Electron microscopy of cestode calcareous corpuscle formation

EXPERIMENTAL

24, 279-289

PARASITOLOGY

Electron

Microscopy

(1969)

of Cestode

Michael

1. Nielandl

Calcareous and Theodor

Corpuscle

Formation

von Brand

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Department of Health, Education, and Welfare, Bethesda, Maryland 20014 (Submitted

for publication,

15 October

1968)

NIELAND, M. L. AWJ VON BRAND, T. 1969. Electron microscopy of cestode calcareous corpuscle formation. Experimental Parasitology 24, 279-289. The formation of calcareous corpuscles in the common cat tapeworm, Taenia taeniaeformis, is described. The corpuscle forms in a cytoplasmic cavity, and the corpuscle-forming cell appears to expend itself in the production of one corpuscle. Two corpuscular components were recognized: a homogeneous matrix that may correspond to one of the organic moieties known to be present in corpuscles, and a granular substance that may represent inorganic material. The corpuscle enlarges by the accretion of these two components in concentric layers with progressive compression of the surrounding cytoplasm. INDEX

DESCRIPTORS:

Taenia

taeniaeformis,

calcareous

corpuscle

formation,

electron

microscopy.

Mineral concretions formed of concentric layers have been described in several invertebrate phyla including the Protozoa ( And& and Faur&Fremiet, 1967)) Platyhelminthes (Erasmus, 1967; Martin and Bils, 1964; von Brand et al., 1960), and Arthropoda ( Gouranton, 1968). Though these mineralized bodies share a morphological resemblance, there is no certainty that they have a common physiological function. Chemical assays have demonstrated a heterogeneity of constituents within the concretions of individual organisms, and variation where different organisms can be compared (Chowdhury et al., 1962; Gouranton, 1968; Scott et al., 1962; von Brand et al., 1960, 1965; Waterhouse, 1950). It appears that the concretions of insects are excretory in nature (Waterhouse, 1950). Among platyhelminths, concretions of trematodes are formed in the excretory system (Erasmus, 1 Present address: Sub-Department of Dermatology, The Johns Hopkins Hospital, 601 N. Broadway, Baltimore, Maryland 21205. 279

1967; Martin and Bils, 1964), but there is no evidence that those of cestodes have an excretory role. Cestode concretions, referred to as calcareous corpuscles, occur in vast numbers in the strobilar parenchyma of both juvenile and adult stages of most species of tapeworms. They range in diameter up to 30 ~1 ( Diamare, 1930), and may constitute as much as 417’ of the dry weight of the organism (Schopfer, 1932). A number of functions have been suggested for calcareous corpuscles. Their large carbonate content may serve to neutralize the acidic products of metabolism, or protect the organism as it passes through the stomach of the definitive host. Corpuscles have also been considered to protect the tissue stage of the organism against calcification by localizing the considerable amounts of calcium that diffuse into it (Desser, 1963). It is quite likely that calcareous corpuscles, whether or not they have any of the abovementioned functions, also have an active role in the organism’s metabolic processes.

280

NIELAND AND VON BRAND

Von Brand and Weinbach (1965) concluded that calcareous corpuscles may constitute a phosphate reservoir for the metabolic needs of tapeworms which, because of their dependence on carbohydrate utilization (Read and Simmons, 1963), would require large amounts of phosphate for the phosphorylation of hexoses and for other phosphorylating processes. Previous studies differ in regard to the formation of calcareous corpuscles (Chowdhury et al., 1962; Logachev, 1951; Timofeyev, 1964). Whether they arise by a kind of metastatic calcification or by cellular formation has not been clear. The present study was undertaken to answer this question. MATERIAL

AND METHODS

Anterior Taenia segments of adult taeniaeformk, obtained from the intestine of an experimentally infected cat, were selected for this study. In the anterior region new segments are formed and increase in size rather rapidly, suggesting that corpuscles in the process of formation would be found more readily here than in mature or gravid proglottids. The segments were fixed overnight at 4°C in 6.25y0 glutaraldehyde buffered with 0.1 M phosphate at pH 7.4, washed with 0.1 M phosphate buffer, and postfixed for 3 hours at 4°C with 2% osmium tetroxide buffered with 0.056 M Verona1 acetate. After ethanol dehydration the tissue was infiltrated overnight in an equal mixture of Epon (Luft, 1961) and propylene oxide, embedded in Epon, and polymerized for 48 hours at 60°C. Sections cut on an LKB Ultrotome with a diamond knife were collected on carbon-Formvar membranes, stained with lead citrate (Reynolds, 1963), and examined in an RCA EMU-3G electron microscope.

OBSERVATIONS

Paraffin sections showing the corpusclerich parenchyma are illustrated in Figs. 1 and 2. The tegument and layers of muscle lie on either side of this region. The corpuscles vary in size and degree of calcification, and there is a suggestion of an annulate structure in some with a densely stained center and peripheral margin (Fig. 2). In electron micrographs the parenchyma appears as expanses of glycogen-containing cytoplasm belonging to highly ramified and interdigitated cells. Embedded in the parenchyma are flame cells, collecting ducts, and cells in various stages of corpuscle formation. The typical appearance of a cell in an early stage of corpuscle formation is shown in Fig. 3. The cytoplasm of this cell contains a cavity in which the corpuscle is forming, a prominent eccentric mitochondria, nucleus, rough endoplasmic reticulum, free particles resembling ribosomes, and Golgi membranes. Although cytoplasm containing glycogen rosettes closely adheres, corpuscle-forming cells do not contain definitely recognizable glycogen particles. The membrane bordering the corpuscle appears irregular and folded, and the clefts between the folds are continuous with the cavity containing the corpuscle (Figs. 3 and 4). Occasional vacuoles are noted that contain material of a density comparable to that of the homogeneous component of the corpuscle (Fig. 4). The corpuscle appears to have two morphologically distinct components : a homogeneous matrix of intermediate density, and a granular material that frequently is deposited in whorls or in patterns that resemble vesicles (Figs. 3 and 4, 6-8). Figure 7 shows a young corpuscle containing vesicles which, when present, may be sites for deposition of the

CESTODE CALCAREOUS

CORPUSCLE FORMATION

Frc. 1. Light micrograph of the anterior segments showing the very densely stained calcareous corposcles in the center of the parenchyma. Layers of muscle lie on either side. Von Kossa and safranin. x40. FIG. 2. Higher magnification view of the parenchymal zone with a suggestion of concentricity in some corpuscles with a dense center and peripheral margin (arrows). Von Kossa and safranin. X 160.

granular material as suggested by Fig. 8. As the corpuscle enlarges, the granular material is deposited more uniformly in concentric rings that appear to alternate with layers of the homogeneous matris

(Figs. 9 and 10). With enlargement, the corpuscle impinges on and indents the nucleus which becomes progressively compressed toward the periphery of the cell (Figs. 5 and 9). The cytoplasm sur-

FIG. 3. Early stage in corpuscle formation. The corpuscle (C) appears to contain two components: a homogeneous substance and a granular material. Arrows indicate the folded membrane lining the cavity containing the corpuscle. The cytoplasm contains a prominent eccentric nucleus (N ), mitochondria (M), free particles resembling ribosomes, and rough-surfaced endoplasmic reticulum in addition to the cavity containing the corpuscle. ~24,000. 282

CJZSTODE

CALCAREOUS

CORPUSCLE

FORMATION

283

FIG. 4. A corpuscle-forming cell with eccentric nucleus (N) and corpuscle (C). The Golgi membranes ( G) and vacuoles (arrows) containing a homologons material may be involved in the synthesis of the corpuscular matrix. ~28,100.

rounding the larger corpuscles becomes extremely thin and contains few recognizable structures (Fig. 10). Eventually, the granular material may be heavily deposited in closely spaced dense concentric rings (Fig. 11). The corpuscles illustrated are in the lower to middle range in size (up to 7 cl) and probably would enlarge still further. As the corpuscles increase in size, and become increasingly mineralized, sectioning becomes singularly difficult either because of their hardness or lack of penetration of the embedding medium, a property noted bv Gouranton (1968).

DISCUSSION

The formation of calcareous corpuscles described here differs from that previously reported. Logachev ( 19X), in a light microscope study, claimed that petrification begins in the nucleus of a parenchymal cell and that deposition of concentric rings proceeds until the cell is completely filled. The rim of protoplasm breaks down, and the corpuscle is set free. Chowdhury et al. (1962), also using the light microscope, felt that certain mesenchymal cells had a selective affinity for calcium, allowing the gradual accumulation of the latter around them in

284

NIELAND

AND

VON

BRAND

CXSTODE

CALCAREOUS

CORPUSCLE

FORMATION

285

FIG. 5. Corpuscle-forming cell probably somewhat later in corpuscle formation than Figs. 3 and 4. Corpuscle (C) is beginning to impinge on and indent the nucleus ( N ). ~24,400. FIG. 6. Central portion of a corpuscle showing the granular material deposited in whorls. The arrow points to the thin rim of cytoplasm stretched around the corpuscle. ~20,000. FIG. 7. Vesicles lying in the center of a corpuscle. The granular component of the corpuscle may be d eposited on these to give them the appearance shown in Fig. 8. ~55,000. FIG. 8. Center of a corpuscle with the granular material deposited in a pattern resembling vesicles. x37,500.

FIG. 9. An older corpuscle showing the nucleus (N) beginning to be deposited in concentric rings. ~21,300.

displaced

to the side of the granular

material

286

FIG. 10. The cytoplasm structures. X 21,200.

NIELAND

surrounding

AND

this corpuscle

successive layers, ultimately rendering the cell indistinguishable. In the only previous electron microscope study, Timof eyev (1964) reported that a large intracellular

VON

BRAND

is quite attenuated

and contains

few recognizable

cavity bordered by a double membrane formed initially by the coalescence of cytoplasmic vacuoles, and gradually filled from with concentrically deposited the center

CESTODE

CALCAREOUS

FIG. 11. Central Dortion of a corpuscle dense layers (arrows): x20,900.

showing

rings. Although cells containing large dilated cisternae of rough-surfaced endoplasmic reticulum were seen in the present study, no stages could be found that suggested a transformation of these cells into corpuscle-forming cells. The cavity in which the corpuscle forms is lined by a single smooth membrane and enlarges only as the corpuscle increases in diameter. It probably forms by the coalescence of Golgi vesicles. Corpuscle-forming cells could not be identified prior to corpuscle formation. The formation of calcareous corpuscles differs in some respects from that described for the mineral concretions of protozoa (Andre and Faurk-Fremiet, 1967)) arthropods (Gouranton, 1968), and other platyhelminths (Erasmus, 1967; Martin and Bils, 1964). In arthropods the concretions form within cisternae of endoplasmic

CORPUSCLE

the granular

287

FORMATION

material

deposited

in closely

spaced

reticulum (Gouranton, 1968) and many concretions may form in a single cell. In protozoa, concretions form in “concretion vacuoles” which may be continuous with ergastoplasmic sacs (Andre and FaurCFremiet, 1967). In the metacercariae of the trematode Acanthoparyphium spinulosum the concretion material reaches the excretory vessel in a flocculent state and the corpuscle becomes organized only in the lumen of the excretory canal (Martin and BiIs, 1964). In the cestode Taenia taeniaeformis the corpuscles are formed intracellularly, and only one corpuscle is formed in a given cell, the latter apparently expending itself in its formation. Furthermore, the cestode corpuscle may attain a size much larger than that described for the other groups of invertebrates. The Golgi membranes and vesicles that contain a homogeneous material, and

288

NIELAND

AND

which are frequently noted in corpuscleforming cells, may contribute one component of the corpuscular matrix. The vesicular remnants shown in Fig. 7 may be derived from these. Saccules of endoplasmic reticulum which appear to communicate with the corpuscle cavity through clefts in the bordering cytoplasm may contribute another moiety. The granular material that is deposited in dense rings possibly corresponds to the mineral component of the corpuscle. It is electrondense in unstained sections also. Cestode calcareous corpuscles contain protein and mucopolysaccharide as part of their organic moiety (Chowdhury et al., 1962; von Brand et al., 1960). Corpuscles that have been incinerated to remove their organic components retain the concentric rings (Scott et al., 1962). Possibly the homogeneous material most prominent in young corpuscles corresponds to the mucopolysaccharide that has been located in the center of the corpuscles (Chowdhury et al., 1955; von Brand et at., 1960). The protein, histochemically demonstrated as concentric rings in paraffin sections (von Brand et al., 1960), may provide the structural framework or substrate for deposition of the mineral components. ACKNOWLEDGMENT The authors express their thanks to Dr. Harley G. Sheffield for the use of his laboratory, and to him and Dr. Marie U. Nylen of the National Institute of Dental Research for their kind assistance in reviewing the manuscript. REFERENCES AND&, J. AND FAUR&FREMIET, E. 1967. Formation et structure des concr&ons calcaires chez Prorodon morgani Kahl. Journal de Microscopic 6, 391-398. CHOWDHURY, A. B., DASGUPTA, B., AND RAY, H. N. 1955. ‘Kemechtrot’ or nuclear fast red in the histochemical determination of calcareous corpuscles in Taenia saginata. Nature 176, 701-702. CHOWDHURY, A. B., DASGUPTA, B., AND RAY, H. N. 1962. On the nature and structure

VON

BRAND

of the calcareous corpuscles in Taenia saginata. Parasitology 52, 153-157. DESSER, S. S. 1963. Calcium accumulation in larval Echinococcus multiloculuris. Canadian Journal of Zoology 41, 10551059. DIAMARE, V. 1930. Note d’istofisiologia sui cestodi II. Sui corpuscoli calcarei dei cestodi. Rinascenza Medica 7, 315-316. ERASIMUS, D. A. 1967. Ultrastructural observations on the reserve bladder system of Cyathocotyb bushiensis Khan, 1962 (Trematoda: Strigeoidea) with special reference to lipid excretion. Journal of ParasitoZogy 53, 525-536. structure, GOURANTON, J. 1968. Composition, et mode de formation des conc&ions min&ales dans l’intestin moyen des Homopt&es Cercopides. Journal of Cell Biology 37, 316-328. the strucLOGACHEV, E. D. 1951. Concerning ture and development of calcareous corpuscles in tapeworms. Doklady Akademii Nauk SSSR (In Russian) 80, 693-696. LUFT, J. H. 1961. Improvements in epoxy resin embedding methods. Journal of Biophysical and Biochemical Cytology 9, 409414. MARTIN, W. E. AND BILS, R. F. 1964. Trematode excretory concretions: formation and of Parasitology 50, Journal fine structure. 337-344. READ, C. P. AND SIMMONS, J. E. 1963. Biochemistry and physiology of tapeworms. Physiological Reuiews 43, 263-305. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 208-212. 1932. Recherches physicoSCHOPFER, W. H. chimiques sur le milieu int6rieur de quelques parasites. Revue Suisse de Zoologie 39, 59194. SCOTT, D. B., NYLEN, M. U., VON BRAND, T., AND PUGH, M. H. 1962. The mineralogical composition of the calcareous corpuscles of Taenia taeniaeformis. Experimental Parasitology 12, 445-458. TIMOF’EYEV, V. A. 1964. Electron microscope studies on the calcareous corpuscles of the plerocercoid and the sexually mature form of Schistocephalus pungitii. Dokludy Akademii Nauk SSSR. (In Russian) 156, 12441247. VON BRAND, T., MERCADO, T. I., NYLEN, M. U., AND SCOTT, D. B. 1960. Observations on function, composition, and structure of

CESTODE

VON

VON

CALCAREOUS

cestode calcareous corpuscles. Experimental Parasitology 9, 205-214. BRAND, T., SCOTT, D. B., NYLEN, ht. U., AND PUGH, M. H. 1965. Variations in the mineralogical composition of cestode calcareous corpuscles. Experimental Parasitology 16, 382-391. BRAND, T., AND WEINBACH, E. C. 196.5. Incorporation of phosphate into the soft

CORPUSCLE

FORMATION

289

tissues and calcareous corpuscles of larval Taenia taeniaeformis. Comparatil;e Biochemistry and Physiology 14, 11-20. WATERHOUSE, D. F. 1950. Studies of the physiology and toxicology of blowflies. XIV. The composition, formation, and fate of the granules in the Malpighian tubules of Lncilia cuprina larvae. Australian Journal of Scientific Research Series B, 3, 76-112.