- Email: [email protected]
Studies on the sparganum of Spirometra erinacei—I. The histology and histochemistry of the scolex
International
Journal for Parasitology,
STUDIES ERINACEI-1.
1972, Vol. 2, pp, 23-28. Pergamon Press. Printed in Great Britain
ON THE SPARGANUM* OF SPIROMET&! THE HISTOLOGY AND HISTOCHEMISTRY OF THE SCOLEX B. H. KWAt
Department
of Zoology, Australian National University, Canberra, A.C.T. 2600, Australia (Received
2 September
1971)
Abstract KWA B. H., 1972. Studies on the sparganum of Spirometra erinacei-I. The histology and histochemistry of the scolex. International Journal for Parasitology, 2, 23-28. A histological and histochemical study of the plerocercoid (sparganum) scolex of S. erinacei was made at the light microscope level. It was intended to ascertain if histolytic glands are present, as this might explain the sparganum’s efficacy in penetrating the gut of the intermediate host. The results of this study, however, show the absence of any visible gland cells in the sparganum scolex. Amorphous bodies, thought to be probably deposits of excretory material, were described. The tegument was shown to be especially rich in protein, lipid and neutral polysaccharide, and an active zone for protein synthesis. Alkaline phosphatase was also present in the tegument. INDEX KEY WORDS: Spirometra erinacei; phosphatase; histology; histochemistry.
sparganum ; plerocercoid ; scolex ; alkaline
INTRODUCTION IT HAS been shown that the plerocercoids or spargana of spirometrid cestodes (Pseudophyllidea) are able to rapidly penetrate the gut of an intermediate host following ingestion and become established as tissue parasites (Li, 1929; Mueller, 1938 a,b; Bearup, 19.53; Sandars, 1953; Gordon, Forsyth & Robinson, 1954; Takahashi, 1959 a,b). However, the mechanism of penetration has not yet been established. The remarkable migration of the sparganum is accomplished despite the fact that it lacks any striking modifications of the scolex which might facilitate mechanical penetration through the relatively thick muscular and collagenous layers of the host intestine. Thus, it would seem that any mechanical effects exerted by the scolex might have to be augmented by some other mechanism to effect penetration. Smyth & Heath (1970) have suggested that histolytic ‘cephalic glands’ may be present in spargana which are possibly involved in the process of penetration. However, evidence for the existence of such glands in spirometrid plerocercoids is lacking. Glands have been described in the plerocercoid scolex of several other species of pseudophyllidean cestodes, including Diphyllobothrium latum, D. vogeli, D. osmeri, D. dendriticum (Kuhlow, 1953), D. dalliae, D. norvegicum (Vik, 1964), as well as in the adult scolex of Dibothriocephalus wilsoni, Adenocephalus pacificus and Glandicephalus antarticus (Wardle and McLeod, 1952), and Abothrium gadi (Williams, 1960). ‘Histolytic glands’ have also been described in the procercoid of Spirometra erinacei by Li (1929). A study of the morphology and histochemistry of the scolex of the sparganum of S. erinacei, at the light microscope level, was therefore undertaken to establish whether recognizable gland cells are present in this region which might be linked with penetration.
* Sparganum-Some
workers confine the use of this term to plerocercoids parasitic in man; others (e.g. Wardle & McLeod) use it in the sense it is used here, namely, for ‘the larval stage of any species of Spiro-
metra’. Editor. t Present address:
School of Biological Science, University of Malaya, Kuala Lumpur, Malaysia. 23
24
B. H. KWA
I.J.P. VOL. 2. 1972
The penetration of this species through the gut of the intermediate host has been studied by Bearup (1953), Sandars (1953) and Gordon, Forsyth & Robinson (1954), but detailed histological studies have not been undertaken previously. MATERIALS
AND
METHODS
Spargana of Spirometra erinacei were collected from two different hosts: Queensland cane toads, Bufo marinus, from Innisfail, Queensland, and the black snake, Demansia text& textilis, from the Lake George area, N.S.W. The spargana used for histological and histochemical studies were either collected directly from the natural hosts, or were fed to laboratory mice (Quakenbush strain) and then collected after penetration of the gut. They were washed in Ringer’s (B.D.H.), and 3-5 mm of the scolex was cut off and processed. For histological purposes spargana were fixed in Bouin or Carnoy overnight, and embedded in paraffin in the usual manner. Sections were cut at 6-8 pm and stained with haematoxylin and eosin. Some sections were stained with Heidenhain’s Azan (Conn, Darrow & Emmel, 1960). For histochemical purposes, material was fixed in Bouin fixative, 70 per cent alcohol, 4 per cent formol-saline at 4°C or Carnoy. For enzyme tests, the scoleces of spargana were fixed in cold formol-saline, embedded in 10 per cent gelatine, frozen on to the chuck of an International model CTD cryostat and sections cut at 20 pm. For other histochemical tests, the variously fixed scoleces were embedded in paraffin and sections cut at 6-8 pm. The following histochemical tests were carried out using procedures given by Pearse (1961): (i) for carbohydrates, the periodic acid Schiff (PAS), alcian blue (AB) and toluidine blue metachromasia tests; (ii) for lipids, the Sudan black B test; (iii) for proteins, the mercury-bromo-phenol blue (Hg BPB) test; (iv) for RNA and DNA, the methyl green pyronin (MGP) test; (v) for enzymes, the Gomori methods for acid and alkaline phosphatase, the Nachlas, Crawford and Seligman method for leucine amino-peptidase, and the Holt and Withers method for esterase. In addition, the acridine orange method for RNA and DNA as detailed by Culling (1963), and the Karnovsky & Roots (1964) techniques for esterases were carried out. Appropriate controls were used for all histochemical tests. These included, for the PAS test, treatment with diastase (Sigma) to remove glycogen; for the MGP and acridine orange method, the examination of sections treated with 1% RNA-ase in PO, buffer, pH 7.3 (Sigma) before application of the test; for the enzyme tests, heat treatment and incubations in media lacking substrates were carried out. Where appropriate, mammalian tissues known to contain specific substances were processed simultaneously. RESULTS
Histology of the sparganum scolex The external surface of the sparganum scolex is covered by microtriches 2.3 pm long, which arise from the tegument (about 10 pm thick). Under the light microscope the tegument appears fairly homogenous and clear (Fig. 1). Immediately below the tegument are the tegumental cells which appear as a single layer of closely packed cells with darkly staining nuclei. Their cytoplasmic connections with the distal cytoplasm are just faintly discernible under the light microscope (see Fig. 1).
FIG. 1. L.S. of sparganum FIG. 2. Test
for
FIG. 3. T.S.
of sparganum
FIG. 4. Sparganum
scolex. (Azan) tegumental
carbohydrates.
x 500. Note that cells are visible.
(PAS) x 500. C = calcareous scolex.
(Hg PBP) longitudinal
cytoplasmic
Note strong corpuscles.
reaction
in distal
x 300. A ; muscles.
amorphous
bodies;
Note that penetrating duodenum of mouse. localized and minor. (H & E) x 10.
FIG. 5. Test for lipids.
(Sudan
Black
extensions
B) x 100. Note strong careous corpuscles.
damage
reaction
to gut
in tegument
of the
tegument. arrows
z
tissue and
is cal-
[I.J.P. f.p. 2.rt
FIG. 6. Test for glycogen (PAS) x 500. Arrows show glycogen granules in parenchyma.
Dark areas are the amorphous bodies. FIG. 7. Control after diastase action (PAS) ,h_500. Note absence of glycogen granules in parenchyma. Persistent reaction in amorphous bodies indicate presence of other polysacchar~des. FE. 8. Test for nucleic acids. (MGP) :.’50. Strong reaction for RNA in perinuclear cytoplasm of tegumental cells. FE. 9. Test for alkaline phosphatase. (Gomori) \ 100. Strong reaction in tegument; reaction in calcareous corpuscles is a false positive probably due to reaction with inorganic phosphate deposits. FE. 10. Control for alkaline phosphatase. (Gomori) ;-~200. Note persistence of reaction in calcareous corpuscles.
I.J.P. VOL.
2. 1972
HISTOLOGYOF
25
SpirO??Wtf'~PLEROCERCOID
Below the tegument and comprising most of the scolex is the parenchyma. Cell membranes are not distinct in this region so it appears syncytial. Calcareous corpuscles, about 10-15 pm in diameter, are distributed irregularly throughout the parenchyma (see Fig. 2). A conspicuous ring of longitudinal muscles lies within the parenchyma (Fig. 3). Enclosed by these muscles and distributed randomly, are irregularly shaped masses of material, referred to as amorphous bodies. These appear to be extracellular deposits since nuclei could not be detected within them. Sometimes these bodies are not distinctly demarcated and they merge gradually with the parenchyma. They are composed of masses of granules which stain a reddish-orange with Heidenhain’s Azan. Two lateral excretory canals are present in the posterior half of the scolex, but in the more anterior regions, these anastomose. Conspicuous nerve cords are situated lateral to each of the excretory canals. No gland cells, which might be involved in secreting material to the exterior, could be detected in the scolex (see Fig. 4). Histochemical study of the sparganum scolex Table 1 presents the results of the histochemical tests on the sparganum scolex of S. erinacei. No differences could be detected between scoleces of spargana taken from infected cane toads and those which had recently penetrated the gut of mice, and the following remarks thus apply equally to spargana recovered from both sources.
TABLED-TABLE + + +
heavy positive
ILLUSTRATING RESULTS OF HJSTOCHEMICAL
reaction;
Method
PAS Glycogen AB Toluidine Blue* Sudan Black B Hg BPB MGP Acridine Orange Esterase LAP Acid Phosphatase Alk. Phosphatase
TESTS ON THE SPARGANUM
+ + medium positive reaction; + light positive reaction; ? generalized diffuse reaction)
D
T
+++ 0 0 0 +++ ++ 0 0 ? 0 0 +++
+ 0 0 0 0 + +++ +++ ? 0 0 ++
P + ++ 0 0 0 0 + + ? 0 0 0
Sites in the scolex M E ++ ++ 0 0
0 0 0 0
+z+ 0 0 ? 0 0 0
:+ 0 0 ? 0 0 0
SCOLEX
reaction;
0 negative
N
C
A
0 0 0. 0 0 + 0 0 ? 0 0 0
i-f 0 0 0 ++ -t-f 0 0 ? 0 0 0
+++ 0 0 0 + +++ 0 0 ? 0 0 0
* Metachromasia D = distal tegument T = tegumental cells P = parenchyma M = muscles
Distribution strong reaction excretory canals, associated with
E N C A
= = = =
excretory canals nerves calcareous corpuscles amorphous bodies
of non-enzymatic substance&. The Sudan black B method for lipids gave a in the tegument. Small amounts of lipid were present in the muscles, and the amorphous bodies mentioned earlier. Slightly larger amounts were the calcareous corpuscles (see Fig. 5).
26
B. N. KWA
I.J.P.
VOL.
2. 1972
For carbohydrates, the PAS reaction was strongly positive in the tegument (Fig. 2) and smaller amounts of carbohydrates were evident in the muscles, amorphous bodies and calcareous corpuscles. A very light and diffuse reaction was also observed in the parenchyma and sub-tegument (Fig. 6). The PAS reaction in the parenchyma and muscles was clearly due to the presence of glycogen since it was abolished following diastase treatment (Fig. 7). PAS reactions in other locations, however, persisted after diastase and thus showed that other types of polysaccharide were present in the sparganum scolex (see Fig. 7). The AB test was negative and there was no evidence of metachromasia with the toluidine bIue test, thus showing that acid mucopolysaccharides were absent. Thus, the mucopolysaccharides were neutral nlucopolysaccharides. With the Hg BPB test, the muscles and amorphous bodies stained especially strongly indicating higher concentrations of protein in these regions than in other zones of the scolex (Fig. 3). DNA was demonstrated in the nuclei in the scolex. RNA was confined almost entirely to the cytoplasm of the tegumental cells; only small scattered amounts were detected in the parenchymal cells (Fig. 8). It was noted, with the acridine orange technique, that the normal green fluorescence seen in nuclei due to DNA was masked by intense red fluorescence of RNA. Green fluorescence in the nuclei was established, however, following ribonuclease treatment. Distribution of enzymatic substances. The tegument and the tegumental cells of the sparganum scolex showed a very distinct reaction for alkaline phosphatase (see Fig. 9). There was no apparent reaction for the enzyme in any other regions. Fafse positive reactions (i.e., persistence of reaction product in controls) were seen in the calcareous corpuscles (Fig. lo), probably due to the presence of insoluble inorganic phosphate known to occur in these structures (von Brand, 1966). Acid phosphatase was absent in the scolex of the sparganum. Again, a false positive reaction was given by the catcareous corpuscles. The test for leucine a~no-peptidase was negative in the sparganum scolex. The limited number of tests for esterase which were carried out gave inconclusive results. A weak and diffuse reaction was given throughout entire sections of the scolex, and no localized concentrations of reaction product were detected. DEXUSSION
The rationale for this examination of the scolex of the sparganum of Spirometra erinaeei was to find if glands and glandular secretions were present which might facilitate the process of penetration by the sparganum through the gut wall of the intermediate host. However, glands have not been detected in this species and, as far as can be determined, in any other species of Spirometra previously studied. The suggestion that cephalic glands may be present in spargana (Smyth & Heath, 1970) was possibly made on the basis of the fact that frontal glands occur in the plerocercoids of several species of the pseudophyllidean genus Diphyllobothrium (see Kuhlow, 1953). These glands predominate in the medullary layer and vary in distribution and number in the different species. Furthermore, Kuhlow noted that the glands had a crystalline content and discharge to the exterior by means of ‘tortuous’ canals. He suggested that their function was to enable the plerocercoid to digest host intestinal tissue during penetration of the fish host, and cited as evidence the fact that adult individuals of the various species do not possess the glands; if they were involved only in penetration, they would presumably be unnecessary in the adult. In the present study the only structures which resemble the glands described by Kuhlow
I.J.P. VOL. 2. 1972
HISTOLOGY OF Spirornetra
PLER~c~~RCO~O
27
(1953) are the amorphous bodies. These bodies appear in cross-section to be roughly in the same position in the plerocercoid scolex and to have the same irregular pattern and size as indicated in Kuhlow’s figures (see Fig. 9, p. 201; Kuhlow, 1953). It was first thought that the amorphous bodies in the sparganum scolex of S. erinace~ were the same as the glands described by Kuhlow (1953). However, present evidence suggests strongly that in S. erinacei they are not glands but rather extracellular deposits of organic material. In the first place, they do not have a discrete cellular structure and no nuclei have been seen within them; they appear amorphous and irregular and frequently blend into the surrounding parenchyma. On staining with Heidenhain’s Azan, they appear reddishorange, whereas the glands described by Kuhlow appeared bright blue when stained similarly. Secondly, no canals or ducts could be detected which connected them to the exterior. Thirdly, they also appear further posteriorly in the strobila of the sparganum and are not limited to the scolex. Fourthly, no reduction in size, or change in their histochemical properties, could be detected in them after the sparganum had penetrated the mouse host, and thus they do not appear to be related to the mechanism of penetration. Finally, no RNA was detected in them which indicates that they are unlikely to be involved in protein synthesis. The function of the amorphous bodies is not known. They contain substantial amounts of neutral mucopolysaccharide and protein but relatively little lipid. One possible explanation is that they are deposits of excretory products possibly inactivated by association with protein. The sparganum lives within the tissue of the host and is therefore in direct and intimate contact with the host. The parasite might be expected, therefore, to minimize the amount of antigenic material being released into the host tissue so as not to provoke a host reaction against it. From the results of penetration experiments (Kwa, unpublished data), it is evident that the sparganum can remain in the mouse host for at least 2 months without causing any significant inflammatory reaction. For an internal parasite the size of the spargan~, this would be unexpected if all excretory products were released into host tissues. It is worth noting in support of this view that when the adults are established in an ‘external’ environment such as the intestine, the amorphous bodies are no longer present in the scolex. Takahashi (1959 b) detected both acid and alkaline phosphatase in the ‘cuticle and subcuticle cells’ of S. mansoni spargana. In this study of S. erinacei spargana, only alkaline phospha~se was detected. All the other histochemical results obtained for S. mansoni by Takahashi (1959 b) and Saraki (1961) agree substantially with those for S. erinacei. The alkaline phosphatase would seem to be more related to absorption rather than secretion (Halton, 1967) and the associated presence of RNA in the tegumental cells suggests that this enzyme is synthesized in this region. Of course the presence of other enzymes being synthesized in the tegument and secreted, cannot be ruled out by results obtained so far. At this stage it is uncertain why distinct glands are present in the procercoid (Li, 1929) but not in the plerocercoid of S. erinacei. It is certain, however, that there is great variability among species of Pseudophyllidea since in Diphyllobothrium osmeri, D. vogeli, D. dendriticum and D. la&m, glands are present in the plerocercoid but not in the adults (Kuhlow, 1953), whereas glands are present in the adults of Dibothriocephalus wilsoni, Adenocephalus parzjkus, GIandice~halus antartic~s (Wardle & McLeod, 1952) and Abothrium gadi Williams, 1960). The absence of glands visible with the light microscope could mean that there are no histolytic glands of any kind present in the sparganum of S. erinacei, thus implying that the mechanism of penetration is by some means other than with histolytic enzymes. It could also
B. H. KWA
28
I.J.P. VOL.2. 1972
mean that enzyme-producing cells may be structurally organized in such a way that they are not visibly apparent as glands under the light microscope. Results to be described elsewhere indicate that the latter case is more likely, since proteolytic enzyme(s) have been found in the scolex of the sparganum. Ackrzowledgements-The
supervision of this work by Dr. M. J. Howell is gratefully acknowledged.
REFERENCES BEARUPA. J. 1953. Life history of a spirometrid tapeworm, Veterinary
causing sparganosis
in feral pigs. Australian
Journal 29: 217-224.
CONN H. J., DARROWM. A. & EMMELV. M. 1960. Staining Procedures. Williams & Wilkins, Baltimore. CULLINGC. F. A. 1963. Handbook of Histopathological Techniques. Butterworths, London. GORDON H. M., FORSYTHB. A. & ROBINSONM. 1954. Sparganosis in feral pigs in New South Wales. Australian Veterinary Journal 30: 135-138. HALTOND. W. 1967. Studies on phosphatase activity in trematoda. Journal of Parasitology 53: 46-54. KARNOVS~YM. J. & ROOTSL. 1964. A ‘direct-coloring’ thiocholine method for cholinesterases. Journal of
Histochemistry
and Cytochemistry
12: 219-221.
KUHLOWV. F. 1953. (Structure and differential diagnosis of Diphyllobothrium plerocercoids.) (in German: English summary.) Zeitschrift fiir Tropenmedizin unt Parasitologic 4: 186-202. LI H. C. 1929. The life histories of Diphyllobothrium decipiens and D. erinacei. American Journal of Hygiene 10:527-550. MUELLERJ. F. 1938a. The life history of Diphyllobothrium munsonoides Mueller, 1935, and some considerations with regard to sparganosis in the United States. American Journalof Tropical Medicine 18: 41-66. MUELLERJ. F. 1938b. Studies on Sparganum mansonoides and Sparganum proliferum. American Journal of Tropical Medicine
18 : 303-328.
PEARS~A. G. E. 1961. Histochemistry: Theoreticai and Applied. J. & A. Churchill, London. SANDARSD. F. 1953. A study of Diphyllobothriidae (Cestoda) from Australian hosts. Proceedings Royal Society,
Queensland
of the
63: 65-70.
SARAKIT. 1961. (A histochemical study on the nucleic acid in Manson’s tapeworm.) (in Japanese: English summary.) Acta Scholae medicinalis in Gifu 9: 176-189. SMYTH J. D. & HEATH D. D. 1970. Pathology of larval cestodes in mammals. Helminthological Abstracts 39: l-23. TAKAHASHI
T. 1959a. (Studies on Diphyllobothrium mansoni. I. Life cycle and host specificity.) (in Japanese: English summary.) Japanese Journal of Parasitology 8: 567-574. TAKAHASHI T. 1959b. (Studies on Diohyllobothrium mansoni. II. Histochemical studies on plerocercoid.) (in Japanese: English summary.) Japanese Journal of Parasitology 10:669-676. VIK R. 1964. The genus Diohvllobothrium. An example of the interdependence of systematics and experimental biology~Experi&ental Parasitology 15: 361-380. VON BRANDT. 1966. Biochemistry of Parasites. Academic Press, New York. WARDLER. A. & MCLEODJ. A. 1952. The Zoology of Tapeworms. Minnesota Press. WILLIAMS H. H. 1960. Some observations on Parabothriumgadipollachii(Rudolphi, 1810) and Abothriumgadi van Beneden 1870 (Cestoda: Pseudophyllidea) including an account of their mode of attachment and of variation in the two species. Parasitology 50: 303-322.
Journal for Parasitology,
STUDIES ERINACEI-1.
1972, Vol. 2, pp, 23-28. Pergamon Press. Printed in Great Britain
ON THE SPARGANUM* OF SPIROMET&! THE HISTOLOGY AND HISTOCHEMISTRY OF THE SCOLEX B. H. KWAt
Department
of Zoology, Australian National University, Canberra, A.C.T. 2600, Australia (Received
2 September
1971)
Abstract KWA B. H., 1972. Studies on the sparganum of Spirometra erinacei-I. The histology and histochemistry of the scolex. International Journal for Parasitology, 2, 23-28. A histological and histochemical study of the plerocercoid (sparganum) scolex of S. erinacei was made at the light microscope level. It was intended to ascertain if histolytic glands are present, as this might explain the sparganum’s efficacy in penetrating the gut of the intermediate host. The results of this study, however, show the absence of any visible gland cells in the sparganum scolex. Amorphous bodies, thought to be probably deposits of excretory material, were described. The tegument was shown to be especially rich in protein, lipid and neutral polysaccharide, and an active zone for protein synthesis. Alkaline phosphatase was also present in the tegument. INDEX KEY WORDS: Spirometra erinacei; phosphatase; histology; histochemistry.
sparganum ; plerocercoid ; scolex ; alkaline
INTRODUCTION IT HAS been shown that the plerocercoids or spargana of spirometrid cestodes (Pseudophyllidea) are able to rapidly penetrate the gut of an intermediate host following ingestion and become established as tissue parasites (Li, 1929; Mueller, 1938 a,b; Bearup, 19.53; Sandars, 1953; Gordon, Forsyth & Robinson, 1954; Takahashi, 1959 a,b). However, the mechanism of penetration has not yet been established. The remarkable migration of the sparganum is accomplished despite the fact that it lacks any striking modifications of the scolex which might facilitate mechanical penetration through the relatively thick muscular and collagenous layers of the host intestine. Thus, it would seem that any mechanical effects exerted by the scolex might have to be augmented by some other mechanism to effect penetration. Smyth & Heath (1970) have suggested that histolytic ‘cephalic glands’ may be present in spargana which are possibly involved in the process of penetration. However, evidence for the existence of such glands in spirometrid plerocercoids is lacking. Glands have been described in the plerocercoid scolex of several other species of pseudophyllidean cestodes, including Diphyllobothrium latum, D. vogeli, D. osmeri, D. dendriticum (Kuhlow, 1953), D. dalliae, D. norvegicum (Vik, 1964), as well as in the adult scolex of Dibothriocephalus wilsoni, Adenocephalus pacificus and Glandicephalus antarticus (Wardle and McLeod, 1952), and Abothrium gadi (Williams, 1960). ‘Histolytic glands’ have also been described in the procercoid of Spirometra erinacei by Li (1929). A study of the morphology and histochemistry of the scolex of the sparganum of S. erinacei, at the light microscope level, was therefore undertaken to establish whether recognizable gland cells are present in this region which might be linked with penetration.
* Sparganum-Some
workers confine the use of this term to plerocercoids parasitic in man; others (e.g. Wardle & McLeod) use it in the sense it is used here, namely, for ‘the larval stage of any species of Spiro-
metra’. Editor. t Present address:
School of Biological Science, University of Malaya, Kuala Lumpur, Malaysia. 23
24
B. H. KWA
I.J.P. VOL. 2. 1972
The penetration of this species through the gut of the intermediate host has been studied by Bearup (1953), Sandars (1953) and Gordon, Forsyth & Robinson (1954), but detailed histological studies have not been undertaken previously. MATERIALS
AND
METHODS
Spargana of Spirometra erinacei were collected from two different hosts: Queensland cane toads, Bufo marinus, from Innisfail, Queensland, and the black snake, Demansia text& textilis, from the Lake George area, N.S.W. The spargana used for histological and histochemical studies were either collected directly from the natural hosts, or were fed to laboratory mice (Quakenbush strain) and then collected after penetration of the gut. They were washed in Ringer’s (B.D.H.), and 3-5 mm of the scolex was cut off and processed. For histological purposes spargana were fixed in Bouin or Carnoy overnight, and embedded in paraffin in the usual manner. Sections were cut at 6-8 pm and stained with haematoxylin and eosin. Some sections were stained with Heidenhain’s Azan (Conn, Darrow & Emmel, 1960). For histochemical purposes, material was fixed in Bouin fixative, 70 per cent alcohol, 4 per cent formol-saline at 4°C or Carnoy. For enzyme tests, the scoleces of spargana were fixed in cold formol-saline, embedded in 10 per cent gelatine, frozen on to the chuck of an International model CTD cryostat and sections cut at 20 pm. For other histochemical tests, the variously fixed scoleces were embedded in paraffin and sections cut at 6-8 pm. The following histochemical tests were carried out using procedures given by Pearse (1961): (i) for carbohydrates, the periodic acid Schiff (PAS), alcian blue (AB) and toluidine blue metachromasia tests; (ii) for lipids, the Sudan black B test; (iii) for proteins, the mercury-bromo-phenol blue (Hg BPB) test; (iv) for RNA and DNA, the methyl green pyronin (MGP) test; (v) for enzymes, the Gomori methods for acid and alkaline phosphatase, the Nachlas, Crawford and Seligman method for leucine amino-peptidase, and the Holt and Withers method for esterase. In addition, the acridine orange method for RNA and DNA as detailed by Culling (1963), and the Karnovsky & Roots (1964) techniques for esterases were carried out. Appropriate controls were used for all histochemical tests. These included, for the PAS test, treatment with diastase (Sigma) to remove glycogen; for the MGP and acridine orange method, the examination of sections treated with 1% RNA-ase in PO, buffer, pH 7.3 (Sigma) before application of the test; for the enzyme tests, heat treatment and incubations in media lacking substrates were carried out. Where appropriate, mammalian tissues known to contain specific substances were processed simultaneously. RESULTS
Histology of the sparganum scolex The external surface of the sparganum scolex is covered by microtriches 2.3 pm long, which arise from the tegument (about 10 pm thick). Under the light microscope the tegument appears fairly homogenous and clear (Fig. 1). Immediately below the tegument are the tegumental cells which appear as a single layer of closely packed cells with darkly staining nuclei. Their cytoplasmic connections with the distal cytoplasm are just faintly discernible under the light microscope (see Fig. 1).
FIG. 1. L.S. of sparganum FIG. 2. Test
for
FIG. 3. T.S.
of sparganum
FIG. 4. Sparganum
scolex. (Azan) tegumental
carbohydrates.
x 500. Note that cells are visible.
(PAS) x 500. C = calcareous scolex.
(Hg PBP) longitudinal
cytoplasmic
Note strong corpuscles.
reaction
in distal
x 300. A ; muscles.
amorphous
bodies;
Note that penetrating duodenum of mouse. localized and minor. (H & E) x 10.
FIG. 5. Test for lipids.
(Sudan
Black
extensions
B) x 100. Note strong careous corpuscles.
damage
reaction
to gut
in tegument
of the
tegument. arrows
z
tissue and
is cal-
[I.J.P. f.p. 2.rt
FIG. 6. Test for glycogen (PAS) x 500. Arrows show glycogen granules in parenchyma.
Dark areas are the amorphous bodies. FIG. 7. Control after diastase action (PAS) ,h_500. Note absence of glycogen granules in parenchyma. Persistent reaction in amorphous bodies indicate presence of other polysacchar~des. FE. 8. Test for nucleic acids. (MGP) :.’50. Strong reaction for RNA in perinuclear cytoplasm of tegumental cells. FE. 9. Test for alkaline phosphatase. (Gomori) \ 100. Strong reaction in tegument; reaction in calcareous corpuscles is a false positive probably due to reaction with inorganic phosphate deposits. FE. 10. Control for alkaline phosphatase. (Gomori) ;-~200. Note persistence of reaction in calcareous corpuscles.
I.J.P. VOL.
2. 1972
HISTOLOGYOF
25
SpirO??Wtf'~PLEROCERCOID
Below the tegument and comprising most of the scolex is the parenchyma. Cell membranes are not distinct in this region so it appears syncytial. Calcareous corpuscles, about 10-15 pm in diameter, are distributed irregularly throughout the parenchyma (see Fig. 2). A conspicuous ring of longitudinal muscles lies within the parenchyma (Fig. 3). Enclosed by these muscles and distributed randomly, are irregularly shaped masses of material, referred to as amorphous bodies. These appear to be extracellular deposits since nuclei could not be detected within them. Sometimes these bodies are not distinctly demarcated and they merge gradually with the parenchyma. They are composed of masses of granules which stain a reddish-orange with Heidenhain’s Azan. Two lateral excretory canals are present in the posterior half of the scolex, but in the more anterior regions, these anastomose. Conspicuous nerve cords are situated lateral to each of the excretory canals. No gland cells, which might be involved in secreting material to the exterior, could be detected in the scolex (see Fig. 4). Histochemical study of the sparganum scolex Table 1 presents the results of the histochemical tests on the sparganum scolex of S. erinacei. No differences could be detected between scoleces of spargana taken from infected cane toads and those which had recently penetrated the gut of mice, and the following remarks thus apply equally to spargana recovered from both sources.
TABLED-TABLE + + +
heavy positive
ILLUSTRATING RESULTS OF HJSTOCHEMICAL
reaction;
Method
PAS Glycogen AB Toluidine Blue* Sudan Black B Hg BPB MGP Acridine Orange Esterase LAP Acid Phosphatase Alk. Phosphatase
TESTS ON THE SPARGANUM
+ + medium positive reaction; + light positive reaction; ? generalized diffuse reaction)
D
T
+++ 0 0 0 +++ ++ 0 0 ? 0 0 +++
+ 0 0 0 0 + +++ +++ ? 0 0 ++
P + ++ 0 0 0 0 + + ? 0 0 0
Sites in the scolex M E ++ ++ 0 0
0 0 0 0
+z+ 0 0 ? 0 0 0
:+ 0 0 ? 0 0 0
SCOLEX
reaction;
0 negative
N
C
A
0 0 0. 0 0 + 0 0 ? 0 0 0
i-f 0 0 0 ++ -t-f 0 0 ? 0 0 0
+++ 0 0 0 + +++ 0 0 ? 0 0 0
* Metachromasia D = distal tegument T = tegumental cells P = parenchyma M = muscles
Distribution strong reaction excretory canals, associated with
E N C A
= = = =
excretory canals nerves calcareous corpuscles amorphous bodies
of non-enzymatic substance&. The Sudan black B method for lipids gave a in the tegument. Small amounts of lipid were present in the muscles, and the amorphous bodies mentioned earlier. Slightly larger amounts were the calcareous corpuscles (see Fig. 5).
26
B. N. KWA
I.J.P.
VOL.
2. 1972
For carbohydrates, the PAS reaction was strongly positive in the tegument (Fig. 2) and smaller amounts of carbohydrates were evident in the muscles, amorphous bodies and calcareous corpuscles. A very light and diffuse reaction was also observed in the parenchyma and sub-tegument (Fig. 6). The PAS reaction in the parenchyma and muscles was clearly due to the presence of glycogen since it was abolished following diastase treatment (Fig. 7). PAS reactions in other locations, however, persisted after diastase and thus showed that other types of polysaccharide were present in the sparganum scolex (see Fig. 7). The AB test was negative and there was no evidence of metachromasia with the toluidine bIue test, thus showing that acid mucopolysaccharides were absent. Thus, the mucopolysaccharides were neutral nlucopolysaccharides. With the Hg BPB test, the muscles and amorphous bodies stained especially strongly indicating higher concentrations of protein in these regions than in other zones of the scolex (Fig. 3). DNA was demonstrated in the nuclei in the scolex. RNA was confined almost entirely to the cytoplasm of the tegumental cells; only small scattered amounts were detected in the parenchymal cells (Fig. 8). It was noted, with the acridine orange technique, that the normal green fluorescence seen in nuclei due to DNA was masked by intense red fluorescence of RNA. Green fluorescence in the nuclei was established, however, following ribonuclease treatment. Distribution of enzymatic substances. The tegument and the tegumental cells of the sparganum scolex showed a very distinct reaction for alkaline phosphatase (see Fig. 9). There was no apparent reaction for the enzyme in any other regions. Fafse positive reactions (i.e., persistence of reaction product in controls) were seen in the calcareous corpuscles (Fig. lo), probably due to the presence of insoluble inorganic phosphate known to occur in these structures (von Brand, 1966). Acid phosphatase was absent in the scolex of the sparganum. Again, a false positive reaction was given by the catcareous corpuscles. The test for leucine a~no-peptidase was negative in the sparganum scolex. The limited number of tests for esterase which were carried out gave inconclusive results. A weak and diffuse reaction was given throughout entire sections of the scolex, and no localized concentrations of reaction product were detected. DEXUSSION
The rationale for this examination of the scolex of the sparganum of Spirometra erinaeei was to find if glands and glandular secretions were present which might facilitate the process of penetration by the sparganum through the gut wall of the intermediate host. However, glands have not been detected in this species and, as far as can be determined, in any other species of Spirometra previously studied. The suggestion that cephalic glands may be present in spargana (Smyth & Heath, 1970) was possibly made on the basis of the fact that frontal glands occur in the plerocercoids of several species of the pseudophyllidean genus Diphyllobothrium (see Kuhlow, 1953). These glands predominate in the medullary layer and vary in distribution and number in the different species. Furthermore, Kuhlow noted that the glands had a crystalline content and discharge to the exterior by means of ‘tortuous’ canals. He suggested that their function was to enable the plerocercoid to digest host intestinal tissue during penetration of the fish host, and cited as evidence the fact that adult individuals of the various species do not possess the glands; if they were involved only in penetration, they would presumably be unnecessary in the adult. In the present study the only structures which resemble the glands described by Kuhlow
I.J.P. VOL. 2. 1972
HISTOLOGY OF Spirornetra
PLER~c~~RCO~O
27
(1953) are the amorphous bodies. These bodies appear in cross-section to be roughly in the same position in the plerocercoid scolex and to have the same irregular pattern and size as indicated in Kuhlow’s figures (see Fig. 9, p. 201; Kuhlow, 1953). It was first thought that the amorphous bodies in the sparganum scolex of S. erinace~ were the same as the glands described by Kuhlow (1953). However, present evidence suggests strongly that in S. erinacei they are not glands but rather extracellular deposits of organic material. In the first place, they do not have a discrete cellular structure and no nuclei have been seen within them; they appear amorphous and irregular and frequently blend into the surrounding parenchyma. On staining with Heidenhain’s Azan, they appear reddishorange, whereas the glands described by Kuhlow appeared bright blue when stained similarly. Secondly, no canals or ducts could be detected which connected them to the exterior. Thirdly, they also appear further posteriorly in the strobila of the sparganum and are not limited to the scolex. Fourthly, no reduction in size, or change in their histochemical properties, could be detected in them after the sparganum had penetrated the mouse host, and thus they do not appear to be related to the mechanism of penetration. Finally, no RNA was detected in them which indicates that they are unlikely to be involved in protein synthesis. The function of the amorphous bodies is not known. They contain substantial amounts of neutral mucopolysaccharide and protein but relatively little lipid. One possible explanation is that they are deposits of excretory products possibly inactivated by association with protein. The sparganum lives within the tissue of the host and is therefore in direct and intimate contact with the host. The parasite might be expected, therefore, to minimize the amount of antigenic material being released into the host tissue so as not to provoke a host reaction against it. From the results of penetration experiments (Kwa, unpublished data), it is evident that the sparganum can remain in the mouse host for at least 2 months without causing any significant inflammatory reaction. For an internal parasite the size of the spargan~, this would be unexpected if all excretory products were released into host tissues. It is worth noting in support of this view that when the adults are established in an ‘external’ environment such as the intestine, the amorphous bodies are no longer present in the scolex. Takahashi (1959 b) detected both acid and alkaline phosphatase in the ‘cuticle and subcuticle cells’ of S. mansoni spargana. In this study of S. erinacei spargana, only alkaline phospha~se was detected. All the other histochemical results obtained for S. mansoni by Takahashi (1959 b) and Saraki (1961) agree substantially with those for S. erinacei. The alkaline phosphatase would seem to be more related to absorption rather than secretion (Halton, 1967) and the associated presence of RNA in the tegumental cells suggests that this enzyme is synthesized in this region. Of course the presence of other enzymes being synthesized in the tegument and secreted, cannot be ruled out by results obtained so far. At this stage it is uncertain why distinct glands are present in the procercoid (Li, 1929) but not in the plerocercoid of S. erinacei. It is certain, however, that there is great variability among species of Pseudophyllidea since in Diphyllobothrium osmeri, D. vogeli, D. dendriticum and D. la&m, glands are present in the plerocercoid but not in the adults (Kuhlow, 1953), whereas glands are present in the adults of Dibothriocephalus wilsoni, Adenocephalus parzjkus, GIandice~halus antartic~s (Wardle & McLeod, 1952) and Abothrium gadi Williams, 1960). The absence of glands visible with the light microscope could mean that there are no histolytic glands of any kind present in the sparganum of S. erinacei, thus implying that the mechanism of penetration is by some means other than with histolytic enzymes. It could also
B. H. KWA
28
I.J.P. VOL.2. 1972
mean that enzyme-producing cells may be structurally organized in such a way that they are not visibly apparent as glands under the light microscope. Results to be described elsewhere indicate that the latter case is more likely, since proteolytic enzyme(s) have been found in the scolex of the sparganum. Ackrzowledgements-The
supervision of this work by Dr. M. J. Howell is gratefully acknowledged.
REFERENCES BEARUPA. J. 1953. Life history of a spirometrid tapeworm, Veterinary
causing sparganosis
in feral pigs. Australian
Journal 29: 217-224.
CONN H. J., DARROWM. A. & EMMELV. M. 1960. Staining Procedures. Williams & Wilkins, Baltimore. CULLINGC. F. A. 1963. Handbook of Histopathological Techniques. Butterworths, London. GORDON H. M., FORSYTHB. A. & ROBINSONM. 1954. Sparganosis in feral pigs in New South Wales. Australian Veterinary Journal 30: 135-138. HALTOND. W. 1967. Studies on phosphatase activity in trematoda. Journal of Parasitology 53: 46-54. KARNOVS~YM. J. & ROOTSL. 1964. A ‘direct-coloring’ thiocholine method for cholinesterases. Journal of
Histochemistry
and Cytochemistry
12: 219-221.
KUHLOWV. F. 1953. (Structure and differential diagnosis of Diphyllobothrium plerocercoids.) (in German: English summary.) Zeitschrift fiir Tropenmedizin unt Parasitologic 4: 186-202. LI H. C. 1929. The life histories of Diphyllobothrium decipiens and D. erinacei. American Journal of Hygiene 10:527-550. MUELLERJ. F. 1938a. The life history of Diphyllobothrium munsonoides Mueller, 1935, and some considerations with regard to sparganosis in the United States. American Journalof Tropical Medicine 18: 41-66. MUELLERJ. F. 1938b. Studies on Sparganum mansonoides and Sparganum proliferum. American Journal of Tropical Medicine
18 : 303-328.
PEARS~A. G. E. 1961. Histochemistry: Theoreticai and Applied. J. & A. Churchill, London. SANDARSD. F. 1953. A study of Diphyllobothriidae (Cestoda) from Australian hosts. Proceedings Royal Society,
Queensland
of the
63: 65-70.
SARAKIT. 1961. (A histochemical study on the nucleic acid in Manson’s tapeworm.) (in Japanese: English summary.) Acta Scholae medicinalis in Gifu 9: 176-189. SMYTH J. D. & HEATH D. D. 1970. Pathology of larval cestodes in mammals. Helminthological Abstracts 39: l-23. TAKAHASHI
T. 1959a. (Studies on Diphyllobothrium mansoni. I. Life cycle and host specificity.) (in Japanese: English summary.) Japanese Journal of Parasitology 8: 567-574. TAKAHASHI T. 1959b. (Studies on Diohyllobothrium mansoni. II. Histochemical studies on plerocercoid.) (in Japanese: English summary.) Japanese Journal of Parasitology 10:669-676. VIK R. 1964. The genus Diohvllobothrium. An example of the interdependence of systematics and experimental biology~Experi&ental Parasitology 15: 361-380. VON BRANDT. 1966. Biochemistry of Parasites. Academic Press, New York. WARDLER. A. & MCLEODJ. A. 1952. The Zoology of Tapeworms. Minnesota Press. WILLIAMS H. H. 1960. Some observations on Parabothriumgadipollachii(Rudolphi, 1810) and Abothriumgadi van Beneden 1870 (Cestoda: Pseudophyllidea) including an account of their mode of attachment and of variation in the two species. Parasitology 50: 303-322.