Oncospheral hook morphogenesis in the cestode Dilepis undula ...

Oncospheral hook morphogenesis in the cestode Dilepis undula(Schrank, 1788) (Cyclophyllidea, Dilepididae)

Abstract. Ultrastructural aspects of the oncospheral hookmorphogenesis in the dilepidid cestode, Dilepis undula(Schrank, 1788), are described. Oncospheral hook primordiaappear within specialised cells, so-called oncoblasts, at thepreoncospheral stage of the embryogenesis. Extended Golgiregions, numerous free ribosomes and mitochondria areinvolved in the hook development. During the hook growth,the blade and base gradually protrude outside the oncoblastplasma membrane. The nucleated oncoblasts persist aroundthe handles of the fully formed hooks even in the matureoncospheres. The mature hooks in the cross-section consistof two to four layers of different electron density, dependingon the level of the section; two of them, namely the highlyelectron-dense cortex and a moderately electron-dense core,

are observed in all parts of the hook. A circular, septatedesmosome and two electron-dense rings on its both sidesare formed around the exits of the hook blades from theoncospheral tegument. Each hook blade has a protectivemoderately electron-dense "cap" on its tip. A hook regionmembrane, similar to that described in other cyclophyl-lideans, forms on the surface of the oncosphere a cavity cov-ering the hook blades. The hook muscle attachment zones atthe hook guard and base are represented by a relatively thicklayer of fibrous material. The peculiarities of the oncospher-al hook formation in Dilepis undula are compared with theresults of our previous studies and literature data on othercestodes so far studied in this respect.

Key words: Dilepis undula, Cestoda, Dilepididae, ultrastructure, oncospheral hook differentiation, oncoblasts

Introduction

Oncospheral hooks, along with the penetration glands, playan important role in the process of the cestode larvae pene-tration through the walls of the digestive tube of the inter-mediate hosts. Their morphogenesis is characterised by apeculiar intracellular mode of formation (Ogren 1961;Moczoń 1971; Swiderski 1972, 1973, 1976; Swiderski andTkach 1997b; Kornakova 1999) within the specialised hook-forming cells, so-called oncoblasts. It represents one of themost interesting example of the cell specialisation towardsthe formation of the dense structures. Several light micro-scope studies on this subject were published (Ogren 1955,1957, 1958, 1961; Rybicka 1966; Swiderski 1967; Moczoń1971; Euzet and Mokhtar-Maamouri 1975, 1976). Morerecently, application of TEM technique facilitated detailedexamination of the oncospheral hook morphogenesis at theultrastructural level (Swiderski 1973, 1976; Swiderski and

Tkach 1997b; Kornakova 1999). These studies demonstratedsome differences observed in the fate of the oncoblasts, pres-ence/absence of a special hook region membrane coveringthe hook blades and final structure of hooks in representa-tives of different cestode families. The number of speciesand diversity of supraspecific taxa, so far examined in thisrespect is, however, insufficient for wider comparativeanalysis.

The family Dilepididae is one of the largest groups ofCyclophyllidea and is placed in the recent morphological andmolecular phylogenetic studies (Hoberg et al. 1997, 1999;Mariaux 1998) among the most derived cestode families.Gabrion (1981) provided some data on the ultrastructure ofmature oncospheral hooks in two bird dilepidids, Ano-motaenia constricta (= Spiniglans constricta - see Bona1994) and Paricterotaenia porosa, while Swiderski et al.(2000) presented data on the hook ultrastructure inHepatocestus hepaticus, a parasite of mammals. In both

Zdzisław Świderski1,2, Yasyl V. Tkach1,3 and Ruslan V. Salamatin1,3

]W. Stefański Institute of Parasitology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warszawa, Poland; 2Department of GeneralBiology and Parasitology, University of Medical Sciences, Chałubińskiego 5, 02-004 Warszawa, Poland; 3Institute of Zoology, UkrainianNational Academy of Sciences, 15 B. Khmelnitsky Str., Kiev, MSP, 01601 Ukraine

Acta Parasitologica, 2000, 45(4), 322–331ISSN 1230-2821Copyright © 2000 W. Stefański Institute of Parasitology, PAS

Morphogenesis of oncosphcral hooks in Dilepis undula 323

cases, the description of the hook morphogenesis was lack-ing. The aim of this paper is to describe the ultrastructuralaspects of the oncospheral hook formation in the cestodeDilepis undula, a type species of the genus Dilepis, which is,in turn, a type genus in the family Dilepididae.

Materials and methods

Adult specimens of Dilepis undula (Schrank, 1788), wereremoved from the intestine of a fieldfare (Turdus pilaris),from the vicinity of Kiev, Ukraine. The proglottids werequickly rinsed in saline, cut into small pieces and fixed in 4%glutaraldehyde in sodium cacodylate buffer (pH 7.4). Afterwashing in the same buffer and postfixation for 2 h in 1%OsO4, they were dehydrated in an ethanol series, infiltratedwith propylene oxide and embedded in Spurr’s resin.Ultrathin sections, mounted on uncoated copper grids, dou-ble stained with lead citrate and uranyl acetate, were exam-ined under a JEM-1200EX electron microscope operated atan accelerating voltage of 80 kV.

Results

Six oncospheral hooks begin their differentiation at the pre-oncospheral stage of the oncosphere development.Oncoblasts are localised in the anterior hemisphere of thedifferentiating oncosphere and are characterised by the largesemilunar interphase nuclei, situated at one of the cell poles(Fig. 1). The opposite, cytoplasmic pole, is occupied by a so-called “hook-forming centre” which is characterised by adense accumulation of mitochondria, free ribosomes,polysomes and extensive Golgi complexes containing vesi-cles of different size and electron density (Fig. 2A). Vesiclesof the Golgi complex contain a dense compact material.

Later, electron-dense granule appears in the oncoblastcytoplasm. It gradually elongates and forms a young hookletentirely embedded in the oncoblast cytoplasm (Figs. 2B and4-6). With the growth of the hook handle, the blade pro-trudes outside the oncoblast (Figs. 2C and 4-13). At thisstage the hook blade is surrounded by a layer of cytoplasmforming a characteristic “cytoplasmic cap” at the tip of thehook (Figs. 6 and 12).

Simultaneously, formation of a septate desmosome andtwo desmosome rings takes place around the each hookguard. Apparently, they play a role as a rigid hook attach-ment structure and also prevent the oncosphere tegumentfrom self-injury during the hook movement (Figs. 2D, 3C, 7,12 and 13). At the sites of the hook exits, the oncospheraltegument is much thicker and forms deep infoldings (Figs. 8,11 and 12) surrounding the hook blades.

At this stage, an early differentiation of the myofibrilsfrom myoblasts, is observed. The wide bands of the hookmuscles surround the oncoblast and later attach at the site ofthe bulb-shaped hook base (Fig. 2D) and at the hook guardregion (Figs. 2D, 3C, 4, 6, 8, 11, 12 and 14) where the hookmuscle attachment (HMA) zones are formed. The HMA

zones appear as a thin tendon-like fibrous layer of low elec-tron-dense material. With their opposite ends, the hook mus-cle bands are attached to the inner surface of the tegumentalbasal membrane (Figs. 11 and 12).

At the time when the hook formation is completed, a spe-cial so-called hook region membrane is formed at the placewhere the hook blades protrude from the oncosphere surface(Fig. 12). This structure is well defined in D. undula and con-sists of a membrane-bound cytoplasmic layer which forms acavity covering in a cap-like manner only one pole of theoncosphere and surrounding the hook blades. The microvilliare especially well-developed on the surface of the onco-spheral tegument covered by the hook region membrane(Fig. 12).

In the cross-sections, the fully formed oncospheral hookshave a heterogeneous structure. Two, three or sometimeseven four layers could be observed, depending on the sectionlevel, e.g. blade, collar, handle or handle base (Figs. 4-14).In general, there are two main layers of hook material ob-served in all regions: a highly electron-dense outer layer orcortex and moderately electron-dense inner layer or core.Additionally, in some part of the hooks, more electron-lucentlayers may be observed between the core and cortex and/oron the hook surface.

The nucleated cell bodies of oncoblasts remain visiblearound the hook handles of mature oncospheral hooks (Figs.2D, 11 and 14). Sometimes, on the same section both nucle-ated and anucleated oncoblasts around mature hook handlewere observed (Fig. 14) suggesting that the absence of thenuclei in the oncoblasts, due to the section level, can be mis-leading.

Discussion

Swiderski (1973) in his work on the oncospheral hook mor-phogenesis in Catenotaenia pusilla (Catenotaeniidae) point-ed out that some data concerning this process presented inthe early light microscopical works, are incorrect due to thelimitations of light microscopy resolution. Since then, ultra-structural investigations of the embryonic hook morphogen-esis have been done on a three species only: Intermicapsi-fer madagascariensis (Anoplocephalidae), Nematotaeniadispar (Nematotaeniidae) and Passerilepis crenata (Hy-menolepididae) (Swiderski 1976, Swiderski and Tkach1997b, Kornakova 1999).

In all cases the hook formation took place in the spe-cialised cells - oncoblasts. They are characterised by a highsynthetic activity which is proved by the accumulation offree ribosomes, Golgi complexes and mitochondria. Theenergy supplied by mitochondria, is necessary for the syn-thesis of the organic substances which are used as a con-structing material in the processes of hook formation(Nieland 1968, Swiderski 1976). Thus, the viewpoint ofKornakova (1999) who suggested that mitochondria do nottake part directly in the formation of the hook material,seems to us unsound.

Zdzisław Świderski et al.

Morphogenesis of oncosphcral hooks in Dilepis undula 325

Ogren (1961) postulated that the cell membrane acts as atemplate for hook blade formation. However, in all the abovementioned species, as well as in D. undula, in the earlystages the hooks are always situated in the close spatial rela-tionship with the invaginated part of the nucleus and usuallytouch the cell membrane only in one point which suggeststhat the cell membrane does not influence, at least directly,the shape of the future hook at it was suggested by Ogren(1961). Probably, in D. undula and other studied so farspecies, the hook shape is determined genetically and com-ponents of the secretory pathway (such as Golgi bodies) incombination with the cytoskeleton play main role in the for-mation of the hook material and hook shape.

Despite the limited number of studied taxa, some prelim-inary comparative analysis of the oncospheral hook develop-ment in different cestodes can be made. Among the mostprominent differences that should be noticed is a fate of theoncoblast in various species. The complete oncoblast nucle-us disintegration in the mature oncosphere is occurring in thetaeniid E. granulosus, anoplocephalid I. madagascariensis,nematotaeniid N. dispar and dilepidid from mammals H.hepaticus (Swiderski 1976, 1983; Swiderski and Tkach1997b; Swiderski et al 2000). A situation when, similarly toDilepis undula, a nucleated oncoblast or its anucleated rem-nants persist around the hook handle, has been found inthe catenotaeniid C. pusilla, taeniid Taenia crassiceps, andhymenolepidids Rodentolepis nana, Hymenolepis diminuta,Hymenolepis citelli, Staphylocystoides stefanskii, Pseud-hymenolepis redonica, Ditestolepis tripartita (Collin 1968,Moczoń 1971, Furukawa et al. 1977, Chew 1983, Swiderskiand Tkach 1997a, Tkach and Swiderski 1997). It is interest-ing, that both cases have been reported for representatives ofthe families Taeniidae and Dilepididae. Such differenceswithin one family should be considered, however, with somecaution, because they may be a result of immature conditionof the oncosphere or lack of the representative sectionsthrough the hook handle.

Taking under consideration the fact that the hook forma-tion in all six oncoblasts is always synchronous within thesame embryo, observation of both nucleated and anucleatedoncoblasts around mature hook handles on the same section(Fig. 14) suggests that insufficient amount of data (lack ofthe nuclei on some sections) may lead to misleading conclu-sions.

Kornakova (1999) reported an unusual situation observedin Passerilepis crenata, a hymenolepidid from passerinebirds. According to her interpretation, in this species theoncospheral hooks never protrude through the oncoblastplasma membrane and the entire cell always remains intact.In our opinion, the Kornakova’s arguments that the penetra-tion of the hook blade through the cell plasma membranewould lead to destroying of the membrane potential andionic balance of the cell resulting in the immediate death ofthe cell, is not well grounded. The process of the hook bladeprotrusion is a gradual process and does not lead to the for-mation of a “hole” in the plasma membrane as suggested byKornakova (1999). According to our observations (forinstance, see Swiderski 1976, Fig. 5 and Fig. 11 in the pres-ent paper), at the point of the blade exit the oncoblast plasmamembrane is always tightly attached to the hook surface dueto formation of cytoplasmic collar and circular septatedesmosome.

Besides, it is hard to explain how the hook-muscle sys-tem would function in the case when the whole hook isembedded in the oncoblast including the hook muscle attach-ment points, namely the hook guard and base. As shown onthe figure 5 of Kornakova’s paper, there is no place for thehook muscle attachment at the hook base of P. crenata.According to our own (Swiderski 1973, 1976; Swiderski andTkach 1997b; Tkach and Swiderski 1997; Swiderski et al2000) and literature data (Collin 1968, Chew 1983, Ubelaker1983, Coil 1991, Conn 1991), the hook-muscle attachmentzones do exist around the hook guard and base in a form ofthick tendon-like structures consisting of a fibrous substance.

Fig. 1. General topography of the anterior pole of the embryo Dilepis undula in the stage of hook formation (preoncospheral phase ofembryogenesis). Note six hook-forming cells (HFC) or oncoblasts with differentiating hook primordia (HP) and bilaterally symmetricalpattern of blastomeres. Fig. 2. Diagram of four consecutive stages of hook development: A – early oncoblast with hook anlage formation,B – early oncoblast with intracellular outline of blade, C – late oncoblast with the blade protruding outside and early handle formation,D – mature oncosphere hook with degenerating oncoblast surrounding the handle. Fig. 3. Diagrammatic drawing illustrating the formationof circular septate desmosome during oncoblast degeneration: A – early oncoblast with the hook blade embedded in the cytoplasm, B – lateoncoblast with the hook blade surrounded by a newly formed desmosome and dense rings (DR1 and DR2) on both sides of the desmosome,C – mature oncospheral hook with enlarged guard and hook muscle attachment; note two dense rings on each side of the desmosome.Key to abbreviations used in all figures: B – hook base, Bl – hook blade, BM – basal membrane of the oncospheral tegument, C – hookcollar or guard, CC – cytoplasmic cap, Cr – cortex or outer sheath of the hook, D – circular septate desmosome, DR1 and DR2 – denserings situated on both sides of circular septate desmosome in the region of blade exit, Em – embryophore, H – hook, Hd – hook handle orshank, HFC – hook-forming cells or oncoblasts, HM – hook muscles, HMA – hook muscle attachments, HP – hook primordium or hookanlage, HRM – hook region membrane, IC – inner core of the hook, IC* – outer layer inner core of the hook, IE – inner envelope, L – lipiddroplets, Mv – microvilli, N – nucleus, OE – outer envelope, OM – oncospheral membrane, r – ribosomes or polyribosomes, SC – somat-ic cells, T – oncospheral tegument, TI – invagination of the tegument

326 Zdzisław Świderski et al.

Figs. 8-10. Details of hook infrastructure. Note: a deep invagination in the oncospheral tegument around the hook exit from the oncospheral tegument and a desmosome and dense rings (DR1 and DR2) surrounding the hook exit (8); different number of layers of hook mate-rial observed on the cross-sections through the handle (9) and blade (10)

Figs. 4-7. Intermediate stages of oncospheral hook morphogenesis showing some details of hook differentiation on longitudinal andoblique sections. Note: the concave kidney-shaped form of oncoblast nucleus (4), robust newly formed blade of hook (5), the hook basewith hook muscle attachment zone protruding from the oncoblast (6 and 7); the protective “cap” on the tip of the hook blade (6) and anearly stage of the circular desmosome formation in the oncoblast with an elongated nucleus (7)

Morphogenesis of oncosphcral hooks in Dilepis undula 327

328 Zdzisław Świderski et al.

Fig. 11. Longitudinal, slightly oblique section through the hook at the late stage of differentiation. Note: the deep invagination of the onco-spheral tegument; beginning of the formation of the hook region membrane and; a nucleated oncoblast around the hook handle; numerousmuscle bands in the cytoplasm of the myoblasts (somatic cell) undergoing differentiation into hook muscles

Figs. 12 and 13. Details of the collar region and blade of the mature hook protruding from the oncosphere. Note: the circular septate desmo-some and two rings consisting of electron-dense material adjacent to the desmosome; tendon-like hook muscle attachment zones composed

fibrous substance of low electron density and electron-dense attachment point; numerous wide bands of hook muscles; a well definedhook region membrane attached to the oncosphere surface and the numerous microvilli on the tegument in the cavity formed by the HRM

Morphogenesis of oncosphcral hooks in Dilepis undula 329

Fig. 14. Longitudinal and cross-sections through the mature hooks. Note nucleated oncoblast on the cross-section and anucleated region ofthe oncoblast cytoplasm around the handle on the longitudinal section

Thus, the above viewpoint of Kornakova (1999) is in evi-dent contradiction with the functional aspects of the hook-muscle system.

It is generally accepted that the complex hook-musclesystem of the cestode oncospheres characterised by robustmuscle attachments and numerous interconnections betweendifferent muscle bands (protraction, abduction and retractionmuscles – see Ogren 1972, Swiderski 1983), provides astructural basis for coordinated hook action.

Electron-dense threads observed during the hook elon-gation in I. madagascariensis, have not been noticed inC. pusilla, N. dispar and D. undula. The nature of the struc-tures, described by Kornakova (1999) in P. crenata as micro-tubules, remains unclear. In our opinion, they resemble theparallel streaks of dense material, described in I. madagas-cariensis by Swiderski (1976) and interpreted as tonofibrilsassociated with keratinisation in other systems (Mercer1961).

The hook region membrane observed in D. undula, ismorphologically very similar to that described by Gabrion(1981) in two other dilepidids from birds, Paricterotaeniaporosa and Anomotaenia constricta. Initially described inC. pusilla (Swiderski 1972), this structure was also described

in other cyclophyllideans (Swiderski 1975, 1983, 1992). Itshould be noted that in D. undula the microvilli in the cavityformed by the hook region membrane, are better developedthan in the neighbouring surface of the oncospheral tegument(Fig. 12), similar to the situation demonstrated by Gabrion(1981) in P. porosa and A. constricta. The functional impor-tance of this feature is, however, unclear.

The formation of the oncospheral hooks differs from thatof the rostellar hooks (Mount 1970) which are formed as aresult of specialisation of tegumental microtriches and super-position of the protein on the hook surface.

Acknowledgements. This study was financially supported by thegrant No. 6 PO4C 01314 from the Polish Committee for ScientificResearch (KBN) and by a fellowship from the J. MianowskiFoundation, Warszawa, Poland to R. Salamatin. The authors wish tothank Dr Yuri Kuzmin (Institute of Zoology, Kiev) for his technicalassistance. The research was carried out in the framework of the sci-entific co-operation between the Institute of Parasitology of thePolish Academy of Sciences and the Institute of Zoology of theUkrainian National Academy of Sciences. Ruslan Salamatinreceived EFP Young Scientist Award for presentation of the pre-liminary results of this research at the EMOP-8 in Poznań, Polandin September 2000.

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(Accepted August 25, 2000)