DOI: 10.2478/s11686-006-0029-1 © 2006 W. Stefañski Institute of Parasitology, PAS Acta Parasitologica, 2006, 51(3), 182–193; ISSN 1230-2821
Ultrastructural and cytochemical studies on vitellogenesis in trypanorhynch cestode Dollfusiella spinulifera Beveridge, Stefañski Neifar et Euzet, 2004 (Eutetrarhynchidae) Zdzis³aw Œwiderski1,2*, Jordi Miquel3, Daniel M³ocicki1, Lassad Neifar4, Barbara Grytner-Ziêcina2 and John S. Mackiewicz5 1W. Stefañski Institute of Parasitology, Polish Academy of Sciences, 51/55 Twarda Street, 00-818 Warsaw, 2Department of General Biology and Parasitology, Warsaw Medical University, 5 Cha³ubiñskiego Street, 02-004 Warsaw, Poland; 3Laboratori de Parasitologia, Departamento de Microbiologia i Parasitologia SanitBries, Facultat de FarmBcia, Universitat de Barcelona, Av. Joan XXIII sn, 08028 Barcelona, Spain; 4Laboratoire de Bioécologie Animale, Departement des Sciences de la Vie, Faculté des Sciences de Sfax, 3018 Sfax, Tunisia; 5Department of Biological Sciences, State University of New York at Albany, Albany, N.Y. 12222, U.S.A.
Abstract The first description of vitellogenesis in the Trypanorhyncha is presented in this paper. Though the type of vitellogenesis and mature vitellocyte in Dollfusiella spinulifera appear to be unique among the Eucestoda, to some extent they resemble that observed in the lower cestodes, namely the Tetraphyllidea and Pseudophyllidea. Maturation is characterized by: (1) an increase in cell volume; (2) extensive development of large, parallel, frequently concentric cisternae of GER that produce proteinaceous granules; (3) development of Golgi complexes engaged in packaging this material; (4) continuous enlargement of proteinaceous granules within vesicles and their transformation into shell globule clusters; and (5) progressive fusion of all vesicles, with flocculent material containing the proteinaceous granules and shell globule clusters, into a single very large vesicle that characterises mature vitellocytes of this tapeworm. Cell inclusions in and around the large vesicle consist of flocculent material of a very low density, a few shell globule clusters, moderately dense proteinaceous granules and numerous large droplets of unsaturated lipids. A new previously unreported mode of transformation of proteinaceous granules into shell globule clusters, that evidently differs from that of pseudophyllideans and tetraphyllideans, is described. Cytochemical staining with periodic acidthiosemicarbazide-silver proteinate for polysaccharides indicates a strongly positive reaction for membrane-bound glycoproteins in all membranous structures such as GER, mitochondria, Golgi complexes, nuclear and cell plasma membranes. Similar staining revealed β-glycogen particles scattered in the cytoplasm of maturing vitellocytes. Typical cytoplasmic β-glycogen particles appear mainly during early vitellocyte maturation but it is characteristic for this species that they are only seldom visible in mature cells. Some working hypotheses concerning the interrelationship between this particular pattern of vitellogenesis, possible mode of egg formation in D. spinulifera, its embryonic development and trypanorhynchean life cycle, are drawn and discussed.
Key words Trypanorhyncha, Dollfusiella spinulifera, vitellogenesis, ultrastructure, cytochemistry, proteinaceous shell granules, shell globule clusters, unsaturated lipids, membrane-bound glycoproteins, β-glycogen
Introduction Skóra Review of the literature indicates that there are no published data on the vitellogenesis in trypanorhynch cestodes, a group much neglected in ultrastructural and cytochemical studies. The only accessible, useful information on this subject is in an unpublished PhD thesis on the fine structure and physiology of a trypanorhynch tapeworm Grillotia erinaceus (McKerr 1985).
*Corresponding
As described in detail in the extensive review on cestode vitellogenesis (Œwiderski and Xylander 2000), vitellocytes have 2 important functions in embryogenesis: (1) formation of hard eggshell (e.g. Pseudophyllidea) or a delicate capsule (e.g. Cyclophyllidea), and (2) supplying nutritive reserves for the developing embryos. During cestode evolution, any of these two functions have been intensified or reduced in different taxa, depending on the type of their embryonic development, degree of ovoviviparity and life cycles (Œwiderski and Mo-
author:
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Fig. 1. Part of the vitelline follicle showing three consecutive stages of vitellogenesis (I–III) in Dollfusiella spinulifera: I – immature vitellocyte of gonial type; II – early stage of cytodifferentiation; III – advanced stage of vitellocyte maturation. Note: (1) a large nucleus with prominent electron-dense nucleolus, few mitochodria and short profiles of GER in the immature cell of gonial type; (2) few electron-dense lipid droplets in the peripheral cytoplasm (right upper corner) visible in the early stage II of vitellocyte differentiation; and (3) a large amount of three types of cell inclusions: (a) large osmiophilic lipid droplets, (b) membrane-bound vesicles containing a core or single proteinaceous granule embedded in amorphous, flocculent electron-lucent material filling the remaining part of the vesicle; and (c) membrane-bound vesicles containing “shell globule cluster” type of inclusions. Inset: high power magnification showing spherical mitochondrion inside the immature vitellocyte and well-developed, numerous profiles of GER between two large osmiophilic lipid droplets. Abbreviations to all figures: β gl – β-glycogen particles; cv – central very large, single vesicle; GC – Golgi complex; GER – granular endoplasmic reticulum; IC – interstitial cell; L – lipid droplet; m – mitochondria; mbg – membrane-bound glycoproteins; n – nucleolus; N – nucleus; pg – proteinaceous granule; sgc – shell globule cluster; vfm – vesicles of a fine textured filamentous or flocculent material containing a core or single proteinaceous granule; WL – washed out lipid droplets with empty vesicles representing artifacts of Thiéry’s method; I – immature vitellocyte; II – early differentiation; III – advanced maturation of vitellocyte; IV – mature vitellocyte
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Stanis³a khtar 1974; Œwiderski and Mackiewicz 1976; Œwiderski and Subilia 1978; Œwiderski and Xylander 2000; Œwiderski et al. 2000, 2004, 2005). The aim of this study is to provide missing information on the ultrastructural and cytochemical aspects of vitellogenesis in the trypanorhynch cestode Dollfusiella spinulifera, a parasite of the spiral valve of the rhinobatid ray Rhinobatos typus, with particular emphasis on vitellocyte differentiation, shell
globule formation, and synthesis of nutritive materials for the developing embryos.
Materials and methods Mature specimens of Dollfusiella spinulifera (Beveridge et Jones, 2000) Beveridge, Neifar et Euzet, 2004 (Trypanorhyn-
Fig. 2. Electron micrograph of the vitelline follicle showing mature vitellocyte (stage IV) surrounded by immature (stage I) and early maturation stages. Note: a large central vesicle filled with an amorphous, flocculent electron-lucent material and showing at its periphery numerous dark lipid droplets and proteinaceous granules
Vitellogenesis in Dollfusiella spinulifera: TEM and cytochemistry
Roborzyñski
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cha, Eutetrarhynchidae) were obtained from the spiral valve of the rhinobatid ray Rhinobatos typus collected on reef flats at Heron Island (Queensland, Australia). Living cestodes were dissected in a 0.9% NaCl solution and different portions of mature proglottids containing testes and the external seminal vesicle were routinely processed for TEM examination. Specimens were fixed in cold (4°C) 3% glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in a 0.1 M sodium cacodylate buffer at pH 7.2, postfixed in cold (4°C) 1% osmium tetroxide in the same buffer, dehydrated in an ethanol series, and finally embedded in Epon resin. Ultrathin sections were obtained using a Reichert-Jung Ultracut E ultramicrotome, placed on copper grids and double-stained with uranyl acetate and lead citrate. Ultrathin sections were examined using a JEOL 1010 transmission electron microscope operated at 80 kV. For cytochemistry of glycogen and other polysaccharides, such as membrane-bound glycoproteins, the ultrathin sections were collected on gold grids. The periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) technique of Thiéry (1967) was applied to determine cytochemical localisation of glycogen and other polysaccharides at the ultrastructural level. These grids were also examined in a JEOL 1010 transmission electron microscope operated at an accelerating voltage of 80 kV.
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Results General topography of vitelline system in D. spinulifera The vitellaria of Dollfusiella spinulifera consist of an extensive system of numerous oval or elongated vitelline follicles enclosed by the cortical parenchyma that form a continuous sleeve around all internal organs. Ultrastructure The TEM results in this paper represent the first published accounts of vitellogenesis in the Trypanorhyncha. Mature vitelline follicles (Figs 1–7) consist of vitelline cells in various stages of development, progressing from immature cells of gonial type situated usually near the periphery to mature vitellocytes towards the centre and of irregularly shaped interstitial cells (Fig. 4). Long projections of the interstitial cells enclose the vitellocytes and extend as a cytoplasmic sheath at the periphery of vitelline follicles. As the general pattern of vitellocyte development in D. spinulifera is similar to that of other cestodes, the terminology used follows that of Œwiderski and Xylander (2000). Although vitellocyte cytodifferentiation constitutes a continuous process, which was arbitrarily subdivided into four dis-
Fig. 3. Consecutive stages of the proteinaceous granule formation, growth and differentiation into shell globule cluster. Scale bars = 1 µm
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crete stages to facilitate its description. Four stages of vitellocyte maturation in D. spinulifera can be distinguished: (I) immature cell of gonial type; (II) early differentiation; (III) advanced maturation; and (IV) mature vitellocyte (Figs 1–7). Maturation is characterized by: (1) increase in cell volume; (2) rapid synthesis and storage of fat as a concentrated energy source in the form of large, highly osmiophilic lipid droplets which appear for the first time in stage II (Figs 1, 2, 4, 5); (3) extensive development of large parallel cisternae of granular endoplasmic reticulum GER that produce vitelline material remaining in close association with Golgi complexes engaged in its packaging (Figs 1–5); and (4) continuous enlargement
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and fusion of small vesicles into larger vesicles and finally into a single, very large vesicle, characteristic for mature vitellocytes of this tapeworm (Figs 1–5). Immature vitellocyte (stage I) The undifferentiated cells of gonial type (Figs 1, 2, 5–8) representing the precursors of vitelline cells, show a high nucleocytoplasmic ratio and a large concentration of free ribosomes in a relatively thin layer of granular cytoplasm. These immature cells, generally at the periphery of the follicle, are spherical or ovoid. The nucleus measures about 5–7 µm in diameter while
Fig. 4. Ultrathin section of vitelline follicle illustrating early (stage II) and advanced (III) stages of vitellocyte maturation, anuclear part of the mature vitellocyte (stage IV) and perikaryon of the interstitial cell with long cytoplasmic extensions
Vitellogenesis in Dollfusiella spinulifera: TEM and cytochemistry
the diameter of the entire cell is approximately 8–9 µm. The large nuclei contain distinct spherical nucleoli of a homogeneous type (Figs 1, upper part; 2). However, it appears that such spherical homogeneous nucleoli change their shapes and become transformed into somewhat irregularly shaped nucleoli (Fig. 1, lower part) of more or less heterogeneous type with few projections of nucleolar material adjacent to the nuclear membrane. Their granular cytoplasm, rich in free ribosomes and polysomes, contains several mitochondria that have numerous
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long cristae (Figs 1, 2 and 6). Sometimes, individual long profiles of sparse GER have also been noticed (Figs 1, 2, 7, 8). Early phase of vitellocyte maturation (stage II) During the early phase of maturation the increase in cell size is accompanied by nuclear transformation and cytodifferentiation of cytoplasmic organelles involved in synthesis, transport and packaging of secretory products such as GER, Golgi
Fig. 5. Electron micrograph of a vitelline follicle showing four consecutive stages of vitellocyte maturation from the immature gonial cell (right lower corner), through early (stage II) and advanced (stage III) stages of differentiation to mature vitellocyte situated in the left upper corner (stage IV). Observe three types of cell inclusions at the periphery of a large central vesicle of the mature vitellocyte filled with amorphous flocculent material. Inset: high power magnification showing large, concentric rings of GER surrounded by mitochondria and vesicles of Golgi complexes
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complexes and mitochondria (Figs 2, 5–7). At the onset of maturation, the nuclear pores (Figs 4 and 6) in a perforated nuclear envelope become more evident and more numerous. During nucleolar transformation, initiated in the previous phase of cytodifferentiation, the homogeneous type of nucleolus (Fig. 1, lower left corner), becomes progressively irregular in outline and appears as an electron-dense network considered homologous with the nucleolonema and the less dense areas, with the pars amorpha (Figs 4 and 5).
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Advanced stage of maturation (stage III) This stage is characterised by a greater increase in cell size. A thick layer of cytoplasm is rich in numerous organelles and inclusions such as large mitochondria, ribo- and polyribosomes, concentric rings of GER profiles, Golgi complexes, numerous lipid droplets of different sizes and a very high electron density, moderately electron-dense proteinaceous-like granules of different sizes embedded in membrane-bound ves-
Fig. 6. Results of the cytochemical test for polysaccharides according to Thiéry’s method showing polysaccharides concentration during different stages of vitelline cell development. Note: empty spaces corresponding to large lipid droplets washed out during cytochemical procedure, well-visible proteinaceous granules situated in the vesicles of flocculent material and highly contrasted layer of polysaccharides on all membranous structures
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Fig. 7. Cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for polysaccharides showing strongly positive reaction for membrane-bound glycoproteins in all membranous structures such as GER, mitochondria, Golgi complex, nuclear and cell plasma membranes, as well as for β-glycogen particles scattered in the cytoplasm of maturing vitellocytes. Fig. 8. High power electron micrograph illustrating details of cytochemical test for polysaccharides. Note the differences in size and number of glycoprotein particles bound to the cell and nuclear membranes as well as membranes of different cell organelles
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icles containing a low electron-dense, apparently amorphous, flocculent material (Figs 1 and inset, 4, 5 inset, 6). The consecutive stages of the development of proteinaceous-like granules and their transformation into shell globule clusters are illustrated in Figure 3A-I. At the beginning (Fig. 3A), a very small spherical granule of Golgi origin – probably proteinaceous in nature – appears inside the vesicle filled with a flocculent material. It still remains moderately electron-dense, grows and reaches the size of about 1.5 µm in diameter occupying more or less an important percentage of the vesicle volume (Fig. 3B-D). Then, it starts further differentiation; probably still growing and progressively changing its initial spherical shape to a more or less irregular one (Fig. 3E). The next step is a transformation (or disintegration) of compact, more electron-dense material of individual granules into numerous small globules forming together the so-called “shell globules cluster” (Fig. 3F-I). The cluster is composed of numerous loosely packed globules of various sizes and of moderate electron density (Fig. 3G-I). Progressive fusion of numerous vesicles of flocculent material containing proteinaceous-like granules and various stages of their transformation into shell globule clusters results in formation of a single, large central vesicle. In the advanced stage of vitellocyte maturation (Figs 1 and 5; stage III) and in mature vitellocytes (Figs 2, 4 and 5; stage IV) different stages of the development of proteinaceous-like granules and their transformation into shell globule clusters, as well as numerous lipid droplets, are always at the periphery of the large single vesicle occupying the central part of the vitelline cell. Mature vitellocytes (stage IV) The majority of large membrane-bound vesicles contain a single, large moderately electron-dense proteinaceous granule embedded in a large amount of apparently amorphous, flocculent material (Figs 1 and 3). Some smaller vesicles, however, show ultrastructurally different type of inclusion composed of numerous small proteinaceous granules grouped together and forming some kind of “shell globule clusters” (Figs 1 and 3). Figures 2–5 illustrate well the ultrastructural variety of different types of cell inclusions situated in and around a single large vesicle of mature vitellocytes of D. spinulifera filled with almost amorphous, flocculent material of a very low density. These inclusions comprise very numerous moderately electron-dense proteinaceous granules, and highly osmiophilic lipid droplets, as well as apparently less numerous “shell globule clusters” (Figs 2–5). During fixation with osmium tetroxide, droplets of triglyceride rich in unsaturated fatty acids became blackened by reduction of osmium (Figs 1–5). Cytochemistry All membranous structures such as GER, Golgi complexes, mitochondria, nuclear and cell plasma membranes indicate strongly positive cytochemical reaction for membrane-bound
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glycoproteins (Figs 6–8). Typical, cytoplasmic β-glycogen particles appear mainly during early vitellocyte maturation (Fig. 7), but, they are only seldom visible in mature cells. After Thiéry’s test with periodic acid-thiosemicarbazidesilver proteinate (PA-TSC-SP) for polysaccharides, the preservation of lipid droplets is always unsatisfactory and results in extraction of lipid droplets in later steps of preparation. As a result of this test, therefore, very electron-lucent washed-out areas remain in place of the lipid, which represent artifacts of this cytochemical procedure (Fig. 6, WL). However, as one can see in Figure 6, the moderately electron-dense proteinaceous granules embedded in the larger amorphous vesicles of flocculent material always remain intact “in situ” after Thiéry’s method.
Discussion Vitellogenesis in cestodes in relation to polylecithal and oligolecithal egg types Two types of ectolecithal eggs, according to the number of vitellocytes per oocyte and amount of vitelline material in each vitellocyte, can be distinguished in cestodes: polylecithal and oligolecithal (Janicki 1918, Jarecka 1975). In monozoic and lower eucestodes such as caryophyllideans, pseudophyllideans and trypanorhynchean D. spinulifera, polylecithal eggs are formed. They are characterised by a very high amount of vitelline material because of two factors: (1) a large number of vitellocytes with each fertilised oocyte per egg, and (2) high content of vitelline material in each vitellocyte. In proteocephalideans (Œwiderski and Subilia 1978; Bruòanská 1997, 1999), and cyclophyllideans (Œwiderski 1968, 1973; Œwiderski et al. 1970a, b, 1978, 2000, 2005) there is generally one vitellocyte per one fertilised oocyte, although exceptions exist (e.g., Mesocestoides spp.; see Œwiderski and Conn 1999, Œwiderski and Xylander 2000). Comparison of vitellogenesis pattern in D. spinulifera and other cestodes The vitellogenesis pattern in D. spinulifera resembles to some extent that reported for tetraphyllideans (Mokhtar-Maamouri and Œwiderski 1976), pseudophyllideans (Œwiderski and Mokhtar 1974; Œwiderski and Xylander 1998, 2000; Korneva 2001), gyrocotylideans (Xylander 1987), amphilinideans (Xylander 1988), nippotaeniids (Korneva 2002) and spathebothriideans (Bruòanská et al. 2005). The common features with vitellogenesis in tetraphyllidean Echeneibothrium beauchampi (Mokhtar-Maamouri and Œwiderski 1976) are: (1) nutritive reserves composed predominantly of numerous lipid droplets and almost no glycogen in mature vitellocytes; (2) reduced amount of eggshell forming material, as the eggs of both E. beauchampi and D. spinulifera lack thick, hardened eggshells. Less evident are similarities with vitellocytes of
Vitellogenesis in Dollfusiella spinulifera: TEM and cytochemistry
pseudophyllideans (Œwiderski and Mokhtar 1974; Œwiderski and Xylander 1998, 2000; Korneva 2001) and spathebothriideans (Bruòanská et al. 2005) that have a great number of large lipid droplets, a great amount of α- and β-glycogen, and a great number of heterogeneous shell globule clusters. Differences vary between vitellocytes of D. spinulifera and those of the monozoic cestodes: amphilinideans, gyrocotylideans and caryophyllideans. All of these have eggs with hard eggshells and require a large amount of nutritive reserves for a long period of embryonation in water. Both amphilinideans (Xylander 1988) and gyrocotylideans (Xylander 1987) have little glycogen but have large accumulations of lipids scattered through out the cyton. Caryophyllidean vitellocytes contain a great amount of α- and β-glycogen both in their cytoplasm and nuclei (Mackiewicz 1968, Œwiderski and Mackiewicz 1976, Œwiderski et al. 2004). The process of vitellogenesis and ultrastructure of mature vitellocytes in D. spinulifera is quite similar to that described in another trypanorhynch Grillotia erinaceus in an unpublished PhD thesis (McKerr 1985). However, there is an important difference between vitellogenesis of these two trypanorhynch species. In G. erinaceus the lipid droplets of low saturated chemical nature show a moderate electron density but proteinaceous granules, or so-called “core of membranebound vesicles” (McKerr 1985), are highly electron-dense. On the other hand, in D. spinulifera the osmiophilic lipid droplets are unsaturated and the proteinaceous granules are of moderate electron density. Origin of egg capsule forming material and development of a single central vesicle of mature vitellocytes In spite of the similar origin of the smallest vitelline granules synthesized in the GER cisternae and packed in Golgi complexes in the form of membrane-bound vesicles, their further differentiation and transformation into shell globule clusters are quite different from those observed in other cestode species (Œwiderski and Xylander 2000). In D. spinulifera (see Fig. 3A-I) there is a continuous enlargement of proteinaceous granules inside of the vesicles of flocculent material and subsequent transformation into shell globule clusters. Progressive fusion of such vesicles, containing both proteinaceous granules and shell globule clusters results in the formation of a single, very large central vesicle, which is characteristic for mature vitellocytes of this tapeworm. In contrast to this mode of shell globule formation, in the vitellocytes of gyrocotylideans, amphilinideans, caryophyllideans and pseudophyllideans (see Œwiderski and Xylander 2000) the membranebound shell globule clusters originate from a progressive fusion of small shell globule vesicles which in mature vitellocytes are composed of numerous individual shell globules grouped together and embedded in an electron-lucent matrix substance. These clusters of shell globules exhibit a great variety in their size, shape and appearance. For example, in gyrocotylideans and amphilinids the individual shell globules within the clusters are not as tightly packed as in caryophyl-
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lideans and pseudophyllideans. In some cyclophyllideans from the family Anoplocephalidae (Œwiderski 1973, Œwiderski et al. 2005), there is a single large vesicle with a very condensed, highly electron-dense vitelline material that differs entirely from the large central vesicle of D. spinulifera in mode of development, differentiation and chemical nature of vesicle content. Histochemical research shows the presence of acid mucopolysaccharides in the vesicles of cyclophyllidean vitellocytes (for review see Rybicka 1966). At the ultrastructural level, the vitelline material appears as a homogeneous and moderately electron-dense secretion and differs from that of D. spinulifera, Glaridacris catostomi or Bothriocephalus clavibothrium. In those species, the vitelline material is in the form of the so-called “shell globules” of heterogeneous type, composed probably of two different substances: an electrondense phenolic protein and an electron-lucid phenolase, the enzyme that brings about the tanning of the shell material (Smyth and McManus 1989). Functional ultrastructure of D. spinulifera vitellocytes in relation to embryonic development of trypanorhynchs Little is known about embryogenesis in Trypanorhyncha. As summarized by Mattis (1986), Chervy (2002) and Palm (2004), there are two types of eggs in different taxa of this cestode order: (1) unembryonated eggs composed of only 2 blastomeres, surrounded by a thick and hard operculate eggshell, which are released into sea water where within 5 to 8 days they differentiate and produce infective hexacanths that are surrounded by the ciliated envelopes of the coracidium and involved in continuous swimming for a few days; (2) apparently embryonated eggs, surrounded only by a relatively thin non-operculate egg capsule, which do not produce a ciliated membrane surrounding hexacanth nor do they hatch, but must be eaten in order to infect the copepod first intermediate host. According to McKerr (1985), the eggs of G. erinaceus belong to the first type while those of D. spinulifera belong to the second. As described by Mattis (1986) in his unpublished PhD thesis, the egg capsules of Dollfusiella (Prochistrianella) have two sticky, long polar filaments that adhere to different objects in the benthos where they are eaten by bottom-living copepods as first intermediate hosts, especially in shallow water localities. According to Mattis (1986) the capsule consists of two layers: (1) outer layer thin, transparent, flexible, lacking operculum, extending from opposite ends as single tapering filaments, and (2) inner layer thick, tanned, lacking operculum. This second type of trypanorhynch egg contains relatively small amounts of nutritive reserves, as a major part of embryonic development apparently takes place in the uterus of gravid segments. This fact indicates that both important functions of vitellocytes, namely eggshell formation and nourishment of the early embryo, may be somewhat reduced in this species. The amount of lipids in vitellocytes is highly variable in different groups of cestodes. The highest concentration was
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described in the vitellocytes of Tetraphyllidea (Mokhtar-Maamouri and Œwiderski 1976), where accumulations of lipid droplets were observed not only in their cytoplasm, but frequently also in the karyoplasm. A large number of osmiophilic, unsaturated lipid droplets was also observed in the vitellocytes of D. spinulifera. In D. spinulifera, lipids may serve as an energy source for the developing embryos. In trypanorhynch eggs a cellular economy, based initially upon autolysed ribonucleoproteins and subsequently upon large lipid reserves, would be better disposed to support longer-term cellular divisions and differentiation (McKerr 1985, Mattis 1986). Additional information on the important role of vitellocytes during embryogenesis of D. spinulifera may be better formulated from a comparison of egg development in trypanorhynchs and caryophyllideans. The ultrastructure of the vitellocytes in the trypanorhynch cestode D. spinulifera, on one hand, and caryophyllideans G. catostomi (Œwiderski and Mackiewicz 1976) and Khawia armeniaca (Œwiderski et al. 2004) on the other, reveals great differences, both in the type, quality, and quantity of eggshell globules and in the type and chemical character of nutritive reserves. Vitellocytes of D. spinulifera, as shown in the present study, contain a great number of unsaturated lipid droplets and only a very small amount of polysaccharides, mainly connected with membranous structures. Vitellocytes of caryophyllideans, on the other hand, are characterised by a great amount of nuclear and cytoplasmic glycogen and the absence of lipid droplets (Œwiderski and Mackiewicz 1976, Œwiderski and Xylander 2000, Œwiderski et al. 2004). Such differences may be associated with the different environmental conditions required for egg maturation in both cestode orders. For example, an aerobic environment is necessary for the development of Trypanorhyncha intermediate hosts, and nearly anaerobic environment (mud) for Caryophyllidea (Œwiderski and Mackiewicz 1976, Œwiderski and Xylander 2000, Œwiderski et al. 2004). To the nutrient factor must be added the morphology of the egg as an important adaptive factor in the successful transmission of any cestode (Jarecka 1961, Mackiewicz 1988). Clearly, the type of vitellogenesis present in D. spinulifera, along with nutrient reserves and egg morphology, are all related to the ecology and successful life cycle of this species. Furthermore, the nature of the nutritive reserves accumulated in the vitellocytes may also reflect the parallelism and analogies in adaptations to the parasitic way of life in different groups of cestodes (Œwiderski and Xylander 2000). Acknowledgements. Authors wish to thank Dr Malcolm Jones (Centre for Microscopy and Microanalysis, University of Queensland, Australia) for providing the embedded specimens of D. spinulifera and Dr Ian Beveridge (Department of Veterinary Science, University of Melbourne, Australia) for confirming the identification of these specimens. We also wish to thank the “Serveis CientificotPcnics” (University of Barcelona, Spain) for their support in the preparation of samples. This study was financially supported by the Project 2005-SGR-00576 from the “DURSI, Generalitat de Catalunya” and by the Project A/2390/05 from the “Programa Inter-
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campus de Cooperación Científica e Investigación Interuniversitaria entre EspaZa y Túnez, AECI, Ministerio de Asuntos Exteriores” of Spain. Dr George McKerr (Faculty of Life and Health Science, University of Ulster, North Ireland, U.K.) and Dr T.E. Mattis (University of Southern Mississippi U.S.A.) kindly provided a copy of his PhD thesis for comparative purposes. The completion of results, analysis of TEM micrographs, and preparation of this manuscript for publication was supported by a Sabbatical Grant (SAB2005-0068) from the “Secretaría de Estado de Universidades e Investigación, Ministerio de Educación y Ciencia” of Spain for ZŒ.
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