A study of the cytoplasmic inclusions of various cells in the alimentary tract of molluscs, with special reference to changes during secretory and digestive processes

Cytological, histochemical and electron microscopical studies were made on the digestive gland cells of certain gastropods.
The digestive gland tubule epithelium of the pulmonate Helix aspersa is made up of four types of cells: thin, digestive, calcium and excretory cells. Thin cells are narrow and undifferentiated. They divide by mitosis and are believed to develop into the other types of cells. Digestive cells are highly vacuolated. The most apical, and smallest, vacuoles have no contents which are visible with the light microscope; those further down the cell contain colourless granules. The nucleus is basal, and immediately apical to it are a number of small yellow granules of lipofuscin. Most of the cytoplasm of calcium cells is occupied by spherules which contain calcium; these spherules can be discharged from the cells. The cytoplasm contains much RNA, and there is a conspicuous Golgi apparatus; apically there are protein granules which contain much tryptophan. Excretory cells consist chiefly of a large vacuole, surrounded by a little cytoplasm. The vacuole contains one or more granules of lipofuscin; these, and the lipofuscin granules of digestive cells, are cast out of the cells and appear in the faeces; they are thus excretory. Excretory cells are degenerate, and probably derived from calcium cells.
Three other species of gastropods were studied for comparison with Helix. These were Pila ovata, a tropical prosobranch; Succinea putris, a primitive stylommatophoran pulmonate; and Testacella maugei, a carnivorous stylommatophoran pulmonate. In spite of the differences in diet and taxonomic position, the tubule epithelia of the digestive glands of these three species show a fundamental similarity to that of Helix.
Pila has protein granules in its digestive cells, as well as the inclusions normally present in such cells. Calcium is absent from the cells which correspond to the calcium cells of Helix. The excretory cells contain large brown granules which consist partly of lipofuscin, and partly of an iron-containing pigment.
In the digestive cells of Succinea there is a gradation between the colourless and the yellow granules; all these granules lie in vacuoles. Many of the spherules in the calcium cells lack calcium.
Testacella has no yellow granules in its digestive cells; these cells have large quantities of lipid in the basal half of the cell. The protein granules of the calcium cells are more numerous than in the other forms studied. Testacella shows no differences from Helix which can be attributed to a carnivorous diet.
When examined in the electron microscope Helix, Succinea and Testacella show similar fine structure in their digestive gland cells. The structure is similar whether the fixative is osmium tetroxide or formaldehyde-sucrose. There is a good correlation between the results of light and electron microscopical studies.
The cytoplasm of thin cells contains ergastoplasm, and there is a typical lamellar-vacuolar field apical to the nucleus. The cytoplasm of digestive cells consists of a finely granular ground substance in which lie numerous small vesicles, particularly apically. These vesicles may be pinocytotic, and pinocytosis channels are sometimes visible epically. Further down the cell there are the large vacuoles visible with the light microscope. The lipofuscin granules in the more basal vacuoles have the same fine structure as mammalian lipofuscin granules. Digestive, calcium and excretory cells bear microvilli on their free surfaces.
The calcium cells contain much ergastoplasm and several lamellar-vacuolar fields, in between the calcium spherules. In Testacella secretory granules are visible apically in the cell.
Electron microscopy shows that there is an intermediate stage between calcium and excretory cells. The fine structure of these intermediate or early excretory cells and of the excretory cells shows clearly that they are degenerating calcium cells.
The digestive gland cells of the gastropods studied are connected together by septate desmosomes.
The results of these morphological studies show that digestive cells have the characteristics of phagocytic cells, and calcium cells have the characteristics of protein secretory cells.
Experiments were carried out to study the sites of phagocytosis and lipid absorption, and to follow the fates of ingested materials, in the alimentary system of Helix. Carmine particles and lipids are taken up by the digestive cells of the digestive gland. These substances are taken up by phagocytosis, and the apical vacuoles and the vacuoles containing colourless granules are phagocytosis vacuoles. Carmine eventually becomes incorporated into the small yellow granules of digestive cells, showing that they are derived from the colourless granules. Carmine and lipids are not taken up by any other cells of the digestive gland.
Lipids are also taken up by the epithelia of the digestive gland ducts, intestine and rectum. Phagocytosis does not occur in these sites.
Since the calcium cells of Helix have the morphological characters of protein secretory cells, experiments were carried out to determine whether the granules of these cells could be discharged into the luman of the gland. This was done by studying the effects of the drugs pilocarpine, atropine, adrenaline and 5-hydroxy-tryptamine on the protein granules of calcium cells. The effects of these drugs on the mucous cells of the alimentary system and on the intestinal secretory cells (see page vii) were also studied.
Pilocarpine causes discharge of mucus throughout the alimentary system, discharge of protein granules from calcium cells, and slight discharge of the granules of the intestinal secretory cells. Its effects are inhibited by atropine. Adrenaline produces similar effects to pilocarpine, but its action on the intestinal secretory granules is stronger. 5-hydroxy-tryptamine causes discharge of mucus and of protein granules, but not of intestinal secretory granules. It was concluded that the protein granules of calcium cells, and the granules of intestinal secretory cells (as well as mucus) are secretory products which are discharged into the lumen of the alimentary system.
For comparison with gastropods, cytological, histochemical and electron microscopical studies were made on the directive gland cells of certain lamellibranchs. The species studied by light microscopy were Anodonta anatina, Sphaerium corneum and Unio tumidus. The digestive gland tubule epithelium of these species consists mainly of two types of cells: digestive and basiphil cells. Digestive cells are highly vacuolated, and closely resemble the digestive cells of gastropods. Basiphil cells have a high concentration of RNA In the cytoplasm, and a conspicuous Golgi apparatus. Apically they have small granules of protein. These cells are believed to be secretory, and the apical granules to be the secretory product. Basiphil cells are the cells considered by previous workers to be undifferentiated. Sphaerium and possibly the other forms studied, have groups of cells bearing very long flagella, among the groups of basiphil cells at the blind end of the tubules.
Electron microscopical observations on the digestive gland cells of Anodonta confirm the results of light microscopy. The fine structure of digestive cells resembles very closely that of the digestive cells of pulmonates. The basiphil cells contain much ergastoplasm and have numerous lamellar-vacuolar fields. There are membrane-bounded bodies in the apical cytoplasm which represent the protein granules seen with the light microscope. The mitochondria in the digestive gland cells of Anodonta usually have few cristae and a matrix of low electron density. Towards the apical surface of the epithelium, the cells are joined together by septate desmosomes; basally there are considerable intercellular spaces into which amoebocytes can penetrate.
Since the functions of the digestive glands of molluscs include absorption and intracellular digestion of food, and formation of secretory products, histochemical tests were carried out on certain gastropods and lamellibranchs to localize certain enzymes involved in these processes. The enzymes studied were alkaline phosphatase, acid phosphatase, and thiamine pyrophosphatase.
Alkaline phosphatase was found to occur in the brush border and basement membrane of digestive inland cells. Acid phosphatase was found to occur mainly in association with the lipofuscin granules of the digestive cells, and occasionally in other sites; it has not been found with certainty associated with intracellular digestion. Thiamine pyrophosphatase has been found in the Golgi apparatus and plasma membranes of digestive gland cells, but the largest amounts were found in or near the phagocytic vacuoles of digestive cells in the carnivorous slug Testacella.
The intestinal epithelium of pulmonate gastropods is usually stated to consist of ciliated and mucous cells. A third type of cell, the intestinal secretory cell, has been found in the final segment of the intestine, immediately preceding the rectum. In this study, such cells have been found in many species of pulmonates. In the part of the intestinal epithelium where they occur, intestinal secretory cells are as numerous as ciliated cells, and mucous cells are scarce.
The intestinal secretory cells have much RNA in the cytoplasm, a conspicuous Golgi apparatus, and numerous secretory granules. The secretory granules consist largely of protein, with possibly a little phospholipid.
Alkaline phosphatase occurs in the free border of all the intestinal epithelial cells, and in the pigmented granules which are found in ciliated cells. Acid phosphatase occurs in the pigmented granules of the ciliated cells, and in the immature secratory granules of the intestinal secretory cells.