisterns, but not in the Golgi apparatus.
The location of the reaction product in this ceil type is very specific. It appears in the form of large electrodense stains inside the central vacuole that occupies almost all of the cytoplasm. There is no PhA activity in the few remaining organellae nor in the apical brush border. Calcium cells
The dist~bution of the enzyme in these cells is completely restricted to the calcium granules. The reaction product is localized in the membranes consti-
Fig. 1. General view showing an electron-dense reaction product in some digestive vesicles (arrow), 2178x .
Phosphatase activity in Helix aspersa
Fig. 2. PM activity in the apical membrane and brush border of the digestive cell (arrow). Negative reaction in apical vesicles and clear sacs, 13,860 x
tutmg the ring growths and, quite evidently, in the centre of the calcium granules (Fig. 4). The calcium granules that appear to be empty show no ring growths because the salts in them resisted slicing and slipped out whole during the cutting process, leaving an empty-shell structure. Therefore they only show signs of reaction product in the external membrane. The small sacs, described above as primary lysosomes, also contain reaction product. The control snails show a negative cytochemical reaction for PhA in the cells of the digestive gland.
DISCUSSION
Our results show that phosphatase acid activity takes place in all the cell types that constitute the hepatopancreas of the snail Helix aspersa. It is known that this type of hydrolithic activity is characteristic of lysosomes (Novikoff et al., 1971). De Table 1. The distribution of PhA activity in the different organellae and cell types of the hepatopancreas of the snail Cell types Brush border Apical vesicles Lysosomes Lytic vesicles Endoplasmic reticulum Golgi Calcium granules
Digestive
Excretory
+++ ---
--
+++ +++
++ +++
+i-
-+I-
Calcium
+++ _ +++
Strong reaction (+ + +); medium reaction (+ +); moderate reaction (+/-); negative activity (-).
Duve and Wattiaux (1966) defined lysosomes cytochemically as being characterized by a surrounding membrane and showing a positive reaction to phosphatase acid. Fawcett (1986) further specify that, apart from being bounded by a membrane, lysosomes contain acid hydrolases and are extremely varied in appearance. From a functional point of view, it is known that digestion in molluscs is both extra- and intra-cellular, the latter having evolved more recently (Sumner, 1969). The distribution of PhA in the digestive cells revealed by our experimental technique coincides with the distribution described by Bowen and Davies (1971) for Arion hortensis. The activity we found that the brush border is similar to that reported by Ugolev (1960) for the intestine of vertebrates. This indicates that the enzyme is located in the plasmatic membrane of the brush border which, given the excretory tubules of this gland, leads us to suspect that the enzyme is involved in extracellular digestive processes (as Bielawski suggests for Helix pomatia). The majority of the vacuolar formations, which are very abundant in this type of cell, seem to play a role in lysosome activity. In our study we have found enzyme activity to be distributed mainly in these vacuolar structures. These results confirm that the digestive cell is responsible for the majority of both intra- and extra-cellular digestive processes in the hepatopancreas of these molluscs. Phosphatase acid activity would be quite important in hibernating snails since it must play a key role in
4.58
A. ALMENUROS and D. PORCEL
Fig. 3. Detail of electron-dense precipitate in brush border, 23,760 x
Fig. 4. Calcium granule, showing an intense PhA reaction in its core and growth rings, 39,600 x
Phosphatase activity in Helix aspersa autophagic processes. Thus, the images obtained in this study of active snails mainly depict heterophagosomes; we found no autophagosomes. The excretory cells show PhA activity in the large cytoplasmic vacuoles. The presence of the electrodense precipitate may be due to the aggregation of numerous digestive vesicles. If this is the case, it would point to the conclusion that this cell type is derived from a digestive cell which has undergone an extreme intensification of lithic digestive processes. This explanation is given by a number of authors who have carried out structural studies (Fretter, 1952; Billet and McGee-Russell, 1955; Sumner, 1965, 1966a,b, 1969; Navascues et al., 1978; and Almendros et d., 1988), and contrasts with the explanation given by Thiele (1953) who considers them to derive from the calcium cells. (Calcium cells have marked PhA activity, which is associated with the calcium granules. The precipitate apoears both in the growth rings and in the centre of thr cells. These results agree with those found for other molluscs such as Arion (Sumner, 1969). We have concluded that PhA is widely distributed in ,.he different cell types forming the hepatopancreas of the snail, but that it is found especially in the digestive cells. It is least evident in the excretory cells and is found only at one specific site in the calcium cells. All this leads us to the conclusion that the digestive cells play the major role in processes of storage and digestion. The excretory cells would thus be a final stage in the cycle of the digestive cell, responsible for expelling digestive residue by apocrima or holocrinia. Finally, the calcium cell would seem to be a different type of stem cell, involved primarily in processes related with the metabolism of calcium and cell detoxification (Howard et al., 1981).
REFERENCES Ab;idia Fenoll F., Rois A., Almendros A. and Navascues J. ( 1982) An electron microscopic study on the muscle cells
and nerve fibres in the digestive gland of the snail ~iyptotnphailus aspersa. 2. L&zig. 96, 873-884. Abolins-Krogis A. (1965) On the
Mikrosk,
Anat.
Forsch
protein stabilizing substance in the isolated g-granules and in the regenerating membranes on the shell of Helix pomatia. Arkiu. Zool. 15,475.
AbcJlins-Krogis A. (1970) Electron microscope observations on calcium cells in the hepatopancreas of the snail Helix pomatia 1. Arkiv. Fiir. Ziolo&
18, 85-91.
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