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Biol. Res. v.35 n.3-4 Santiago 2002
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Biol Res 35: 365-371, 2002
Echinococcus granulosus protoscolex formation in
natural infections
MARIO GALINDO, M JULIETA GONZALEZ AND NORBEL GALANTI*
Program of Cellular and Molecular Biology, Institute of Biomedical Sciences (ICBM), Faculty of
Medicine, University of Chile, Santiago, Chile.
ABSTRACT
Echinococcus granulosus is a parasitic platyhelminth that is responsible for cystic hydatid disease. From the inner, germinal layer of hydatid cysts protoscoleces are generated, which are are the infective forms to the dog. Systematic studies on the cell biology of E. granulosus protoscolex formation in natural infections are scarce and incomplete.
In the present report we describe seven steps in the development of protoscoleces. Cellular buds formed by a clustering of cells emerge from the germinal layer of hydatid cysts. The buds elongate and the cells at their bases seem to diminish in number. Very early on a furrow appears in the elongated buds, delimiting anterior (scolex) and caudal (body) regions. Hooks are the first fully-differentiated structures formed at the apical region of the nascent scolex. In a more advanced stage, the scolex shows circular projections and depressions that develop into suckers. A cone can later be seen at the center of the hooks, the body is expanded and a structured neck is evident between the scolex and the body. During protoscolex development this parasitic form remains attached to the germinative layer through a stalk. When fully differentiated, the stalk is cut off and the infective protoscolex is now free in the hydatid fluid.
Key terms: Echinococcus granulosus, protoscolex formation, hydatid cyst
INTRODUCTION
The cestode Echinococcus granulosus is the causative agent of cystic hydatid disease, or hydatidosis, which is recognized as one of the major zoonoses, affecting both humans and domestic animals in various parts of the world (Gottstein and Hemphill, 1997). The disease is due to the pressure exerted on the viscera of the intermediate hosts by hydatid cysts. The inner germinal layer of these cysts is cellular, and the protoscolex, the larval form of the parasite, is developed from those cells. Mature protoscoleces are liberated into the cyst lumen. Although this flatworm has been studied over the years, basic aspects of its cell biology have received little attention. Specifically, systematic studies on the characterization of protoscolex formation in natural infections are scarce and incomplete.
An early report clearly defined the ultrastructure of the germinal layer in human hydatid cysts and proposed steps in the development of brood capsules and protoscoleces (Bortoletti and Ferreti, 1973). That work was followed by a more detailed description of the formation of those structures using experimental infections in Mongolian jirds. In that work a diagramatic description of the formation of brood capsules and protoscoleces in secondary hydatid cysts was reported (Thompson, 1976). Buds are formed in the germinal layer and grow toward the cyst cavity. The buds later become stalked and vacuolated. A new process of budding is initiated from the inner layer of the cells of these cavities, which leads to the formation of protoscoleces.
The study of protoscolex formation from the germinal layer of hydatid cysts in natural primary infections of E. granulosus is of importance considering that it determines the fertility of the cyst, which should be considered both in diagnosis and therapeutic practice (Eckert et al, 1995).
In this work we describe seven steps in the formation of protoscoleces at the germinative layer of hydatid cysts in natural infections in sheep, considering not only the formation and growth of the buds from the germinal layer of the cysts, but also the timing of the appearance of structural features in the nascent protoscoleces.
MATERIALS AND METHODS
Samples
E. granulosus hydatid cysts were obtained from livers or lungs of sheep killed at the Lo Valledor slaughter house in Santiago and from Coyhaique and Puerto Porvenir in the south of Chile. Cyst fertility was determined by the presence of free protoscoleces in the hydatid fluid and of growing protoscoleces attached to the germinal layer. Other parameters of cyst fertility such as a whitish color and thickness of the germinal layer were also considered (Bortoletti and Ferreti, 1978). Germinal layers joined to laminated layers were dissected from open cysts. When needed, protoscoleces were decanted by gravity from the hydatid fluid, washed in PBS pH 7.2 at 38.5 º C and treated with pepsin 0.1% in Hanks' salt solution pH 2.0 at 38.5º C for 15 min. Pepsine was removed by four washings with Hanks' medium. Viability of the protoscoleces was evaluated on the basis of body movements and flame cell activity as observed under a light microscope.
Light microscopy
For light microscopy studies, protoscoleces and pieces of germinal layer were washed three times in 0.1 M sodium cacodilate (buffer A) at 4º C and fixed in 2.5% glutaraldehyde in buffer A at 4º C for 3 hrs. The samples were then washed three times in buffer A at 4º C, postfixed in 1% OsO4 prepared in buffer A at room temperature for 1 hr, washed three times in buffer A at 4º C and observed directly.
Alternatively, samples fixed in glutaraldehyde and postfixed in OsO4 were embedded in Epon, and 0.5-1.0 µm sections were cut in an automatic Zeiss ultramicrotome. These sections were stained in toluidine blue and observed directly. In some experiments, pieces of germinal layer were fixed in alcoholic Bouin, embedded in paraffin, and 5 µm sections were stained in hematoxylin-eosin. All these specimens were observed under a Nikon light microscope.
Electron microscopy
For scanning electron microscopy work, protoscoleces and pieces of germinal layer fixed in glutaraldehyde and postfixed in OsO4, as described above, were dehydrated in ethanol and acetone, dried in a Polaron E 3000 apparatus and then sputter-coated with gold under a Sputtering Device Polaron E 5000. Samples were observed under a Zeiss scanning electron microscope.
RESULTS
Stages in the formation of protoscoleces
A piece of germinal layer from a fertile hydatid cyst showing different steps in the process of formation of protoscoleces is observed in Figure 1. The earliest step is a spheric bud (a) that elongates, narrowing at its base (b), adjacent to the germinal layer (GL). In a later step (c), a developing protoscolex showing the rostellum (R) and the body (B) is observed. At this stage, hooks (H) are already developed while suckers (S) as well as other body structures are still in formation. A stalk joining the growing protoscolex to the germinal layer can be seen (arrow). In (d), a well-developed evaginated protoscolex with a scolex (Sc) and body (B) is shown. In (e), an invaginated protoscolex is observed. Both protoscoleces are still attached to the germinal layer through stalks (arrows).
FIGURE 1. Light microscopy micrograph of a piece of germinal layer from a fertile hydatic cyst showing different stages during the formation of protoscoleces. (a®) spheric buds. GL, germinal layer. (b®) elongated bud. (c®) protoscolex in differentiation. H, hooks; R, rostellum; S, suckers; B, body; (Þ), stalk. (d®) evaginated protoscolex. Sc, scolex; B, body. (e®) invaginated protoscolex. The sample was fixed in glutaraldehyde and post fixed in osmium tetroxide. Bar = 50 µm.
In Figure 2, six steps in the formation of protoscoleces are shown as observed under a scanning electron microscope. In 2A a bud (double arrowheads) is growing from the germinal layer (GL); a clear difference can be appreciated between the surfaces of both structures, with the bud presenting a more homogeneous aspect than the germinal layer. In 2B, the bud has elongated and stretched at its base (asterisk). A furrow (arrowheads) appears delimiting an anterior and a caudal region. The surface is still homogeneous on the entire growing bud. This structure elongates even further, and a spheric head develops in the anterior region, from which the scolex will be formed (2C, arrowheads). The stretching at its base is more evident (asterisk) and will produce a stalk. The whole structure resembles a club. In a next step (2D), a scolex (Sc) in formation is shown, presenting well-developed hooks (H) at its apical regionand lateral projections (arrows), from which the suckers will eventually form. Clearly, hooks are the first fully-differentiated structures that appear during protoscolex formation. A rostellar base (dot) is already observed between the hooks and the lateral projections while a neck (arrowheads) is visible between the scolex (Sc) and the body (B). The surface of this emerging protoscolex is still homogeneous. At this stage of protoscolex formation, the stalk elongates increasing the distance between the protoscolex and the germinal layer (2D, asterisk). In a more advanced stage (2E), the scolex shows circular projections and depressions in the region where the suckers are forming (small arrows). In this stage the hooks are well developed (H) and a cone (C) can be seen at their center. The rostellar base is more defined (dot). The body (B) has expanded and the neck (arrowheads) between the scolex and the body is now more evident. Finally, in 2F, a fully developed protoscolex is shown, in which the scolex (cone, hooks, rostellar base and suckers) is well defined. The surface of the different structures in the mature protoscolex is now heterogeneous. During all these processes the protoscolex remains attached to the germinal layer through a stalk (asterisks in 2B through 2E).
FIGURE 2. Scanning electron micrographs of progressive stages during the formation of E. granulosus protoscoleces. (A) Undifferentiated spheric bud ( ). GL, germinal layer. (B) Early elongated bud. (>), furrow; (*), base. (C) Late elongated bud. (D) Protoscolex in differentiation. H, hooks; (•), rostellar base; Sc, scolex; (®), presumptive suckers; (>), neck; B, body; (*), stalk. (E) Protoscolex in differentiation. C, rostellar cone; (®), suckers in differentiation. (F) Fully formed protoscolex. C, rostellar cone; H, hooks; (•), rostellar base; S, suckers; (>), neck; B, body. A-B, bar = 20µm; C-F, bar = 40µm.
Cellular organization in the growing
protoscolex
A similar series of events related to protoscolex formation, as observed in paraffin-embedded sections stained in hematoxylin-eosin, is shown in Figures 3A-C. The first of these events is a clustering of cells in the germinal layer (GL). Well-defined limits between this cluster or emerging bud and the adjacent tissue are observed (3A, arrows). The surface of the cluster of cells seems to be thicker than the one of the adjacent germinal layer (3A).
In (3B) the bud has grown, al though it is still deeply embedded in the germinal layer (GL); a stretching at the base of the bud is observed (arrows). No limits are evident between groups of cells in the bud or between the bud and the germinal layer (3B). The bud elongates (3C), and the cells at its base seem to diminish in number as the stalk that attaches the growing protoscolex to the germinal layer (GL) stretches (arrows). At the tip of this bud the cells are concentrated, forming a spheric structure. A border appears separating the cells of the spheric structure from the cells of the body of the growing protoscolex. This border is also evident through a stretching of the tegument (arrowheads). The entire structure resembles the one described in Figures 2B and 2C. The body of the bud shows a lower density of cells and a thicker tegument than the head.
A set of Epon-embedded serial sections of a growing protoscolex, fixed in glutaraldehyde, postfixed in osmium tetraoxide and stained in toluidine blue, is shown in Figures 3D-F. In 3D the scolex (Sc) and the body (B) are clearly distinguishable. Cell density is very low at the stalk (arrows), increasing at the body and at the scolex, where they are found mainly laterally (see also 3E-F). The border between the scolex and the body is evident (long arrowheads). In Figures 3E-F, groupings of cells are found at the place where suckers will be formed (short arrowheads). At this step cell density is very low at the stalk, showing cellular discontinuity between the germinal layer (GL) and the growing protoscolex (3F, arrows).
FIGURE 3. Light microscopy micrographs of sections of progressive stages during the formation of E. granulosus protoscolex. (A) Undifferentiated spheric bud (Þ). GL, germinal layer. (B) Early elongated bud.(Þ), base. (C) Late elongated bud. Note the border (>) between the cells of the anterior and posterior regions of the bud. (Þ), base. (D-F) Serial semi-thin sections of an early stage in the scolex morphogenesis of the protoscolex. Note the cells clustering (>) in the presumptive suckers. Sc, scolex; (>), neck; B, body; (Þ), stalk. Bar = 40 µm.
With this structural information, a sequence of seven steps can be distinguished from the early bud to a mature protoscolex that has broken free from the germinal layer into the hydatid fluid (Fig. 4). As early as step 4 (letter D), differentiation is evident with the hooks being the first defined structure of the protoscolex.
FIGURE 4. Diagrammatic representation of the development of E. granulosus protoscolex in natural infection. LL, laminated layer; GL, germinal layer; Tg, tegument. (A) Spheric bud. (B) Early elongated bud showing an anterior and a posterior region. (C) Late elongated bud with presumptive scolex and body. (D) Protoscolex in development showing rostellum formation. (E) Protoscolex in development showing suckers formation (F) Fully developed protoscolex attached to the germinal layer. (G) Protoscolex free in the cyst cavity.
DISCUSSION
The protoscolex of E. granulosus is central in the biological cycle of that parasite and is of particular interest in primary and secondary infections. It is known that the protoscoleces are formed from buds that emerge from the germinal layer of hydatid cysts; however, the timing of appearance of each of the different structures present in the adult protoscolex has not been reported. Likewise, there are no reports on the structuring of these protoscoleces from undifferentiated cells or from a specific cell line or from a specific cellular territory of the germinal layer.
The present report clearly shows that buds emerge from any region of the germinal layer of hydatid cysts, showing no specific cell types. These buds develop anterior and caudal regions very early in their growth process. The early appearance of two main regions of the protoscolex, the scolex and the body, suggests the presence of a gradient of morphogens in the bud, such as the products of the maternal effect genes and/or the homeotic genes that are active during the embryo's development. Indeed, some genes that may participate in the process of development and maturation of the protoscoleces have been described and their products located in specific cells (Oliver et al, 1992; Esteves et al, 1993; Martinez et al, 1997). Those signalling proteins participating in the generation of spatial organization during growth, such as the homeobox-containing transcription factors, have been identified in most pluricellular organisms (McGinnis et al, 1984; Burglin, 1995)
The appearance of functionally-specialized cell types in a spatial distribution is of importance in Platyhelminthes, which are the first organisms in evolution showing bilateral organization (Reuter and Gustafsson, 1995; Hausdorf, 2000). During protoscolex formation, different cell types develop early, organizing themselves into cellular territories with specific functions (scolex, hooks, suckers, etc). In fact, spatial organization is unique in the evolution of multicellular organisms. Interestingly, POU-domain sequences have been described in the flatworm Dugesia tigrina (Stuart et al, 1995) and in the planarian Dugesia japonica (Orii et al, 1993). Taking all this information into consideration, it is possible that the early appearance of anterior-caudal polarity and bilateral symmetry in the growing protoscoleces described here may be related to the presence and function of homeotic genes in Echinococcus. Despite the obvious importance and appeal of this idea, it remains to be proven.
The growth process of a bud from the germinal layer, as well as the formation and maturation of protoscoleces from the buds, should have proliferative bases; however, increased cell size and secretion of extracellular material are other manifestations of growth that may also be involved during early steps of protoscolex formation. Clearly, cell proliferation is involved in the formation of the bud as well as in bud elongation. On the other hand, the process of differentiation that gives rise to the hooks is accompanied by an increase in size of the involved cells. Thus, increases in cell number and cell size appear to be present during the early development of protoscoleces.
More information is still needed, however, to reach a comprehensive knowledge of the process of formation and growth of the protoscolex, both at the cellular and at the molecular levels. This information may generate new ways to control the parasite through the development of specific pharmacological agents that work against structures emerging from the germinal layer. On the other hand, it may also point to specific markers of cyst fertility, which could be used for diagnosis and therapeutic approaches.
ACKNOWLEDGMENTS
We would like to thank Dr. Jorge Allende and Dr. Remigio López for corrections. This work was supported by grants from FONDECYT-CHILE 1970766 and 1010817, NRTP/SIDA-SAREC, Fundación Andes-Antorchas-Vita and DID-U. de Chile I013-99/2.
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* Corresponding author: Norbel Galanti. Program of Cellular and Molecular Biology. Institute of Biomedical Sciences (ICBM). Faculty of Medicine, University of Chile, Casilla 70061, Correo 7. Santiago, Chile. Telephone: (56-2) 678-6475. Fax: (56-2) 737-3158 . e-mail: ngalanti@machi.med.uchile.cl
Received: June 21, 2002. In revised form: August 19, 2002. Accepted: August 27,2002
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