The Body Wall and Musculature of the Marine Triclad Palombiella Stephensoni
Part 3: Histochemical Observations
Publication of this paper is assisted by a grant from the Victoria University of Wellington Publications Fund.
The histochemistry of the body wall and musculature of the marine triclad Palombiella stephensoni (Palombi, 1938) is investigated. The secretions of the subepidermal basiphil glands contain neutral mucopolysaccharide, and in addition the intraepidermal portion of these glands contains protein. It is suggested that this protein is a product of the epidermal cells. The subepidermal eosinophil glands contain protein. There are two types present; one is associated with the reproductive apertures, and the other is the adhesive type which assists in adhering the animal to the substrate and has been previously described. The subepidermal pigment may be a melanin.
Palombiella stephensoni (Palombi, 1938) is a marine triclad (Phylum Platyhelminthes, Class Turbellaria, Order Tricladida) of the family Bdellouridae. The morphology of the body wall and musculature of this animal as seen by light mocroscopy of histological preparations has been described (Wineera, 1969; 1971). In the present study histochemical methods are employed in an effort to characterize further the various body wall constituents described earlier.
Materials and Methods
The preparation of material for paraffin embedding (collection of animals and their maintenance in the laboratory; anaesthetization; dehydration and embedding) has been described elsewhere (Wineera, 1969). For paraffin embedding in the present study animals were fixed in Lillie's alcohol-acetic acid-formalin (Pearse, 1960, p.788), and in buffered 4% formaldehyde (Pease, 1964, p.52) for 18 hrs. Sagittal, transverse and frontal sections were cut at 5μ on a rotary microtome. For the study of lipids, some worms were fixed in Bakers formol-calcium (Pearse, 1960, p.787), embedded in 20% gelatin and cut at 8μ and 5μ on a freezing microtome. The following histochemical tests were applied:
Tests for Protein:
|1.||The mercury/bromphenol blue test (Pearse, 1960). Staining times of ½ hr. and 2 hrs. were employed.|
|2.||The Ninhydrin/Schiff test for protein bound NH2 (Pearse, 1960).|
|3.||The Sakaguchi reaction for arginine; Baker's 1947 modification (Pearse, 1960).|
|4.||The Millon reaction for tyrosine (Casselman, 1959). The Millon reaction as given by Pearse (1960, p.791) differs from that given by Casselman, although both authors claim it to be the Baker modification of this test. Both authors give the same instructions for the preparation of 200 ml. mercuric sulphate reagent. Pearse then instructs that 0.5 ml. 0.25% sodium nitrate is to be added to the mercuric sulphate reagent, while Casselman says to add 0.5 ml. 0.25% sodium nitrite to 5 ml. of the mercuric sulphate reagent for use. In the present study a positive reaction for tyrosine in tissue sections was obtained with Casselman's method but not with Pearse's.|
|5.||The DMAB-nitrate reaction for tryptophan (Adams, 1957, cited in Pearse, 1960).|
Tests for Lipid:
|1.||Staining with Sudan black B. The stain was used as a saturated solution in 60% isopropyl alcohol (Casselman, 1959). The position of Sudan staining tissue components in some sections were recorded by means of a microscope stage micrometer and the sections were then decoloured by soaking (1 hr. to 16 hrs.) in 60% isopropyl alcohol. They were then restained with Sudan black to test whether tissue components which took the stain initially exhibited "true sudanophilia" (Casselman, 1959, p.74).|
|2.||Some section were extracted with pyridine at 60°C for 24 hrs. and then stained in Sudan black.|
|3.||Unstained frozen sections were examined by phase contrast microscopy.|
Tests for Carbohydrates:
|1.||The periodic acid-Schiff reaction (Casselman, 1959). Oxidation was for 9 min. at room temperature in 0.5% periodic acid. The Schiff's reagent used was a variant of Lillie's, cited in Casselman (p.36). Some sections were treated with malt diastase (1% in distilled water at 37°C for ½ hr.) prior to the PAS test, and others were treated with Schiff's page 3 reagent without periodic acid oxidation. Nuclear counterstains used were Delafield's haematoxylin, and 0.1% Azure A in 30% ethanol.|
|2.||Metachromasia. Sections were stained for 5 min. in 0.01% Azure A in 30% ethanol (Kramer and Windrum, 1953) or in 0.1% toluidine blue in 30% ethanol for 1 min. All sections were examined in distilled water before being dehydrated, cleared and mounted in DPX.|
|3.||Sulphation/metachromasia. The low temperature sulphation of Moore & Schoenberg (1957) was employed. Sections were then stained and examined as in 2 above.|
|4.||Mowry's colloidal iron method for acid mucopolysaccharides (Mowry, 1958).|
|5.||Alcian blue staining for acid mucopolysaccharides (Wagner and Shapiro, 1957).|
|6.||A combination of sulphation/Mowry's colloidal iron technique.|
|7.||Sulphation followed by Alcian blue staining.|
Tests for Nucleic Acids:
|1.||The Feulgen reaction (Pearse, 1960) for deoxyribosenucleic acid. Hydrolysis was for 10 minutes in N. HCl at 60°C; the "Schiff" reagent used was Azure A-Schiff prepared according to Himes and Moriber (1956).|
|2.||The methyl green/pyronin Y method of Kurnick (cited in Pearse, 1960) for deoxyribosenucleic acid and ribosenucleic acid. The proportions of methyl green to pyronin Y, and the staining time were modified. Serial sections were treated with RNase (Sigma(r)), 1 mg./ml. in glass distilled water for 1½ hrs. and 3hrs. at 37°C prior to staining. Controls were placed in distilled water at 37°C for corresponding times.|
|1.||The Allochrome procedure (Lillie, 1951). Although not strictly a histochemical technique, this test successfully distinguished between certain tissue constituents. The picromethyl blue solution employed was 40 mg. methyl blue per 100 ml. saturated aqueous picric acid. The staining time in this solution was 6 min.|
|2.||Feulgen-PAS technique for demonstration of DNA and polysaccharide in the same section.|
|3.||The Himes & Moriber (1956) method for DNA, protein and polysaccharide. This method is a Feulgen-PAS technique followed by staining in naphthol yellow S, a dye which Deitch (1955) has shown is specific for basic protein within a wide range of dye concentrations and staining times.|
|4.||Sudan black B/PAS. Sections were stained in Sudan black as described above (Tests for Lipid, 1). The positions of sudanophil structures were recorded using a microscope stage micrometer, and the sections were then decoloured. The PAS test was then applied, and the positions page 4 of the previous sudanophilia were examined. Some sections were placed in Schiff's reagent without periodic acid oxidation, as controls.|
|5.||Sudan black B/Naphthol yellow S. As for 4 above, but the PAS routine was replaced by staining in naphthol yellow S, 0.02% in 1% acetic acid for 5 min.|
|6.||Mowry colloidal iron method/PAS, for demonstration of acidic and neutral polysaccharides in the same section (Mowry, 1958).|
|1.||Bleaching of pigment. Sections were placed in 10% hydrogen peroxide, and in peracetic acid (Pearse, 1960, p.860) until the pigment was bleached.|
|2.||Whole worms were placed in dilute (5%) hydrochloric acid in an attempt to extract pigment.|
|3.||The ultraviolet/fluorescence method of MacRae (1961) for demonstrating the presence of porphyrins in planarians.|
|4.||Falg technique (Gurr, 1965).|
Tables 1 and 2 summarise the results. Additional features are given below.page 5
The Epidermis: The Feulgen reaction coloured nuclei only. With the methyl green/pyronin test the cytoplasm adjacent to the nucleus often coloured with pyronin (Pl. 1, Fig 1,C). Digestion of sections with RNase for 1½ hrs. reduced pyronin staining, and digestion for 3 hrs. elminated this staining reaction.
The "Basement Membrane": The Allochrome procedure coloured the membrane deep blue.
The Muscles: A small area of cytoplasm around muscle cell nuclei was coloured with pyronin in the methyl green/pyronin test. This colour was removed by digestion of the section with RNase.
The Parenchyma: The parenchyma exhibited an overall dark greyish colour after staining with Sudan black B. Many very small refractile bodies are present throughout the parenchyma, but it is difficult, because of their small size and refractility, to tell whether or not they stain with Sudan black.
A small group of cells situated caudally in the parenchyma immediately behind the penis gave a strong positive result with the Mowry colloidal iron method. These same cells were PAS negative.
The Feulgen reaction demonstrated many nuclei in the parenchyma.
Subepidermal Glands: Two major types of subepidermal gland have been described (Wineera, 1969) for this animal. These are the eosinophil glands and the basiphil glands.
The Eosinophil Glands: Two types are present in P. stephensoni. One type (hereafter termed adhesive glands) was described earlier (Wineera, 1969). The other type (hereafter termed caudal glands) are page 6 located in an area of parenchyma just above the ventral body wall in the caudal region of the worm. The spermathecae and the genital opening also occur in this region of the animal. The glands are intensely eosinophil (Pl. 1, Fig. 6, black material). They contain closely packed granules approximately 1.2μ in diameter, and clusters are found crossing the basement membrane and within the epidermis. With pyronin staining the adhesive gland cells varied from colourless to bright red. The staining could be eliminated by prior treatment of sections with RNase for 3 hrs. Some cells contained colourless secretion granules surrounded by pyronin staining cytoplasm, and others possessed pyroninophil cytoplasmic strands.
The Basiphil Glands: On one occasion basiphil glands displayed metachromasia after staining with toluidine blue and mounting in DPX. The sulphation/Azure A and sulphation/toluidine blue techniques demonstrated metachromasia when the sections were viewed in water. After dehydration and mounting in DPX the colour changed from red to purple. With the allochrome procedure subepidermal portions of the basiphil glands coloured a purplish colour, while the intraepidermal parts coloured red.
Pigment: In paraffin sections the pigment granules are bleached by 10% hydrogen peroxide after 6 days. With 40% peracetic acid made according to Pearse (1960, p.860) the bleaching time was 17 hrs. The colour of the dorsal surface of whole worms was not lessened by extracting them with 5 % hydrochloric acid for 2 weeks. The ultraviolet/fluorescence method of MacRae (1961) for demonstrating the presence of porphyrins in planarians gave negative results. The same test applied to a fresh water triclad gave a positive result.
The "Basement Membrane": It was suggested in an earlier paper by the present author (Wineera, 1969) that the "basement membrane" probably is collagenous. The results of the present study do not contradict this suggestion, but nor do they give it unequivocal support. The "basement membrane" exhibits a variable PAS reaction, and weak positive staining for protein, which could be expected of collagen (Pearse, 1960, p.162). It showed no metachromasia after staining with toluidine blue or azure A either before or after sulphation, suggesting that acidic carbohydrates are not present in this structure. There is the possibility that some are present but that the reactive groups which give rise to the metachromatic phenomenon are not present in sufficient quantity or are not placed at suitable intervals along the polysaccharide molecule. The weak positive staining with Mowry's colloidal iron reagent is best considered as non specific staining by the reagent and not indicative of the presence of acidic muco-polysaccharides. In the allochrome procedure the "basement membrane" coloured deep blue, which Lillie (1951) considers a characteristic of collagen when subjected to this procedure. Skaer (1961) describes in a fresh water triclad a thick basement membrane which he concludes contains collagen.
Parenchyma: The parenchyma of turbellarians continues to be an extremely difficult subject for study. The elements clearly distinguished in the present study were the neoblast cells (Pedersen, 1959) and the page 7 gland cells. As found by Pedersen for neoblast cells in a fresh water triclad, these cells in P. stephensoni contain large amounts of cytoplasmic RNA. The present study sheds no light on the problem of the origin of the "basement membrane", or the fibres continuous with it (Wineera, 1969). The extent to which lipids occur in the parenchyma, and their distribution, cannot be decided until further work involving electron microscopy is undertaken. The cells in the parenchyma which gave a strong positive reaction with the colloidal iron reagent are considered to be glands associated with the penis.
The Eosinophil Glands (Pl. 1, Fig. 3; Pl. 1, Fig. 7, S) : These are clearly protein in nature. The adhesive glands are shown to contain arginine, and the caudal glands tyrosine and tryptophan. They contain no polysaccharide material. The weak staining of the adhesive glands in the Mowry colloidal iron technique is considered non specific staining by the iron reagent. This view is supported by the fact that the glands do not colour with Mowry's reagent after sulphation, and do not exhibit metachromasia with azure A or toluidine blue. The Mowry colloidal iron method is based on the demonstration of bound ferric iron as prussian blue (Mowry, 1958). Wagner & Shapiro (1957) indicate that some proteins bind ferric iron and could give positive results in tests which demonstrate this ferric iron, as for example, the colloidal iron test for acidic polysaccharides. The staining of both types of gland by Sudan black is not considered "true sudanophilia" due to lipids, since staining was not diminished by hot pyridine extraction. The variable staining of the adhesive cells with pyronin indicates variable amounts of RNA in these cells. This can be correlated with the protein nature of the adhesive gland secretion. The variable nature of the reaction may indicate different stages of the secretion cycle in different cells. The cells showing "stranded" pyroninophilic cytoplasm probably represent those described by Wineera (1971) as possessing ergastoplasm. Pedersen (1959, 1963) records the presence of eosinophil adhesive glands in fresh water triclads. The distribution of the glands described by Pedersen is similar to the distribution of the eosinophil glands in P. stephensoni. Pedersen also describes the glands as being protein in nature, but states that they contain, in addition, a phospholipid component. The caudal glands referred to in this study are considered to be associated with the reproductive organs since they are found only in the region of the genital openings.
The Basiphil Glands: The reactions of these gland cells indicate that they contain neutral mucopolysaccharides: They exhibit diastase resistant PAS positivity, and are negative in tests for protein, lipid, and nucleic acid. Also they do not stain in the tests for acidic polysaccharides except after sulphation (Pl. 1, Fig. 5) which is a recognized procedure (Pearse, 1960; Moore and Schoenberg, 1957; Kramer and Windrum, 1954) for forming acidic polysaccharides from neutral ones present in tissue sections. The observed case of metachromatic staining of the basiphil glands with toluidine blue before sulphation seems anomalous in the light of the previous discussion, for it should indicate the presence of acidic mucopolysaccharides (Pearse, 1960; Moore and Schoenberg, 1957). Nucleic acids can display metachromasia under certain conditions (Pearse, 1960; Bergeron and Singer, 1958) but in the present case the gland cells gave page 8 negative results in tests for nucleic acids. It would appear best at this time to attach no significance to this case of metachromatic staining because (i) the staining was weak and (ii) the staining was an isolated case and could not be repeated in several subsequent attempts.
The nature of the basiphil secretion appears to change both in morphology and in staining reaction from the subepidermal parts of the glands to those in the epidermis: The subepidermal parts of these glands are granular and, as has already been mentioned, contain neutral mucopolysaccharides. The intraepidermal portions often appear homogenous, and stain in tests for protein (e.g. mercury/bromphenol blue test) (Pl. 1, Fig. 4, S) as well as in the PAS test. In the allochrome method the distinction between these two parts is seen clearly. The significance of these differences within different parts of the basiphil glands is not known. It is possibly that the protein part of the secretion is a product of the epidermal cells themselves, since these were found to contain varying amounts of RNA adjacent to their nuclear region.
The basiphil glands stain with Sudan black B, but this does not indicate the presence of lipids, as extraction of sections with hot pyridine does not lessen the staining reaction. Presumably the Sudan black acts, in this case, as a weak basic dye (as Casselman (1959) shows it is able to do), and not as a non-ionic fat soluble colourant.
In his studies of fresh water triclads, Pedersen (1959, 1963) describes three types of basiphil subepidermal gland. One type (called type 3) owes its basiphilia to RNA granules in its cytoplasm and is found immediately in front of the pharynx. This type appears to have no counterpart in P. stephensoni. The other two types, taken together, are similar in morphology, distribution, and staining reaction to the basiphil glands in P. stephensoni. In the present study slight differences in the basiphil gland cells occurred. For example the size and degree of packing of secretion granules varied slightly from some gland cells to others. But all basiphil glands exhibited the same staining reactions, and the variations observed were not thought sufficient to justify the division of the basiphil glands into two groups. However, it is possible as Skaer (1961) suggests, that the basiphil glands include more than one histochemically distinct type.
Pigment: The pigment in P. stephensoni may be a melanin. It is slowly bleached by hydrogen peroxide, and is bleached by 40% peracetic acid in a time which is very close to that given by Pearse (1960) for the bleaching of melanin in tissue sections by this substance. Skaer (1961) concludes that the pigment in the triclad Polycelis nigra is a melanin, but Needham (1965) states that P. nigra pigment can be extracted by dilute (4%) hydrochloric acid, which is not characteristic of melanin. In the present study the pigment granules were insoluble in 5% HCl.
I would like to acknowledge the assistance given by Dr. Patricia M. Ralph of the Zoology Department, Victoria University, in constructive criticism of this paper, and for her interest throughout the course of the work.
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Legend to the Plates
Fig. 1: Sagittal section of dorsal body wall in head region. Methyl green/pyronin stain, × 700.
C, cytoplasm of an epidermal cell; E, epidermis.
Fig. 2: Sagittal section of dorsal body wall. Mercury/bromphenol blue stain, × 1,000.
B, basement membrane; C, circular muscles; E, epidermis; L, longitudinal muscles; P, parenchyma; S, intraepidermal basiphil secretion.
Fig. 3: Sagittal section of caudal region showing caudal glands. Mercury/bromphenol blue stain, × 850.
E, ventral epidermis.
Fig. 4: Sagittal section of ventral body wall to show intraepidermal portions of subepidermal basiphil glands. Mercury/bromphenol blue stain, × 1,500. Note increasing intensity of staining towards epidermal surface.
E, epidermis; LM, longitudinal muscle; S, "sacs" of basiphil secretion.
Fig. 5: Sagittal section of ventral body wall in cephalic region. Sulphation/mowry colloidal iron method, × 800.
B, basiphil glands; E, epidermis.
Fig. 6: Sagittal section of ventral body wall in caudal region showing strongly fuchsinophil caudal glands (black). Falg stain, × 500.
B, basement membrane; E, epidermis.
Fig. 7: Sagittal section of cephalic region of animal showing adhesive glands opening through ventral epidermis. Mercury/bromphenol blue stain, × 400.
E, ventral epidermis; P, parenchyma; S, adhesive gland secretion.