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Developmental changes of Echinococcus multilocularis metacestodes revealed by tegumental ultrastructure and lectin-binding sites R. LEDUCQ and C. GABRION Laboratoire de Parasitologie Compare'e, Place Eugene Bataillon, U.S.T.L. Montpellier II, 34095 Montpellier France

cedex 5,

(Received 11 June 1991; revised 28 July 1991; accepted 28 July 1991) SUMMARY

Ultrastructural investigations (SEM, TEM) combined with lectin-binding analysis, have revealed concurrent modifications in tegumentary structure and surface glycoconjugates during the establishment and differentiation of Echinococcus multilocularis metacestodes in jirds. The laminated layer, which is amorphous and rich in polysaccharides when initially secreted by the young cyst, takes on a different appearance and has a different glycoconjugate composition according to whether the cyst becomes fertile or sterile. The laminated layer of fertile cysts transforms into a microfibrillar matrix, the protein content of which may increase while sugar content decreases during protoscolex differentiation. Independently of this structure, brood capsules, from which arise protoscoleces, are formed by invagination of the cyst tegument. The intense secretion of glycoconjugates from the brood capsule wall during invagination may serve to interact with host factors passing through the laminated layer. The combined use of ultrastructural study and lectin labelling has allowed the demonstration of an ultrastructural and biochemical gradient of differentiation of the protoscolex. Seven stages of differentiation have been described. The possibility that the excreted-secreted tegumentary glycoconjugates, revealed by lectin labelling during protoscolex differentiation, might be the gradual biochemical expression of one or several stimuli implicated in the phenomenon of protoscolex maturation, is discussed. Key words: Echinococcus multilocularis, metacestode, tegumentary structures, carbohydrate moities, differentiation, hostparasite relationships.

INTRODUCTION

The ultrastructure of the various developmental stages of Echinococcus metacestodes in rodents has been described by a number of authors (Mankau, 1957; Ohbayashi, 1960; Sakamoto & Sugimura, 1969, 1970; Lascano, Coltorti & Varela-Diaz, 1975; Thompson, 1976; Marchiondo & Andersen, 1983). Differences in tegumentary structures (microvilli and microtriches) and glycocalyx have been observed between the cyst, brood capsule and protoscolex as well as between different regions (scolex and soma) of mature protoscoleces. This diversity of structure suggests a diversity of function of these surface structures (absorption, excretion-secretion, ancillary functions) (Arme, 1988). It can be assumed that sugar moities of glycoconjugates of the metacestode syncytial tegument are of prime importance in host-parasite relationships and in the parasite's interaction with its environment. Indeed, it has been demonstrated that carbohydrates, particularly extracellular polysaccharides, play essential roles in cellular functions (Bennett, 1963; Sharon, 1975; Muramatsu, 1989). T o date, however, there have been no studies concerning changes in excretedsecreted glycoconjugates of Echinococcus larval forms during establishment and differentiation in the Parasitology (1992), 104, 129-141

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intermediate host. This contrasts with other more extensively studied cestodes (Sandeman & Williams, 1984; Joshua, Harrison & Sewell, 1989), trematodes (Dunne, 1990) and nematodes (Kaushal et al. 1984; Ham, Smail & Groeger, 1988). This paper describes the use of lectins to reveal glycoconjugate sugar moities in semi-thin and thin sections of E. multilocularis metacestodes. This technique permits the direct comparison of the tegumental ultrastructure and sugar composition.

MATERIALS AND METHODS

Three-month-old female jirds (Meriones unguiculatus) were infected intraperitoneally according to the method of secondary echinococcosis (Norman & Kagan, 1961). The E. multilocularis isolate was obtained from Savoie (France) (Contat et al. 1983). Jirds were sacrificed 60-90 days post-infection. Infected organs and isolated cysts were rapidly removed and fixed overnight at 4 °C, by immersion in either of the following solutions: (i) fixative solution for lectin labelling, containing 4 % (w/v) paraformaldehyde, 0-1 % (v/v) glutaraldehyde, 0 1 M sucrose in phosphate-buffered saline (PBS) (015 MNaCl, 2-5 mM KC1, 8 mM N a 2 P O 4 and 1 mM

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Fig. 1. For legend see opposite.

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E. multilocularis cyst: sugars and differentiation

KH2PO4), (ii) fixative solution for ultrastructural studies containing 0-5 % (w/v) paraformaldehyde, and 2 % glutaraldehyde in PBS. Following fixation, samples were rinsed 3 times for 10 min in PBS. Morphological and ultrastructural studies Protoscoleces and vesicles were isolated in small plastic tubes covered at each extremity with a plankton-net. They were post-fixed for 1 h with 1 % OsO4 in PBS, then rinsed twice for 10 min with PBS and dehydrated in graded ethanol. Specimens were dried by the critical-point method in a Balzers M 9202 apparatus. Samples were sputtered with gold before observation on a Jeol JSM 35 scanning electron microscope. Parasite labelling with lectins

For light microscopy, the FITC (fluorescein isothiocyanate)-conjugated lectins (except Ricinus communis agglutinin 60 or RCA-II) and specific inhibitory sugars were the same as those used in previous studies (Leducq, Gabrion & Gabrion, 1988; Berrada-Rhkami et al. 1990; Leducq et al. 1990). Wheat-germ agglutinin (WGA) specific for iV-acetyl glucosamine residues (NacGlu), Helix pomatia agglutinin (HPA) with Phaseolus vulgaris agglutinin-E (PHA-E) and Soy bean agglutinin (SBA) specific for iV-acetyl galactosamine residues (NacGal), Concanavalin agglutinin (ConA) specific for mannose (Man) or glucose residues, Ulex europaensis agglutinin-I (UEA-I) specific for fucose residues and Ricinus communis agglutinin 120 (RCAI) specific for galactose residues (Gal) were purchased from Sigma (St Louis, USA). In order to check whether the parasite contains neuraminic acids, we also tested the lectin Limax flavus agglutinin (LFA) conjugated to FITC. Fluorescence was examined under a Zeiss epifluorescence microscope equipped with planapochromatic immersion lenses.

Whole specimen labelling

Isolated protoscoleces were rinsed twice for 10 min with PBS containing 5 mM NH4C1 (PBS-NH4C1) to block free aldehyde groups from host and parasite tissues prior to exposure to lectins. FITCconjugated lectins were diluted to 20/tg/ml in PBS and centrifuged at 43 000 £ for 3 min. Parasites were incubated with diluted lectins at room temperature in a dark moisture chamber for 1 h, then rinsed with PBS 3 times for 10 min. They were placed on slides and mounted with Mowiol 488 (Calbiochem, Hoechst, RFA) prior to observation.

Section labelling

The post-fixation step was omitted since Os0 4 modifies lectin binding to sugars (Suzuki et al. 1981; Roth, 1983). Samples were dehydrated on ice in a graded series of ethanol (15°-95°) and stored at - 2 0 °C for 20 min in 95° ethanol. Dehydrated tissues were embedded in a hydrophilic resin, London Resin White (Hard grade LRW, Touzard & Matignon, Fullam Products, New York) as follows: 1 vol. LRW/1 vol. 95° ethanol at - 2 0 °C overnight, 2 vol. LRW/1 vol. 95° ethanol at - 2 0 °C for 3 h, in LRW at 4 °C twice for 3 h then overnight and finally in LRW at 37 °C in closed capsules during 5 days. Embedded specimens were either cut immediately or stored at 4 °C. Thin (005-0-09/tm thick) and semi-thin (0-5-1/mi thick) sections were obtained on a Reichert Jung ultracut. Labelling of semi-thin sections. This method was previously described for sections of the parasite embedded with Spurr (Leducq et al. 1988). Sections were dried on 0-1 % gelatin-coated slides. They were incubated twice for 10 min at room temperature in PBS-NH4C1. FITC-lectins were diluted to 50/tg/ml in PBS and centrifuged at 43 000 £ for 3 min. Samples were covered with diluted lectins for 45 min at room temperature in a dark moisture chamber then rinsed twice for 10 min with PBS before mounting with Mowiol 4-88. Control tests

Fig. 1. Semi-thin and lectin-labelled thin sections (unstained) of different larval forms and tegumentary structures. (A) Distal cytoplasm (dc) and laminated layer (/) of a young cyst without brood capsules labelled with HPA-gold (5 nm diameter). The lectin mostly labels the slightly fibrous zone above the microvilli (m). v, vesicles. (B) Semi-thin section of a young cyst developing brood capsules (be). I, laminated layer; cm; cellular mass; arrows, invaginating brood capsule wall. (C) Stage 2 protoscolex labelled with WGA-gold (10 nm diameter). The lectin reacts with the clear vesicles of distal cytoplasm (v) and the glycocalyx of microvilli (m) and truncated microtriches (tm). (D) Glycocalyx of brood capsule microvilli (m) labelled with SBA-gold (10 nm diameter). (E) Rostellar tegument of a stage 3 protoscolex, between the first and the second constriction, labelled by SBA-gold (10 nm diameter), bm, broad microtriches; v, clear vesicles of the distal cytoplasm (dc). (F) Sucker tegument of a stage 6 protoscolex labelled with WGA-gold (10 nm diameter); sm; spined microtriches; sc; sensory cell; v; clear vesicles of the distal cytoplasm (dc). (G) Soma tegument of a stage 4 protoscolex labelled with WGA-gold (10 nm diameter), be, blunt elevations; v, clear vesicles of the distal cytoplasm (dc). (H) Cell coat (cec) secreted on the soma tegument surface of a stage 7 protoscolex, labelled by a pre-embedding method with WGA conjugated with ferritin (arrows); be, blunt elevations.

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Fig. 2. For legend see opposite.

E. multilocularis cyst: sugars and differentiation

with sugars were performed as previously described (Berrada-Rhkami et al. 1990). Labelling of thin sections. Sections were collected on Formvar-coated grids. For ultrastructural labelling, the following lectins conjugated to gold particles (A520 = 5) of different diameters (5 to 20 nm; Sigma) were employed: WGA, SBA, HPA, ConA, UEA-I, peanut agglutinin (PNA) and Dolichos biflorus agglutinin (DBA). PNA and DBA are specific respectively for Gal and NacGal residues but they also reveal branched residues of aNacGal and /?Gal, either sugar as terminal (Goldstein & Poretz, 1986). They were diluted to 1/10 or 1/20 (v/v) with 0-1 M PBS containing 0-5 % A7638 albumin (Sigma) and 005 % Tween 20 (Sigma) = PAT. Grids were posed on drops of PAT (twice for 5 min) then on a drop of diluted lectin for 1 h at room temperature in a moisture chamber. Sections were rinsed with PAT (3 times for 5 min) then with distilled water (twice for 5 min). For multiple labellings, the lectins conjugated with the largest gold particles were used first (Roth & Binder, 1978). LRW contrasts specimens so the double staining with uranyl acetate and lead citrate was omitted on most of the samples. Sections were observed with a Jeol 200 CX transmission electron microscope. Some observations were made on sections of Spurr labelled by a pre-embedding method: fixed parasites were rinsed with PBS then with PBS-NH 4 C1; they were incubated overnight in diluted lectins and rinsed with PBS before embedding in Spurr's resin by the classic method. RESULTS

During the establishment and development of cysts in the intermediate host, the morphology and glycoconjugate composition of the metacestode undergo parallel changes. Modifications in host-parasite

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relationships were observed as ultrastructural and biochemical transformations of the laminated layer (and the distal cytoplasm of the cyst) that is in direct contact with host tissues. Transformations of protoscoleces during their differentiation were also analysed. Host—parasite relationships The laminated layer on the cyst surface showed differing appearance and glycoconjugate composition related to the metacestode development. These developmental changes have been split into three stages. (i) Cysts without brood capsules. The laminated layer was thin (2—4 fim), composed of an amorphous, slightly fibrous and apparently viscous material. It was strongly labelled by WGA, HPA (Fig. 1A), ConA, SBA and RCA-1. This labelling pattern remained the same on cysts strongly attacked by host cellular reactions. In degenerating cysts (cells of the germinal layer in contact with host's cells and filled with lysosomes), however, the laminated layer lost lectin labelling. WGA and RCA-1 were the last lectins to label the surface of these degenerative cysts; they were also the most reactive lectins with the extremely fibrous laminated layer of sterile cysts which looked like small hydatids and contained big calcareous corpuscules (Fig. 2 A) (ii) Cysts which developed brood capsules then protoscoleces. The laminated layer became thicker (4—10 /tm) and was divided into an electron-transparent zone adjacent to trie microvilli of the tegument and an electron-dense zone composed of a microfibrillate matrix towards the host. In the densest zone, reactions became very weak with SBA, ConA and WGA (Fig. 2C) but remained relatively strong with HPA. RCA-I labelling of the laminated layer

Fig. 2. In toto isolated protoscoleces and semi-thin sections of cysts in infected organs of jirds labelled with FITClectins. (A) Laminated layer (/) and big calcareous corpuscules (cc) of small hydatid-like sterile cysts strongly labelled by WGA-FITC. (B) Stage 2 protoscolex labelled with RCA-I-FITC; A, part A; B, part B. (C) Young cyst developing brood capsules (be) labelled with WGA-FITC. The densest zone of the laminated layer (/) is sparsely labelled in contrast with the tegumentary cytons (tc) of the invaginating zone. Strong labelling of the cone-shaped cavity (arrow) formed by the first outline of the brood capsule and the zone of granulation (2) towards the host's tissues (h) are partially aspecific. (D) Cyst containing protoscoleces of stage 6 (p6) and 7 (J>7) labelled with HPAFITC. Products of secretion (asterisk) rejected in the brood capsule react aspecifically with the lectin. g, glycogen labelled in cells of the cyst and protoscoleces; /, laminated layer; er, evaginated rostellum ; ir, invaginated rostellum; s, suckers; so, soma; cc, calcareous corpuscules. (E) Young cyst containing stage 1 protoscoleces (/>1) labelled with RCA-I-FITC. be, brood capsule; arrow: infolded channel (cut in section) formed by the invaginated brood capsule wall; /, laminated layer. (F) Stage 5 protoscolex in toio-labelled by SBA-FITC. r, rostellum; s, suckers; so, soma. (G) Cyst containing stage 6 protoscoleces labelled with SBA-FITC. The invaginated protoscoleces (/>) secrete a thin cell coat that is well-labelled by the lectin (arrow). Arrow-head: tegument of the invaginated zone (suckers and rostellum); be, brood capsule; cc; calcareous corpuscle; /, laminated layer. (H) Invaginated protoscoleces labelled with ConA-FITC. /, laminated layer of the cyst; be, brood capsule; arrow: labelled-cell coatjg', glycogen labelled in specialized and muscular cells. (I) Stage 7 invaginated protoscolex labelled with WGA-FITC. Arrow, labelled-cell coat; tc, tegumentary cytons; be, brood capsule; cc, calcareous corpuscules in the protoscolex and the germinal layer of the cyst; bl, basal lamina of suckers (s); r, rostellum.

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Fig. 3. For legend see opposite.

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E. multilocularis cyst: sugars and differentiation

remained strong (Fig. 2E). PNA and DBA were weakly bound to the laminated layer but some vesicles of the distal cytoplasm of the cyst reacted with these lectins. Microvilli of the distal cytoplasm increased in length and number. They developed a truncated spine (web-like structure) and formed small saccules on their tips which appeared to penetrate the laminated layer to reach the electrondense zone. Non-specific reactions with lectins (fluorescence observed in control tests with inhibitory sugars) were observed in a zone of necrosis or granulation in contact with host cells (Fig. 2C). Host cellular reactions were now organized in concentric zones around the fertile cysts. (iii)

When protoscoleces were invaginated.

The

laminated layer appeared as a dense material of regular thickness and devoid of lectin labelling (Fig. 2G and H). The reactive zone in contact with host cells disappeared (or lost lectin binding) when all the protoscoleces were invaginated. Metacestode differentiation

Beneath the laminated layer, and independently of this structure, the germinal layer of the cyst formed brood capsules by invagination of the tegument and adjunction of undifferentiated cells (Fig. 1B). The first outline of the brood capsule appeared as a cone-shaped cavity into which were secreted glycoconjugates that labelled strongly with WGA (Fig. 2C), HPA and RCA-1 (reactions partly non-specific) and less so with SBA and ConA. Clear vesicles of the brood capsule tegument (i.e. distal cytoplasm, covered with sparse microvilli, and tegumentary cytons) revealed a similar labelling pattern (Fig. 2 C) (reactions, this time all specific with the lectins cited above). The tegument of the protoscolex was derived from an up-pushing of the brood capsule wall (Fig. 2E), 1 or 2 days after invagination. During protoscolex development, the invagination zone of the brood capsule increased in size and became spherical. A slender and infolded channel formed by the brood capsule wall was seen between this zone and the

secondary vesicle containing protoscolex. Protoscolex differentiation was divided into several stages. Stage 1 (Fig. 2E). Young protoscoleces appeared as buds covered with sparse microvilli (Fig. 3B). At this stage, lectins reacted with glycoconjugates located on specific parts of brood capsules and immature protoscolex teguments. Lectins specific for iV-acetyl galactosamine ( +glucose) residues reacted with the glycocalyx (Fig. 1 D); those specific for galactose residues reacted with clear vesicles of the distal cytoplasm and the one specific for JV-acetyl glucosamine residues reacted with both of the latter structures (Fig. 1 C). During differentiation of the protoscolex, the brood capsule tegument became thinner, reacted less and less with lectins (Fig. 2E cf. G and H). At this stage, the protoscolex was undergoing important ultrastructural and biochemical modifications. Ultrastructural and lectin-binding site modifications of the tegument of the differentiating protoscolex are summarized in Fig. 4. Stage 2 (Fig. 3 A). The protoscolex bud is divided into two parts. At the base (part B), microvilli were progressively replaced by truncated microtriches (Fig. 1 C) over the whole of the tegument. On the part A (bottom of the protoscolex), numerous protrusions developed and truncated microtriches were restricted to the folds of 3 small constrictions, the second one being the most marked (Fig. 3B). The apical tegument bound more lectins (WGA, RCA-I, HPA, SBA and less with ConA) than the tegument of part B (Fig. 2B). Stage 3 (Fig. 3C). Protoscolex differentiation progressed via the formation of another important constriction dividing the part B into two parts: C and D. The parts A, C and D delimited respectively the future rostellum, suckers and soma. On the future soma, truncated microtriches were progressively replaced by blunt elevations (also called knoblike structures) (Fig. 1 G and H). The tegument of this part was little labelled with lectins. Spined microtriches (Fig. 1F) (0-5-0-8 /im long, 0-3 /tm

Fig. 3. SEM of the protoscolex at several stages. (A) Stage 2. Three small constrictions (arrow heads) appear at the bottom (^4) of the young protoscolex; B, base of the protoscolex. (B) Second constriction of the bottom of stage 2 protoscolex, m, microvilli; pr, protrusions; tm, truncated microtriches. (C) Apex of stage 3 protoscolex. m3, broad microtriches of the third constriction; ph, precursors of hooks; m\-2, broad microtriches from the first to the second constriction; m, microvitli of the future rostellum; sm, spined microtriches of the future suckers. (D) Stage 4. The dome above the hooks is invaginated, m, filamentous microvilli of the rostellum; h, hooks; ml-2, broad microtriches delimiting the base of the rostellum; sm, spined microtriches of suckers. (E) Rostellum of stage 5 protoscolex; m, microvilli; d, invaginable dome above the hooks; h, hooks; tp, tegumentary protrusions covering hooks. (F) Stage 6, sm, slender microtriches developed by the rostellar tegument covering hooks; sm, spined microtriches of suckers. (G) Stage 5 protoscolex, r, (invaginated) rostellum; s, suckers; so, soma. (H) Evaginated stage 6 protoscolex with the dome-shaped rostellum (r) in extension, s, well-formed suckers; sm, spined microtriches of suckers; sm, slender microtriches. (I). Invaginated stage 6 protoscolex, so, soma covered with blunt elevations. (J) Microtriches of the well-formed suckers, posteriorly directed. (K) Blunt elevations (b) and truncated microtriches (tm) covering the soma of stage 5 protoscoleces. (L) Fibrous cell coated secreted on the soma from stage 6.

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Stage 1

Stage 2

Stage 3

Rostellum

Suckers

Soma

Stage 5

•»•»••»,

Stage 6

Stage 7

Fig. 4. Transformation of the tegument of protoscoleces during their differentiation. For each stage uhrastructural characteristics of the tegument are illustrated on the left and the localization of lectin binding sites (WGA HPA SBA and RCA-1) are represented by arrow-heads on the right. The density of the arrow-heads indicates intensity of the reaction with all the lectins above cited. The large arrows indicate that the zone above this symbol is invaginable A) apex of immature protoscoleces or rostellum from the stage 3; (B) base of immature protoscoleces; (C) suckers(D) soma.

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large at the base) developed, instead of truncated mitrotriches, over the future suckers. The tegument of the suckers was labelled almost as intensely (by the same lectins as the stage 2) as the future rostellum. The future rostellum was completely covered by filamentous (long and flexible) microvilli, but also displayed other tegumentary structures. A multiplication of broad and angular spined microtriches was observed at the base of the rostellum. The length and thickness of these microtriches increased progressively up to the second constriction (0-8-3-5 /tm long, 0-3-1 /im large at the base) (Fig. 1 E). A narrow, double row of spined microtriches, that were smooth and slightly curved (3—4 fim long, 1 fim large at the base) appeared on the second constriction. These microtriches were precursors of hooks (without guard and handle). On the third constriction, sparsely distributed broad, angular microtriches (2-3-5 fim long, 1 fim large at the base) developed in a large double row. Stage 4 (Fig. 3D). Microtriches of the third constriction were presumably lost by shedding. In contrast, hooks, filamentous microvilli of the rostellum and blade-like mictrotriches of the suckers increased in length and thickness. On the rostellum and suckers, lectin labelling of the tegument was equally intense with most of the lectins. On the soma, the tegument was thicker and clear vesicles, apparently evolved from infolded channels running from the proximal tegumental membrane to the distal surface, were slightly labelled with WGA, RCA-1, HPA and SBA. In thin sections, labelling was observed up to the surface, on each side of the blunt elevations (Fig. 1 G). Stage 5 (Fig. 3E and G). The rostellar tegument thickened and formed tegumentary protrusions covering hooks. Long microvilli were then replaced by microtriches with long and slender spines. The tegument of the soma was infolded; on its surface, blunt elevations were more protuberant (Fig. 3K). The soma was again less intensely labelled with WGA, RCA-I, HPA and SBA (Fig. 2F) than suckers and the rostellum. Stage 6 (Fig. 3H and I). Because of the increases in length and density of the slender microtriches (up to 4 fim thick) (Fig. 3 F) due to the thick tegument covering the hooks, the dome-shaped rostellum (Fig. 3H) looked like a 'head of hair' of the protoscolex. The glycocalyx of these structures were strongly labelled with WGA and RCA-I, and less so with SBA, HPA (Fig. 2D) and ConA. Hooks, enclosed in the tegument, were completed by a guard and a handle. Suckers were now well-formed (slightly furrowed) and possessed a dense covering of posteriorly directed microtriches (1-6 fim long, 0-3 fim large at the base) (Fig. 3 J). Clear vesicles of

the distal cytoplasm were more intensely labelled in this sucker region than in the rostellar region (WGA Ss RCA-I > HPA = SBA > ConA). A thin, fibrous cell coat was secreted (Fig. 3 L) on to the soma, which was now completely covered with blunt elevations (and not truncated microtriches as before). During the cell coat secretion, in toto labelling revealed a high reactivity of this surface coat with WGA, RCA-I, HPA and SBA, while labelling of suckers (Fig. 1F) and rostellum glycocalyces decreased. Stage 7. The cell coat thickened (up to 4 fim thick) (Fig. 1 H). The protoscoleces remained invaginated from the moment when the surface coat became rigid and uniform. Clear Golgi vesicles of the tegumentary cytons of the rostellum, suckers and soma strongly reacted with lectins (WGA > RCA-I > SBA = HPA); an intense reaction was again seen on the suckers and rostellum glycocalyces (Fig. 21). WGA was the most reactive lectin with the distal cytoplasm of the soma. This lectin also reacted with the syncytium of excretory ducts in the protoscolex and with the basal lamina of suckers (Fig. 21). PNA bound to the basal lamina of the soma tegument and RCA-I to extracellular matrix in the centre of suckers. It is of note that numerous secretory products secreted into the brood capsules had non-specific reactions with lectins (Fig. 2D). Finally, the parasite revealed little labelling with LFA, UEA-I and PHA. The whole calcareous corpuscules of the germinal layer of the cyst were the only structures to be significantly labelled (partly aspecifically) by these lectins. They also reacted with WGA (Fig. 2 A and I) and RCA-I, but less so with HPA, ConA and SBA (Fig. 2G). They increased in number and diameter during maturation of protoscoleces. The matrix of calcareous corpuscules no longer reacted with lectins when protoscoleces were invaginated. In protoscoleces, whole calcareous corpuscules only reacted with WGA (Fig. 21) and RCA-I, less with SBA (Fig. 2G). ConA (Fig. 2H) and, to a much lesser extent, HPA (Fig. 2D) labelled glycogen in specialized and muscular cells.

DISCUSSION

In a preliminary study (Leducq et al. 1988), the sugar composition of E. multilocularis cysts of defined ages (containing developing or all invaginated protoscoleces) was examined by lectin labelling, with the same lectins used in the present paper, on etched, semi-thin, Spurr-embedded sections. The thorough study of ultrastructural transformations and modifications in glycoconjugate composition of the metacestode during its development in jirds required the detailed analysis of

R. Leducq and C. Gabrion tegumentary structures, in parallel with better adapted techniques for ultrastructural labelling of hydrophilic molecules (sugar moities). The hydrophilic London Resin White (LRW) was chosen because of its easy handling (easier than the Lowicryl resin for example, which requires handling at lower temperatures and sometimes displays a reduced preservation of ultrastructural morphology). The use of lectin labelling techniques on LRW sections and in toto, in conjunction with conventional ultrastructural studies by SEM, has enabled a more precise evaluation of the presence of, and developmental changes in, sugars on differentiating vesicles and protoscoleces of E. multilocularis. On the surface of young cysts, an amorphous, slightly fibrous and viscous material, constituting the primary laminated layer, is rich in glycoconjugates, sugar moieties of which contain principally galactose, NacGal, NacGlu and glucose or mannose. This richness in glycoconjugates is a proof of their viability since the laminated layer of degenerating cysts loose lectin labelling. Galactose residues appear as the predominant components of the laminated layer. On fertile young cysts, the proportion of galactose and hexosamines (NacGal and NacGlu) seem to be the same and NacGal appears more abundant than NacGlu. These results are in accordance with those obtained by Kore et al. (1967) for E. granulosus using chromatographic and electrophoretic methods. According to Sakamoto & Sugimura (1970) cysts develop this PAS + laminated layer 14 days after inoculation of E. multilocularis oncospheres in Cotton rats. In parallel with the organization of the host cellular reaction around the metacestode, the laminated layer thickens and develops, distally, an electron-dense microfibrillate matrix. Sakamoto & Sugimura (1970) remarked that these transformations occurred from the 30th day post-inoculation, when brood capsules arise within the cysts. In the present study it has been demonstrated that these ultrastructural transformations correspond to biochemical modifications of the laminated layer. Indeed, the laminated layer of a cyst developing brood capsules react less intensely with lectins (SBA, ConA and WGA) with the exception of RCA-I and HPA. Kilejian & Schwabe (1971) suggested that the laminated layer of E. granulosus was composed of two chemically different subunits. The one richer in galactosamine, resulting from cellular reactions between host and parasite, was preferentially polymerized. Our observations constitute an argument for this hypothesis. Indeed, HPA, specific for highly branched terminal NacGal residues, reacts more intensively with the microfibrillate matrix (polymerized subunit) than with the electron-lucent zone towards the germinal layer of the cyst of E. multilocularis. Harris et al. (1989), cultivating activated oncospheres of E. granulosus, observed that the first

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lamination of the laminated layer was associated with the presence of microvilli, whereas the second lamination was associated with abbreviated microtriches. T h e first lamination of E. granulosus corresponds to the slightly fibrous, amorphous material, surrounding fertile young E. multilocularis cysts before brood-capsule development, while the second lamination corresponds to the microfibrillate matrix constituting the laminated layer of cysts after brood capsule formation. Indeed, it was noted that, on fertile cysts, the microvilli of the syncytium possessed a truncated spine. These structures develop sacs at their tips which may contribute to the formation of the microfibrillate matrix of the laminated layer, as has been suggested by Lascano et al. (1975) for E. granulosus. No more sugar was revealed by lectin labelling of the laminated layer when protoscoleces were invaginated. At this stage, the microfibrillate matrix appearing on cysts, which develop brood capsules, might be a proteincarbohydrate complex, as suggested by all the authors cited above for E. granulosus. The sugar content of this complex decreases with the establishment of cysts in the host together with the differentiation of protoscoleces. When environmental conditions are unfavourable for the development of brood capsules, cysts grow in diameter and remain in a quiescent state or degenerate. In this case, WGA and RCA-I are the last lectins to react with the laminated layer. Kore et al. (1967), considering the molar ratios among the different sugars found in the laminated layer of E. granulosus, raised the question as to whether there was only one macromolecule or two different polysaccharides, one comprised of galactose and NacGal, and the other comprised of galactose and NacGlu. The present results support the hypothesis of a differential expression of two types of polysaccharides on the laminated layer of E. multilocularis relative to the developmental status (fertile or sterile) of the cyst. When cysts become fertile, the laminated layer may transform into an insoluble, microfibrillate structure preferentially containing /?Gal (revealed with RCA-I) and branched aNacGal residue (revealed with HPA) polysaccharides. In contrast, cysts that are becoming quiescent or degenerate may secrete a fibrous laminated layer rich in /?Gal and highly branched /?NacGlu residue (revealed with WGA) polysaccharides. Richards (1984) suggested that the laminated layer of E. granulosus consisted of a protein-carbohydrate-lipid complex equivocally related to chitin. Chitins are polymers of NacGlu residues. WGA has high affinity for these polysaccharides (Goldstein & Poretz, 1986). The present observations might, thus, indicate the presence of carbohydrate complexes related to chitin on the laminated layer of E. multilocularis cysts when they undergo transformation into small hydatids under the influence of specific environmental conditions.

E. multilocularis cyst: sugars and differentiation

The development of brood capsules confirms the progression of the cysts towards fertility. In contrast to the results of Sakamoto & Sugimura (1970) and Thompson (1976) for E. multilocularis and E. granulosus respectively, the present ultrastructural studies demonstrate that E. multilocularis brood capsules are formed by invagination of the cyst tegument and germinal layer independently of the laminated layer. Brood capsules do not become completely autonomous, as hypothesized by Bortoletti & Ferretti (1973) for E. granulosus, but remain in continuity with tegumentary syncytium of the cyst by slender and infolded channels. The process of formation is analogous to the protoscolex formation within cysticerci which had been retained for a long time in their intermediate hosts (Slais, 1966). Lectin labelling showed an intense secretion of glycoconjugates in the cone-shaped cavity formed by the first outline of the brood capsule. Aspecific reactions may reveal that these glycoconjugates are glycoproteins reacting, as endogenous lectins, with sugar moities of FITC-lectins. Lamsam & McManus (1990) suggested that T. crassiceps metacestode releases glycoproteins from its surface. Highly immunogenic carbohydrates of these glycoproteins may serve to divert the host immune response, for example by fixing complement. It may be hypothesized that brood capsules of E. multilocularis secrete such products under the microfibrillate matrix of the laminated layer, to interact with host factors and limit the biological consequences of immune reactions at the host—parasite interface. Alkarmi & Behbehani (1989) found that factors from E. multilocularis inhibit chemotaxis of murine neutrophils and macrophages. At 8 and 10 weeks post-infection, the neutrophils and macrophages lose their response to parasite antigens but retain their ability to migrate to the parasite factors. Considering that the time required for development of the parasite in mice is approximately double that in the cotton rats and jirds (Mankau, 1957; Yamashita, 1960; Sakamoto & Sugimura, 1970; Liance et al. 1990), this loss of response to the parasite might then correspond to the period when the laminated layer transforms into a microfibrillate matrix and when the cone-shaped cavity of developing brood capsules reacts intensely with lectins. Towards the 12th and the 16th week post-infection, neutrophils and macrophages lose their ability to migrate in response to parasite factors. This might correspond to the moment when protoscoleces completely invaginate and secrete a cell-coat that reacts intensely with lectins and about the time that the laminated layer is no longer labelled and the zone of necrosis or granulation is resorbed. The first protoscolex bud arises from the brood capsule wall as the latter forms a secondary vesicle towards the cyst cavity 1 or 2 days after invagination. During protoscolex differentiation, changes in the

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surface ultrastructure of E. multilocularis are very similar to those observed by Rogan & Richards (1987) for E. granulosus. These authors suggested that a 'hook forming stimulus' implying fusions of microtriche spines, caused the formation of broad microtriches observed between the bottom of suckers and the hooks (zone delimiting the base of the rostellum). The length and thickness of these microtriches indeed gradually increase from the first to the second constriction of young E. multilocularis protoscoleces. Other stimuli might be implicated in the formation of other tegumentary structures. It might be proposed that the secretion of particular tegumentary glycoconjugates, revealed by lectin labelling during protoscolex differentiation, represents the biochemical expression of one or several stimuli implicated in the phenomenon of protoscolex maturation and which are progressively expressed. Indeed, the rostellum is the first region intensively labelled by lectins and the first one which differentiates. The sucker region reacts strongly with lectins when its characteristic tegumentary structures (blade-like microtriches) develop. Lectin labelling has also shown up cell-coat secretion on the soma tegument which was little labelled until this time. Thus, the combined use of ultrastructural studies and lectin labelling has permitted the demonstration of a graduated differentiation of the protoscolex. Lectin labelling of different stages of trematodes (Simpson et al. 1983) and nematodes (Kaushal et al. 1984; Ham et al. 1988) has shown the appearance of certain sugar moities and the disappearance of others during differentiation. In contrast, lectin labelling did not reveal the novel appearance of any sugar moities (others than those revealed by WGA, HPA, SB A, RCA-1 and ConA) on E. multilocularis metacestodes during their development in the intermediate host. Changes in sugar composition of the parasite would appear to be essentially quantitative rather than qualitative. It is interesting to note that location of WGA, HPA, SB A and RCA-1 ( ± C o n A ) labelling can be superimposed on the location of antigen-B secretion specific for Platyhelminthes (Olivo, Plancarte & Flisser, 1988). Yarzabal et al. (1977) detected antigen B on the laminated layer of E. granulosus. Antigen B was also located in the distal cytoplasm and tegumentary cytons of brood capsules and the anterior part (rostellum and suckers) of Echinococcus protoscoleces; it was found diffusely in the cell-coat, at the surface of brood capsules and in cells of the germinal layer (Davies et al. 1978). A 105 kDa molecule, very similar to the highly immunogenic antigen B, was shown to be produced by Taenia saginata metacestodes throughout the age range experimentally examined by Joshua, Harrisson & Sewell (1988). Joshua et al. (1989) also revealed glycoprotein antigens of different molecular weights specific for different ages of T. saginata meta-

R. Leducq and C. Gabrion cestodes. Thus, if the same sugar moities are revealed, however, their sequence and/or exposure on different proteins or lipids may differ during differentiation of the E. multilocularis metacestode. Such glycoproteins or glycolipids may be stage or age-specific. Regulation of the stage or age-specific proteins may occur at the post-transcriptional level and depends on developmental and immune phenomena (stimuli) as demonstrated for adult Hymenolepis diminuta by Siddiqui, Karcz & Podesta (1987). This work constitutes the first step in the study of structural and biochemical modifications correlated with variations in exogenous (host) and endogenous (parasite) parameters occurring during the establishment and differentiation of a metacestode in its intermediate host. Further studies with E. multilocularis will concentrate on glycoproteins and glycolipids labelled with lectins in order to determine whether these molecules are stage (or age)-specific and if they are of antigenic nature. The authors are grateful to Professor J. Gabrion and Dr J. Reversat for helpful suggestions, criticisms and technical assistance. Thanks are also due to Dr Y. Robbins for help with the English. This investigation was supported by INSERM (grant No. 85 8015). REFERENCES ALKARMI, T. & BEHBEHANI, K. ( 1 9 8 9 ) . EchillOCOCCUS

multilocularis: inhibition of murine neutrophil and macrophage chemotaxis. Experimental Parasitology 69, 16-22. ARME, c. (1988). Ontogenic changes in helminth membrane function. Parasitology 96, S83—SI04. BENNETT, H. s. (1963). Morphological aspects of extracellular polysaccharides. Journal of Histochemistry and Cytochemistry 11, 14—23. BERRADA-RKHAMI, O . , LEDUCQ, R., GABRION, J . & GABRION,

C. (1990). Selective distribution of sugars on the tegumental surface of adult Bothriocephalus gregarius (Cestoda: Pseudophyllidea). International Journal for Parasitology 20, 285-97. BORTOLETTI, G. & FERRETTI, G. (1973). Investigation on larval forms of Echinococcus granulosus with electron microscope. Rivista di Parassitologia 34, 89-110. COLTORTI, E. A. Sc VARELA-DIAZ, V. M. ( 1 9 7 4 ) . EchinOCOCCUS

granulosus: penetration of macromolecules and their localization on the parasite membranes cysts. Experimental Parasitology 35, 225—31. CONTAT, F . , PETAVY, A. F . , DEBLOCK, S. & EUZEBY, J . ( 1 9 8 3 ) .

Contribution a l'e'tude epidemiologique de l'echinoccocose alveolaire en Haute-Savoie. Bulletin de la Socie'te' Franfaise de Parasitologie 1, 55—8. DAVIES, C , RICKARD, M. D . , BOUT, D. T. & SMYTH, J. D.

(1978). Ultrastructural immunocytochemical localization of two hydatid fluid antigens (antigen 5 and antigen B) in the brood capsules and protoscoleces of ovine and equine Echinococcus granulosus and E. multilocularis. Parasitology 77, 143-52.

140

w. (1990). Schistosome carbohydrates. Parasitology Today 6, 45-8. GOLDSTEIN, i. j . & PORETZ, R. D. (1986). Isolation, physiochemical characterization and carbohydratebinding specificity of lectins. In The Lectins. Properties, Functions, and Applications in Biology and Medicine (ed. Liener, I. E., Sharon, N. & Goldstein, I. J.), pp. 33-248. London: Academic Press. HAM, p. j . , SMAIL, A. j . & GROEGER, B. K. (1988). Surface carbohydrate changes on Onchocerca lienalis larvae as they develop from microfilariae to the infective thirdstage in Simulium ornatum. Journal of Helminthology 62, 195-205. DUNNE, D.

HARRIS, A., HEATH, D. D., LAWRENCE, S. B. & SAW, R. J.

(1989). Echinococcus granulosus: ultrastructure of epithelial changes during the first 8 days of metacestode development in vitro. International Journal for Parasitology 19, 621-9. JOSHUA, G. W. P., HARRISON, L. J. S. & SEWELL, M. M. H.

(1988). Excreted/secreted products of developing Taenia saginata metacestodes. Parasitology 97, 477-89. JOSHUA, G. W. P . , HARRISON, L. J. S. & SEWELL, M. M. H.

(1989). Development changes in proteins and glycoproteins revealed by direct radio-iodination of viable Taenia saginata larvae. Parasitology 99, 265-74. KAUSHAL, N. A., SIMPSON, A. J. G. HAUSSAIN, R. & OTTESEN,

E. A. (1984). Brugia malayi: stage-specific expression of carbohydrates containing iV-acetyl-D-glucosamine on the sheathed surfaces of microfilariae. Experimental Parasitology 58, 182-7. KILEJIAN, A. & SCHWABE, c. w. (1971). Studies on the polysaccharides of the Echinococcus granulosus cyst, with observations on a possible mechanism for laminated membrane formation. Comparative Biochemistry and Physiology 40B, 25-36. KORE, I., HIERRO, J., LASALVIA, E., FALCO, M. & CALCAGNO,

M. (1967). Chemical characterization of the polysaccharide of the hydatid membrane of Echinococcus granulosus. Experimental Parasitology 20, 219-24. LAMSAM, s. & MCMANUS, D. p. (1990). Molecular characterization of the surface and cyst fluid components of Taenia crassiceps. Parasitology 101, 115-25. LASCANO, E. F., COLTORTI, E. A. & VARELA-DIAZ, V. M.

(1975). Fine structure of the germinal membrane of Echinococcus granulosus cysts. Journal of Parasitology 61, 853-60. LEDUCQ, R., GABRION, J. & GABRION, C. (1988).

Caracte'risation des glycoconjugues de surface et intracellulaires dans les formes larvaires d'Echinococcus multilocularis a l'aide de lectines. Comptes Rendus de la Socie'te de Biologie 182, 501-8. LEDUCQ, R., BERRADA-RKHAMI, O., GABRION, J. & GABRION,

c. (1990). Lectin analysis of glycoconjugate distribution during differentiation and strobilization of Bothriocephalus gregarius metacestode in a paratenic host. International Journal for Parasitology 20, 645-54. LIANCE, M., BRESSON-HADNI, S., VUITTON, D., BRETAGNE, S.

& HOUIN, R. (1990). Comparison of the viabilty and developmental characteristics of Echinococcus multilocularis isolates from human patients in France. International Journal for Parasitology 20, 83-6.

E. multilocularis cyst: sugars and differentiation s. K. (1957). Studies on Echinococcus alveolaris (Klemm, 1883), from St. Lawrence Island, Alaska. I. Histogenesis of the alveolar cyst in white mice. Journal of Parasitology 43, 153-9.

MANKAU,

MARCHIONDO, A. A. & ANDERSON, F. L. (1983). F i n e

structure and freeze-etch study of the protoscolex tegument of Echinococcus multilocularis (Cestoda). Journal of Parasitology 69, 709-18. MURAMATSU, T. (1989). Les sucres de la membrane cellulaire. La Recherche 20, 624-34. NORMAN, L. & KAGAN, j . G. (1961). The maintenance of Echinococcus multilocularis in gerbils (Meriones unguiculatus) by intraperitoneal inoculation. Journal of Parasitology 47, 870-4. OHBAYASHI, M. (1960). Studies on Echinococcosis X. Histological observations on experimental cases of multilocular echinococcosis. Japanese Journal of Veterinary Research 8, 134-45. OLIVO, A., PLANCARTE, A. & FLISSER, A. (1988). Presence of antigen B from Taenia solium cysticercus in other Playthelminthes. International Journal for Parasitology 18, 543-5. RICHARDS, K. s. (1984). Echinococcus granulosus equinus: the histochemistry of the laminated layer of the hydatid cyst. Folia Histochemica et Cytobiologica 22, 21-32. ROGAN, M. T. & RICHARDS, K. S. ( 1 9 8 7 ) . EchinOCOCCUS

granulosus: changes in the surface ultrastructure during protoscolex formation. Parasitology 94, 359-67. ROTH, j . & BINDER, M. (1978). Colloidal gold, ferritin and peroxidase as markers for electron microscopic double labeling lectin techniques. Journal of Histochemistry and Cytochemistry 26, 163-9. ROTH, j . (1983). Application of lectin-gold complexes for electron microscopic localization of glycoconjugates on thin sections. Journal of Histochemistry and Cytochemistry 31, 987-99. SAKAMOTO, T. & SUGIMURA, M. (1969). Studies on echinococcosis XXI. Electron microscopical observations on general structure of larval tissue of multilocular Echinococcus. Japanese Journal of Veterinary Research 17, 67-80.

141

& SUGIMURA, M. (1970). Studies on echinococcosis XXIII. Electron microscopical observations on histogenesis of larval Echinococcus multilocularis. Japanese Journal of Veterinary Research 18, 131-H. SANDEMAN, R. M. & WILLIAMS, j . F. (1984). Lectin binding to cystic stages of Taenia taeniaeformis. Journal of Parasitology 70, 661-7. SHARON, N. (1975). Les sucres dans la vie sociale des cellules. La Recherche 6, 16-24. SAKAMOTO, T.

SIDDIQUI, A. A., KARCZ, S. R. & PODESTA, R. B. ( 1 9 8 7 ) .

Developmental and immune regulation of gene expression in Hymenolepis diminuta. Molecular and Biochemical Parasitology 25, 19-28. SIMPSON, A. J. G., CORREA-OLIVERIA, R., SMITHERS, S. R. &

SHER, A. (1983). The exposed carbohydrates of schistosomula of Schistosoma mansoni and their modification during maturation in vivo. Molecular and Biochemical Parasitology 8, 191-205. SLAIS, j . (1966). The importance of the bladder for the development of the cysticercus. Parasitology 56, 707-13. SUZUKI, S., TSUYAMA, S., SUGANUMA, T., YAMAMOTO, N. &

MURATA, F. (1981). Postembedding staining of Brunner's gland with lectin-ferritin conjugates. Journal of Histochemistry and Cytochemistry 29, 946-52. THOMPSON, R. c. A. (1976). The development of brood capsules and protoscoleces in secondary hydatid cysts of Echinococcus granulosus. A histological study. Zeitschrift fiir Parasitenkunde 51, 31—6. YAMASHITA, j . (1960). On susceptibility and histogenesis of E. multilocularis in the experimental mouse with the stade of echinococcosis in Japan. Parasitologia 2, 399^06. YARZABAL, L. A., DUPAS, H., BOUT, D., NAQUIRA, F. &

CAPRON, A. (1977). Echinococcus granulosus: the distribution of hydatid fluid antigens in the tissues of the larval stage. II. Localisation of the thermostable lipoprotein of parasitic origin (Antigen B). Experimental Parasitology 42, 115-20.