The cytology of the digestive and salivary glands of the limpet, Patella ByD. PUGH (From the Biochemistry Department, Institute of Orthopaedics, Stanmore, Middlesex) Summary As reported by earlier investigators, the epithelium of the digestive tubules is com- posed of two cell-types. One type of cell is glandular, the other type is absorptive and digestive, and to a lesser extent secretory. The latter type of cell also contains glycogen and numerous lipid globules, so that the digestive gland as a whole contains a large quantity of reserve food material. The epithelium of the digestive duct possesses a single cell-type; the cells are ciliated and heavily pigmented, and they produce a viscous secretion. The salivary gland is a compound tubular gland. The cells elaborate a secretion containing protein and probably some carbohydrate. Introduction MANY naturalists have included a general description of the limpet in their studies. The shell and radula have been thoroughly investigated, and several works have given accounts of the limpet's internal morphology and histology. The visceral hump of the limpet is brownish green. The pigments respon- sible for this coloration were thoroughly investigated by MacMunn (1886 a, b; 1889) and Newbigin (1898). MacMunn examined the colour of the pigments in frozen sections by low-power microscopy, and conducted spectrophoto- graphic analyses of alcoholic or alcoholic ether solutions. In his earlier paper he concluded that the green pigment was enterochlorophyll, and was elabor- ated by the animal from plant chlorophyll in the food. He also demonstrated that the pigment granules did not contain starch or cellulose, and so did not represent food or symbiotic algae. In a later paper he stated that the pigment was chlorophyll, although the solution was more stable than a normal solution of chlorophyll. Newbigin examined sections of material fixed in formaldehyde solution. She described the pigment granules of the gut cells and the 'typical molluscan vesicles' of the digestive gland cells. She examined alcoholic solutions of digestive glands and faeces and demonstrated enterochlorophyll spectro- photographically. The digestive gland extract turned green on addition of acid, and the spectrum was altered. Unlike chlorophyll the original spectrum could be restored by the addition of alkali, and the process repeated several times. Both investigators recorded that the green solutions of enterochloro- phyll displayed a red fluorescence. Newbigin also found a variable amount of a lipochrome pigment associated with the enterochlorophyll, and MacMunn remarked on the solubility of enterochlorphyll in lipids. The salivary glands were first mentioned by Lankester (1867). Griffiths [Quart. J. micr. Sci., Vol. 104, pt. 1, pp. 23-37, 1963.]
Transcript
The cytology of the digestive and salivary glands ofthe limpet, Patella
ByD. PUGH(From the Biochemistry Department, Institute of Orthopaedics, Stanmore, Middlesex)
SummaryAs reported by earlier investigators, the epithelium of the digestive tubules is com-posed of two cell-types. One type of cell is glandular, the other type is absorptive anddigestive, and to a lesser extent secretory. The latter type of cell also contains glycogenand numerous lipid globules, so that the digestive gland as a whole contains a largequantity of reserve food material. The epithelium of the digestive duct possessesa single cell-type; the cells are ciliated and heavily pigmented, and they produce aviscous secretion.
The salivary gland is a compound tubular gland. The cells elaborate a secretioncontaining protein and probably some carbohydrate.
IntroductionM A N Y naturalists have included a general description of the limpet in theirstudies. The shell and radula have been thoroughly investigated, and severalworks have given accounts of the limpet's internal morphology and histology.
The visceral hump of the limpet is brownish green. The pigments respon-sible for this coloration were thoroughly investigated by MacMunn (1886 a, b;1889) and Newbigin (1898). MacMunn examined the colour of the pigmentsin frozen sections by low-power microscopy, and conducted spectrophoto-graphic analyses of alcoholic or alcoholic ether solutions. In his earlier paperhe concluded that the green pigment was enterochlorophyll, and was elabor-ated by the animal from plant chlorophyll in the food. He also demonstratedthat the pigment granules did not contain starch or cellulose, and so did notrepresent food or symbiotic algae. In a later paper he stated that the pigmentwas chlorophyll, although the solution was more stable than a normal solutionof chlorophyll.
Newbigin examined sections of material fixed in formaldehyde solution.She described the pigment granules of the gut cells and the 'typical molluscanvesicles' of the digestive gland cells. She examined alcoholic solutions ofdigestive glands and faeces and demonstrated enterochlorophyll spectro-photographically. The digestive gland extract turned green on addition ofacid, and the spectrum was altered. Unlike chlorophyll the original spectrumcould be restored by the addition of alkali, and the process repeated severaltimes. Both investigators recorded that the green solutions of enterochloro-phyll displayed a red fluorescence. Newbigin also found a variable amount ofa lipochrome pigment associated with the enterochlorophyll, and MacMunnremarked on the solubility of enterochlorphyll in lipids.
The salivary glands were first mentioned by Lankester (1867). Griffiths[Quart. J. micr. Sci., Vol. 104, pt. 1, pp. 23-37, 1963.]
24 Pugh—Digestive glands of Patella
(1888) gave an account of their position and morphology. In 1885 Gibsonwrote the first comprehensive account of the morphology and anatomy of thelimpet. He stated that the cells of the digestive gland tubules were packedwith 'biliary secretion', and mentioned the salivary gland as being a compoundtubular gland. The tubules were lined with cuboid epithelial cells filled withyellow granules.
Davis and Fleure published a monograph on the limpet in 1903. Then in1932 Graham published a detailed account of the morphology and histologyof the alimentary canal. He showed that the gut epithelium had a differentappearance in different regions of the gut. The paper included a short de-scription of the salivary gland, a more detailed account of the digestive gland,and the results of feeding experiments were recorded.
Rees (1934) wrote a paper on the biology of Cecaria patellae, which in-cluded a description of the digestive gland of normal and parasitized limpets.
The following account is a report of further investigations on the digestiveand salivary glands of the normal limpet.
Material and methodsLimpets [Patella vulgata) were obtained from the Dove Marine Laboratory,
Cullercoats. They were dispatched by train, in a tin with seaweed, receivedthe following day, and stored for a week at 40 C. The limpets ingest smallpieces of stone along with fragments of the algae on which they browse, andthis period of starvation helps to eliminate the gritty material from theanimal's gut. The digestive gland closely surrounds the coils of the intestine,and part of the gut is always sectioned with the digestive gland, so that thepresence of stony material in the lumen of the gut hinders section cutting.
Blocks of digestive gland were cut from the posterior dorsal portion of thevisceral hump. The block included part of the superficial gut coil (fig. 4 inDavis and Fleure, 1903). The salivary glands were removed whole. In sometypes of fixation the entire visceral hump was fixed and dissected afterwards,because the tissues of the limpet are soft and friable; but when fixatives con-taining osmium were used, the visceral hump was dissected before fixation.Of many fixatives investigated it was found that Zenker was the best for thedigestive gland; Susa gave closely similar results. Cytoplasmic inclusions werewell preserved by mixtures containing potassium dichromate, such as Sanfe-lice's and Regaud's fluids. Flemming-without-acetic was the best fixative forthe salivary gland. This and Zenker were the only fixatives that preserved themasses of globules visible in fresh preparations. Generally a strongly acidfixative preserved more of the tissues than a neutral or weakly acid fixative.
Unless otherwise stated, tissue blocks were dehydrated and embedded inparaffin after fixation. Tissues fixed in formaldehyde solution, with and with-out postchroming, were embedded in gelatin (Pearse, i960) for the colorationof lipids and certain other histochemical procedures.
Sections from paraffin blocks were usually cut at 10 /u; 5 /x and 2-5 )u sectionswere used for special methods. Formaldehyde material embedded in gelatine
Pugh—Digestive glands of Patella 25
was sectioned at 15 /x. The sections were soaked individually in 30% ethanoland then floated on water, where they flattened immediately (Holt, 1958).They were mounted on clean, untreated slides. The sections were mountedbefore staining because of the fragility of the material.
Sections from each block were stained as a routine with haematoxylin andeosin, iron haematoxylin, and toluidine blue. Mitochondria were demon-strated by the Kull and Altmann stains. Methyl green / pyronin staining,and Feulgen and plasmal reactions, were used on suitably fixed sections.
Various histochemical methods were used in an attempt to ascertain thenature of the cells' contents, particularly the massed granules of one type ofdigestive gland cell, here called cell type A. Sections were stained by theperiodic acid / Schiff method (PAS), buffered methylene blue, and mucicar-mine to demonstrate compounds containing carbohydrates.
Lipids were stained with Sudan black, Sudan III and IV, Nile blue, oil red,and Baker's acid haematein. The tetrazonium and Millon's reactions wereused to stain protein. Frozen sections were examined for /?-glucosaminidaseand jS-glucuronidase, with AS-LC naphthyl glycosides in a simultaneous-coupling azo-dye technique (Pugh and Walker, i960).
Fresh material was examined in teased preparations and squashes.
ObservationsThe digestive gland
As stated by several authors (Gibson, 1885; Davies and Fleure, 1903;Graham, 1932), the digestive gland occupies the greater part of the visceralhump, closely investing the coils of the intestine. The gland is composed ofa mass of tubules, which are not arranged in definite lobes. The tubules arelined by a single layer of cells surrounded by a thin layer of connective tissue.The epithelial cells of the tubules are of two types, here designated cell typesA and B. Cell type A is more numerous than cell type B; the latter cells fre-quently occur in groups of two or three. The ducts of the digestive glandempty into two main trunks which fuse to give a single duct opening into thestomach. The columnar epithelium lining the ducts is composed of a singlecell-type.
Fresh preparations. Small pieces of digestive gland were examined fresh,teased in saline or in the fluid lying between the shell and the mantle of thelimpet. Cell type A is full of globules, usually lightly tinted green; some mayappear colourless, but the majority are coloured. A large apical granule isbrownish green and appears to consist of a mass of smaller granules. Newbigin(1898) found than an alcoholic solution of digestive gland gave a red fluores-cence in ultraviolet light. The globules of cell type A examined fresh withultraviolet light show a bright red fluorescence; the apical granule appearsdark red, almost brown. The cells are often disrupted, and rounded cellfragments consisting of either a nucleus or apical granule and a fragment ofcytoplasm, occur frequently. Whole cells detached from the basement mem-brane have a tendency to assume a spherical form, and after being removed
26 Pugh—Digestive glands of Patella
from the animal for several minutes, any remaining intact cells will haveassumed this shape. By phase-contrast microscopy the globules of cell type Aappear refractile and the nucleus may be observed. The apical granule can beseen to consist of an aggregate of small granules in each of which the pigmentis concentrated at the periphery. Granules which are possibly mitochondriacan be seen either in or on the apical granule, and in the cytoplasm of the cellfragments. Cilia have not been observed in these gland cells. Cell type Bdoes not contain any of the globules which fill the type A cells. By phase-contrast microscopy the cell may be seen to be packed full of mitochondria,and the nucleus appears as a large refractile body. The cell has a tendencyto round up, but does not fragment as easily as does cell type A. The cells ofthe digestive ducts are ciliated. Each cell possesses four cilia, two of which arelonger than the other pair. These cells have the upper half of the cell filledwith bright green pigmented granules.
Stained preparations: Cell type A. Cells of type A are narrow columnarcells from 18 to 30 [j. in height and from 4 to 5 ju. in breadth, in fixed prepara-tions. The lateral membranes of the cells are well preserved after Zenkerfixation, but difficult to distinguish after some types of fixations, such asformaldehyde. The tips of the cells may be flattened, rounded, or apparentlyin process of being separated from the cell. In the cells with flattened tipsthe apical layer of cytoplasm is finely granular, and often appears verticallystriated; this layer of cytoplasm is denser than that of the remainder of thecell. Distended cell tips show a vacuolated region with an outer rim of densecytoplasm (fig. 1). All gradations between these two extremes occur.
Immediately below this layer, in all cells of this type, there lies a brownish-green Iobed granule (which will be referred to as the apical granule) withina vacuole. This granule is 3 to 4 /u, in diameter. It is removed by fixation inalcohol, or by fixation in formalin and subsequent dehydration. It is wellpreserved by fixatives containing dichromate.
The greater part of the cell is filled with granules of varying size. AfterZenker fixation followed by haematoxylin and eosin staining, the granulesstain pink; usually the largest granules are the palest and the staining intensityincreases with reduction in granule size. Comparison with a formaldehyde-fixed gelatin-embedded section, or a Flemming-without-acetic preparation,shows that there are in addition small granules containing lipid among theother granules. These are particularly numerous at the base of the majorityof the cells and they occur throughout the cytoplasm of a few cells. Mitochon-dria may be stained by iron haematoxylin, Altmann, or Kull stains afterZenker or osmium fixation. They appear as small ovoid bodies scatteredthroughout the cytoplasm between and often clustered around the granules,and a few lie over the apical granule.
The nucleus is situated near the base of the cell. It is 2-5 to 3 ju, in diameter,and contains a central nucleolus and some scattered chromatin, both of whichare Feulgen-positive. The nucleus stains purple with methyl green/pyronin.After Mallory's triple stain the apical cytoplasm stains red, while the remain-
Pugh—Digestive glands of Patella 27
der of the cytoplasm and the apical granule are unstained. The mitochondriastain red, and the cytoplasmic granules blue.
Cell type B. Cells of type B are fewer in number than those of type A.They are triangular. The cell base varies from 7 to 10 /x and the cell tapers toa narrow neck where it reaches the lumen. The cells are from 15 to 20 ̂ i inheight.
secretion droplet
apical granule
cell type A
cell type 8
protein granule
retractile sphere
lipid globule
mitochondria
— nucleus
nucleus
FIG. I . Cells of digestive gland; composite drawing.
In fixed and stained preparations the cytoplasm is foamy and basiphil.After formaldehyde fixation the cytoplasm stains homogeneously, but afterZenker, Susa, or osmium fixation, refractile spheres may be distinguished(fig. i). These spheres stain an intense red with Altmann's acid fuchsin, andKull's triple method; by the latter method the surrounding cytoplasm stainsblue.
The mitochondria of cell type B are more numerous than those of celltype A. They are scattered thickly throughout the cytoplasm. Because of theintensely basiphil staining of the cytoplasm the mitochondria are difficult todistinguish after iron haematoxylin, but can be demonstrated by Altmann'smethod, and by Sudan black staining of material fixed in Zenker and embeddedin paraffin.
The nucleus (4 /x in diameter) is larger than that of cell type A, and has acentral nucleolus 1 to 1-5 /u in diameter. The nucleolus, nuclear membrane,and chromatin granules are strongly Feulgen-positive; the nuclear sap and thebasiphil cytoplasm are weakly positive. Staining with methyl green / pyronincolours the nucleus purple. The cytoplasm stains purple except for the celltip, which has a reddish, vacuolated appearance; the refractile spheres stain inshades of blue, grey, or pink. After Mallory's triple stain the mitochondriastain red, the cytoplasm deep orange, and the refractile spheres pale orange.
28 Pugh—Digestive glands of Patella
secret/on droplet
pigment granule
Digestive duct cells. The digestive ducts are lined by narrow, ciliated,columnar epithelial cells. The walls of the two main ducts are slightly ridged.The epithelium is composed of a single layer of cells; the increased widthof the ridges is caused by the greater length of some individual cells. Thesecells are 3 \x in width and range from 16 to 35 p in height. The cells of the
smaller ducts are of uniform size,measuring 3/A in width and 21 /n inheight, with only slight variations.Apart from the variations in height,all the duct cells are essentially similar(fig- 2).
The cilia may be observed in freshpreparations. They are rarely pre-served in fixed preparations, but thematerial lying outside the cell tips hasa striated appearance as if its positionwas determined by ciliary action. Thisextracellular material consists of debrisand of clear globules, some of whichappear to be in process of being ex-truded from the epithelium (fig. 2).
The apical layer of cytoplasm isgranular and stains deeply with eosin.Below this is a layer of small pigmentgranules, which usually fill about half
mitochondrion
Epithelium of digestive duct; com-posite drawing.
of the cell. These granules appearbright green when examined fresh, and olive green after fixation.
The nucleus lies below the pigment granules in the basal third of the cell.The cells of the main duct have oval nuclei measuring 3 to 3 -5 /n, their longerdiameter lying parallel with the long axes of the cells. Transversely, theyoccupy the width of the cell. The nuclei of the small duct cells are round,3 fj, in diameter, and also take up the entire cell width. All the nuclei have asmall central nucleolus, clear sap, and several chromatin granules. The nucleiare Feulgen-positive. The mitochondria are small and scattered throughoutthe cell. They are rather more numerous in the pigment layer and lie close tothe lateral cell membranes. The nucleus stains purple with methyl green /pyronin. After Mallory's triple stain the apical cytoplasm and mitochondriastain red, the nucleus blue, and the basal cytoplasm orange.
Lipid. Lipid has been detected by examining unstained osmium-fixedsections and formalin-fixed, gelatin-embedded material. 10% aqueous formal-dehyde does not preserve the digestive gland cells very well, so the effect of theaddition of buffers or salts, with and without postchroming, was tried (table 1in appendix, p. 37). Postchroming material fixed with formaldehyde calcium,or adding sea-water to this solution improves the fixation, without altering thestaining properties of the cells.
Pugh—Digestive glands of Patella 29
In frozen sections the small lipid droplets of cell type A colour with Sudanblack in 70% ethanol. These droplets were removed during the dehydrationof blocks fixed in any fluid which did not contain osmium. Sudan blackcolouring of Zenker-fixed paraffin sections coloured black only the mito-chondria and small granules lying on the apical granule (of similar size tomitochondria). Sections fixed by Flemming-without-acetic were treated asdescribed by Wigglesworth (1957); the mitochondria and smaller fat granulescould then be seen more distinctly. Cell type B does not contain any fatgranules; the cell is unstained in frozen sections treated with Sudan black. Informalin-fixed frozen sections and Zenker-fixed paraffin sections the pigmentgranules of the digestive duct cells were coloured black with an alcoholicsolution of Sudan black. These granules were also blackened by osmium.
The picture obtained by colouring with Sudan III, Sudan IV, and oil red(Pearse, i960) is similar to that obtained with Sudan black. Several methods(Cain, 1950) have been used in an attempt to discover the nature of the lipids(table 2). From the results obtained it seems possible that the fat granules ofcell type A contain a simple lipid which may be unsaturated and that theapical granule contains enterochlorophyll. The granule of the duct cellsappear to contain enterochlorophyll and a lipid, which is probably a mixtureof phospholipid and lipochrome.
Compounds containing carbohydrate. Sections protected by collodion weretreated by the PAS method, either without prior treatment or after digestionwith either diastase (saliva) or hyaluronidase (Benger Ltd., Holmes Chapel,Cheshire). In untreated sections the granules and cytoplasm of cell type Astained intensely, so that the granules were indistinguishable at low magni-fications. Cell type B stained moderately. The apical cytoplasm of the ductcells stained deeply and the remaining cytoplasm lightly; the pigmentgranules were unstained.
Treatment with diastase reduced the amount of colour developed in thecytoplasm of cell type A with PAS. The granules were unaffected by diastase.A reduction of PAS-positive material occurred in the cytoplasm of cell type B.Hyaluronidase digestion removed a greater quantity of PAS-positive materialthan did treatment with diastase. In some parts of a tubule hyaluronidase-labile material was removed from the whole of cell type A; in other partsfrom the distal region only. Material was removed from both granules andcytoplasm. Cell type B stained rather less intensely after hyaluronidase treat-ment than cells of this type in a control section. Treatment with diastase orhyaluronidase slightly reduced the amount of colour developed with PAS inthe duct cells. These results suggested the presence of glycogen and amucoid substance; further tests were made for both compounds.
Mucoid substances. As reported by Graham, it was found that the granulesof cell type A give a negative reaction with mucicarmine. The cell type Agranules stained lightly and the connective tissue deeply with alcian blue.No other part of the digestive gland stained with this dye.
Toluidine blue stained the granules of cell type A red purple, and the
30 Pugh—Digestive glands of Patella
whole of cell type B deep purple in aqueous mounts. This metachromasiawas partially removed by dehydration. The apical cytoplasm of the duct cellappeared pink and the remainder of the cell purple in aqueous mounts; afterdehydration the former appeared purple and the latter blue.
A methylene blue extinction curve was obtained from Zenker-fixedsections at pH 2-4 to 8-o (Pearse, i960). At pH 5-2 and higher the digestivetubules stain an intense blue-black. Below this pH the staining becomes lessintense. At pH 4-0 the cytoplasm of cell type A fails to bind methylene blueand the granules bind the dye weakly. Cell type B binds methylene bluestrongly at pH 4-0 and moderately at pH 2-4. At pH 3-6 and below the apicalcytoplasm of the duct cells was unstained; the basal cytoplasm stainedlightly at pH 2-4. The cells stained deeply at pH 6-o and above. The pigmentgranules failed to bind methylene blue at any pH value.
A water homogenate of digestive gland tested for hexuronic acid by thecarbazole method (Dische and Borenfreund, 1951) gave a negative result.A similar homogenate examined chromatographically (Leaback and Walker,1957) gave a positive reaction for hexosamine.
Glycogen. Attempts to confirm the presence of glycogen by Best's carminemethod have been inconclusive. The cytoplasm of cell type B stained deeplywith the carmine solution and also with the haematoxylin counterstain; thebase of cell type A stained lightly. Iodine stained material in both these sites,but not in the digestive duct cells.
Glycosidases. Cell type B gave a negative result for /J-glucosaminidase,while cell type A showed a high activity. The cytoplasm between the granuleswas deeply, and the apical region of the cell very deeply stained. The ductcells stained lightly and diffusely. Cell type A showed a very high /S-glucuro-nidase activity. There was some variation between cells, but usually the dyewas deposited throughout the cytoplasm, increasing in quantity towards thetip, which stained intensely. Cell type B stained deeply and diffusely. Theapical cytoplasm of the duct cells was deeply, and the remainder of the cyto-plasm lightly stained. The pigment granules were unstained. The additionof the principal salts of sea-water to the incubation mixture markedly increasedthe /S-glucosaminidase activity in sections. Homogenates of digestive glandassayed for this enzyme in the presence of artificial sea-water (Sverdrup,Johnson, and Flemming, 1942) showed an enzyme activity 5 to 10 times thatof the control. Additions of sodium chloride to mammalian tissue homo-genates have been shown to cause a slight increase in enzyme activity (Pugh,Leaback, and Walker 1957). Such additions of salt have no obvious effect onthe activity of j8-glucuronidase.
Protein. In sections stained by the tetrazonium method the nucleus, cellmembrane, and cytoplasm were moderately stained, and the apical granuleand cytoplasm deeply stained. Most of the cytoplasmic granules staineddeeply, the larger granules stained more heavily than the small granules(lipid globules were not preserved in these preparations). The cytoplasmand nucleus of cell type B stained deeply; the refractile spheres were
Pugh—Digestive glands of Patella 31
moderately stained. Sections treated with Millon's reagent stained similarly,but less intensely than those stained by the tetrazonium method.
The salivary glands
The limpet possesses four orange-coloured salivary glands, which lie at thefront of the visceral hump. There is a dorsal and ventral gland lying on eachside of the fore-gut. The salivary gland is a difficult tissue to study histologi-cally because very few fixatives preserve all of its structure, and the appearanceof fixed material varies greatly according to the fixative used. As statedearlier, of the fixatives tried, only Zenker and Flemming-without-acetic pre-served all of the material observed in fresh preparations.
The salivary glands are of similar structure: the only differences observedbetween them are consistent with the individual glands being in a differentfunctional state, and the following account applies to all the salivary glandtissue. The salivary gland is a compound tubular gland. The tubules aresurrounded by a thin layer of connective tissue, and are arranged in lobessurrounded by a thicker layer of connective tissue. The lumen of individualtubules is often difficult to distinguish. The salivary ducts are lined by cuboidepithelium; the cells possess large central nuclei.
After fixation in Flemming-without-acetic followed by staining with haema-toxylin and eosin, the cells show great variation in their contents and stainingproperties. Some cells appear pale yellow with finely granular cytoplasm,others contain small globules which stain pink. The remainder of the salivarygland cells contain globules which range in size from these small ones tolarge globules, one of which may fill an entire cell. Occasionally one ofthese large globules may be seen to be in process of being extruded from thecell. The globules stain increasingly basiphil with increase in size. Thelargest globules stain purple with the haematoxylin.
A single tubule usually contains cells showing all variations, i.e. thosepossessing granular cytoplasm, and cells with all sizes of globules. Individualcells appear to contain only one size of globule. The amount of cytoplasmvisible in a cell varies inversely with the size of the globule that it contains.For convenience the cells which appear granular are referred to as type 1 cells;the others which contain graded sizes of globules are grouped together astype 2 cells (fig. 3).
After Flemming-without-acetic or Zenker fixation, type 2 cells measurefrom 7 to 13 fj, at the base, the majority approximately 11 /x, and have a heightof 10 to 13 fi with an average of 11 to 12 (x\ the cells taper slightly towards thecentre of the tubule. Type 1 cells are more angular in appearance, the lengthsbetween two corners varying from 7 to 13 /x with an average of 9 ju..
Type 2 cells have peripheral nuclei which are small and ovoid, with theirgreater diameter about 3 /x. They appear granular, but do not have nucleoli.The nuclei are Feulgen-positive. The small quantity of cytoplasm usuallypresent in these cells stained lightly with plasma dyes.
The nuclei of type 1 cells were either peripheral, eccentric, or central in
32 Pugh—Digestive glands of Patella
position. They were spherical with a diameter of 3 JX, and resembled thenuclei of type 2 cells by having a granular appearance, and being Feulgen-positive.
After the majority of fixatives the differences between the two types ofsalivary gland cells were markedly increased. Cell type 2 was empty exceptfor a nucleus or contained a small quantity of cytoplasm and possibly a few
granuletype 1 cellnucleus
bosiphil cytoplasm
FIG. 3. Cells of salivary gland; composite drawing.
small globules. The cell had a heavily marked outline and a peripheralnucleus with its long axis lying parallel with the cell membrane. Type 1 cellsstained more deeply basiphil than did the cell type B of the digestive gland,and no nuclei or cytoplasmic structure could be observed.
Altmann and Kull stains after Regaud, Schridde, Zenker, or Flemming-without-acetic fixations showed the mitochondria to be tiny spherical bodiesin both cell types. The largest globules stained intensely red with Altmann'sacid fuchsin. If Kull's triple stain was used, the globules stained in shades ofred, orange, and blue, the largest red and the smallest blue. Type 1 cellsappeared solidly red after Altmann's stain; if highly differentiated the cellcould be seen to contain mitochondria. If Kull's method was used the cyto-plasm stained deep blue or green, and the mitochondria red.
In sections stained by Mallory's triple stain the cytoplasm of type 1 cellsstained blue, and some cytoplasmic granules deeper blue. The large globulesof cell type 2 stained red, the small globules and cytoplasm orange, and thenuclei of both cell types blue.
The nuclei of all the salivary gland cells stain green or purple with methylgreen / pyronin. After formaldehyde fixation the cytoplasm of cell type 2stained pale pink by this method. In material fixed in Zenker the cell type 2globules also stained pink; most of this colour could be removed by ribonu-clease digestion. The cytoplasm of cell type 1 stained intensely pink in
Pugh—Digestive glands of Patella 33
sections fixed by either method; deeply stained granules occurred in thecytoplasm. In sections treated with ribonuclease the type 1 cell appearedlarger than in the control and the cytoplasm stained palely. The granuleswere larger and globular in appearance; they were similar in size to the smallacidophil globules of some 2 cells.
Lipid, After formaldehyde fixation and postchroming the cytoplasm of type1 cells coloured moderately, and scattered cytoplasmic granules deeply, withSudan black. The same result was obtained with material fixed in Zenker andembedded in paraffin. These cells were not blackened by osmium. The otherstaining reactions of the type 1 cell lipid, which are summarized in table 2,suggest that it is phospholipid.
Substances containing carbohydrate. Sections were stained by the PASmethod, either untreated or after enzyme digestion as for the digestive glandsections. In untreated sections the cytoplasm and granules of type 1 cellsstained intensely. The outlines of type 2 cells stained lightly; the globuleswhen present were unstained. Sections treated with hyaluronidase did notshow any difference in staining properties from the untreated sections. Priorincubation with diastase did not affect the staining of type 2 cells. Thegranules of type 1 cells stained as intensely as in the control, but the sur-rounding cytoplasm stained lightly; so a considerable quantity of PAS-positive material had been removed by digestion with diastase.
Glycogen. Type 1 cells were intensely basiphil, and the same difficulty ofstaining with Best's carmine stain for glycogen was encountered as with thedigestive gland, and the results were inconclusive. The presence of glycogenwas confirmed by iodine staining in cell type 1.
Mucoid substances. In sections stained with toluidine blue and examinedwet, type 1 cells stained purple and the cytoplasm of type 2 cells lighter purpleor blue. Mounting in balsam did not destroy the metachromasia or type 1cells, but reduced to some extent that of type 2 cells. In sections mountedin balsam, granules were visible in type 1 cells which stained more deeply thanthe surrounding cytoplasm. The type 2 cell globules did not stain, except foran occasional faint purple coloration. An attempt was made to stain theseglobules by using different concentrations of toluidine blue and by the addi-tion of wetting agents.
Increasing the dye concentration above 0-5% did not cause any part of thegland to stain more intensely. The addition of wetting agents (triton X-100and span 20, from Charles Lennig, London, W.C. 1) at concentrations ofo-i to 10% caused the globules of type 2 cells to stain purplish pink. Celltype 1 stained intensely and uniformly purple.
A methylene blue extinction curve was performed on salivary glandsections. Type 1 cells bound methylene blue strongly at pH 4-0 and moder-ately at pH 2-4. The cytoplasm of type 2 cells stained palely at pH 4-0 anddid not stain at pH 2-4. The globules stained at pH 6-o and higher pH; theywere unstained at more acid pH.
Glycosidases. Neither ^S-glucosaminidase or /?-glucuronidase have been
34 Pugh—Digestive glands of Patella
demonstrated in the salivary glands. Tissue homogenates and histochemicalmethods were used.
Protein. In sections stained by the tetrazonium method, type i cells stainedmoderately after Zenker fixation. After formaldehyde fixation type i cellsstained more deeply and the granules could be distinguished as intenselystained dots. Type 2 cells stained lightly after both types of fixation. Theglobules, which were only preserved by Zenker fixation, also stained, thelarger deeply and the smaller globules moderately. Millon's reagent leftthe cytoplasm and globules of type 2 cells unstained. Type 1 cells stainedorange pink. Granules were visible but did not differ in colour from the sur-rounding cytoplasm.
DiscussionDigestive gland
The staining reactions of cell type A suggest that this cell may have severalfunctions. The presence of glycogen and a large quantity of fat suggests thatthe digestive gland may function as a food depot. Barry and Munday (1959)have reported glycogen storage in limpet tissues. For the visceral hump theygive a mean value of 2% by weight from July to November, decreasing duringNovember to January to a value of 0-3%, which persisted until March whenthe level rose again. The limpets examined have been killed at intervals duringthe year, and there has been no obvious difference in the amount of glycogenfound in individual cells. But there was considerable difference in the sizeof the digestive gland throughout the year. It decreased in size as the gonadincreased, then grew again in size after the gonad had shed its contents.This supports the suggestion that the digestive gland functions as a food depot.
The apical granule is similar in form in cells of type A, and contains a deri-vative of chlorophyll named enterochlorophyll by MacMunn. The remainingcytoplasmic granules may represent absorbed material with mucus from thegut, or droplets of secretion elaborated by the cells to be discharged into thelumen. The staining reactions of these granules suggest that they contain asubstance of high molecular weight which has some of the properties of anepithelial mucin, and a small quantity of enterochlorophyll. The appearanceof the tips of the cells and the rinding of isolated pieces of cytoplasm in thetubule lumen suggest either excretion or secretion by the cells. The resultsobtained from preliminary feeding experiments (unpublished results) sup-ports the latter suggestion.
/3-Glucuronidase is associated with the degradation of mucosubstancesin mammalian tissues (Walker, i960). It is possible that this enzyme may acton ingested mucus. This enzyme has been reported to have a possible diges-tive function in limpets (Corner, Leon, and Bulbrook, i960) and in Helixpomatia (Billet and McGee-Russell, 1955). The possibility of the enzymehaving a digestive function is the more likely as it occurs in both types ofdigestive gland cell; however, the enzyme may possess both functions.j3-Glucosaminidase occurs only in cell type A; this enzyme is stated to be
Pugh—Digestive glands of Patella 35
associated with mucosubstances, but as far as is known it has not been sup-posed to have a digestive function in herbiverous molluscs.
Unlike cell type A, type B cells contain only a small quantity of glycogenand no fat granules. The cell contains numerous mitochondria and has ahigh protein content, both of which are characteristic of glandular cells.The cell also contains a considerable quantity of ribonucleic acid. The refrac-tile spheres contain lipoprotein and possibly DNA. They are probably secre-tion droplets that will be released into the tubule lumen. During the feedingexperiments referred to above, it was found that these spheres increased innumber several hours after the limpet had been fed. The results obtainedduring this investigation of the digestive gland are fundamentally in agree-ment with those of Graham (1932).
The cells of the digestive ducts are of a single type. Each cell is ciliated andalso has a secretory function. The distal part of the duct cell is filled bynumerous pigment granules which contain enterochlorophyll and phospho-lipid. In addition the granules contain a lipid which is probably lipochrome.This pigment was stated to occur in alcoholic extracts of digestive gland(Newbigin, 1898). It is unlikely to be derived from the digestive gland cells,which contain a simple lipid only. The apical cytoplasm contains glycogen,a hyaluronidase-labile material, and much protein; these substances arefound in the scanty basal cytoplasm in smaller quantities. The cell secretesinto the duct lumen a viscous material which is presumably elaborated by theapical cytoplasm. This secretion behaves as an epithelial mucin assistingthe transport of food material along the duct. The secretion is viscous anddiffers markedly from mammalian mucin in its staining properties.
Salivary gland-It has been stated (Graham, 1932) that the salivary gland produces a
lubricating fluid which is poured on to the radula as the limpet feeds. It seemspossible that all or most of the salivary gland cells are engaged in the elabora-tion of a viscous secretion which contains protein, no lipid, and little or nocarbohydrate. However, the cytoplasm of cell type 1 contains a small quan-tity of glycogen. The globules of cell type 2 do not show the staining reactionsof a mucosubstance with any method used. The globules may possess amembrane which is impervious to water, as they were unstained by anaqueous solution of toluidine blue but coloured by a solution containing awetting agent.
The cytoplasm of cell type 1 contains protein, ribonucleic acid, and someglycogen and lipid. After ribonuclease digestion small replacement globulesappear in place of the granules seen in control sections. At high magnifica-tions the original granules may sometimes be seen lying at the periphery of theglobules or between them, and this suggests a close association between thetwo bodies. The whole cell increases in size after ribonuclease digestion inaddition to globule formation. The globules visible after ribonuclease diges-tion are similar in appearance to the small eosinophil globules of some type 2
36 Pugh—Digestive glands of Patella
cells. This supports the suggestion that type 1 and type 2 cells represent twostages in a secretory cycle and do not differ greatly in function. The finalstage of development of the cycle would be the cell containing a single largeglobule with only a narrow lining of cytoplasm and a peripherally displacednucleus. After extrusion of the globule the cell may regenerate and re-enterthe cycle as a type 1 cell, or it may be replaced.
The author wishes to thank Dr. S. M. McGee-Russell of the Virus ResearchGroup, Medical Research Council, and Dr. P. G. Walker, Head of theBiochemistry Department, for help and encouragement during the prepara-tion of this paper. This work forms part of a thesis accepted for the degree ofM.Sc. by the University of London.
ReferencesBARRY, R. J. C, and MUNDAY, F. A., 1959. J. mar. biol. Assoc. U.K. 38, 81.BILLET, F., and MCGEE-RUSSELL, S. M., 1955. Quart. J. micr. Sci., 96, 35.CAIN, A. J., 195°- Biol. Rev., 25, 75.CORNER, E. D. S., LEON, Y. A., and BULBROOK, R. D., i960. J. mar. biol. Assoc. U.K. 39, 51.DAVIS, J. A., and FLEURE, H. J., 1903. Patella (L.M.B.C. Memoir). London (Williams and
Norgate).DISCHE, Z., and BORENFREUND, E., 1951. J. biol. Chem., 92, 583.GIBSON, R. J. H., 1885. Trans, roy. Soc. Edinb., 32, 601.GRAHAM, A., 1032. Ibid., 57, 287.GRIFFITHS, A. B., 1888. Proc. roy. Soc, 44, 325.HOLT, S. J., 1958. General cytochemical methods, x, 375- New York (Academic Press).LANKESTER, E. R., 1867. Ann. Mag. nat. Hist., 20, 334.LEABACK, D. H., and WALKER, P. G., 1957. Biochem. J., 67, 22P.LEE, B., 1950. The microtomist's vade-mecum, n t h ed. London (Churchill).MACMUNN, C. A., 1886a. Phil. Trans, roy. Soc, 177, 235.
18866. Ibid., 177, 267.1899. Proc. roy. Soc. B, 64, 436.
NEWBIGIN, M., 1898. Quart. J. micr. Sci., 41, 391.ORTON, J. H., SOUTHWARD, A. J., and DODDS, J. M., 1956. J. mar. biol. Assoc. U.K., 35, 149.PEARSE, A. G. E., 1960. Histochemistry. 2nd ed. London (Churchill).PUGH, D., LEABACK, D. H., and WALKER, P. G., 1957. Biochem. J., 65, 16P.
and WALKER, P. G., i960. J. Histochem. Cytochem., 9, 103.REES, F. G., 1934. Proc. zool. Soc. Lond., 45, 45.SVERDRUP, H. V., JOHNSON, M. W., and FLEMMINC, R. H., 1942. The oceans. New York
(Prentice Hall).WALKER, P. G., i960. Biochem. J., 75, 4P.WIGGLESWORTH, V. B., 1957. Proc. roy. Soc. B, 147, 185.
Pugh—Digestive glands of Patella
Appendix
TABLE I
Preservation of the granules of digestive gland cell type A
37
Fixative
10% formalin(Baker's fixative)
10% formalin(Baker's fixative) followed
by postchroming10% formalin* in sea-water
solution10% formalin* in sea-water
solution
O I
o-i
O I
0 1
Additions
M CaCl2, pH 70
M CaCl2, pH 7-0
M (CH3COO)2Ca, pH 5-5
M CaCl2, pH 70
Lipid globules
O
+
±
+
Cytoplasmicgranules
0
O
4-
O
+ , preserved. ± , lipid globules preserved but disrupted. O, not preserved. • A solutioncontaining the principal salts of sea-water was used to give a final solution of salinity about3-55%-
