Immature vitellocytes (Figs. 1A, D, G, 2A, 3A, 4A) of all studied species are localized at the peripheral region of the vitelline follicle (Fig. 1A, D, G). They possess a large nucleus with irregular clumps of heterochromatin dispersed in the nucleoplasm and a thin layer of perinuclear cytoplasm (Figs. 2A, 3A, 4A). The nucleus of immature vitellocytes in C. namaquensis and S. solidus possesses distinct nucleolus (Figs. 2A, 4A). Free ribosomes and a few large mitochondria are scattered in the cytoplasm. Granular endoplasmic reticulum (GER) is visible at this stage in the cytoplasm of immature vitellocytes of C. namaquensis and D. expansa (Figs. 2A, 3A).

During this stage of development (Figs. 2B, 3B, 4B), the volume of perinuclear cytoplasm increases. The vitellocytes are characterized by a period of high secretory activity. Mitochondria, free ribosomes, developing granular endoplasmic reticulum (GER) and Golgi complexes are involved in the synthesis and packaging of vitelline material. The nucleus exhibits several irregular clumps of heterochromatin and nucleolus. The increased volume of perinuclear cytoplasm contains a few profiles of GER confined to the nuclear membrane and several mitochondria, mainly in the periphery of the cells (Figs. 2B, 3B, 4B). Preceding the formation of shell globule clusters, several individual shell globules inside the Golgi-derived vesicles are observed in the vitelline cytoplasm (Figs. 2B, 4B). Subsequently, the vesicles increase in size and aggregate, forming shell globule clusters composed of globules of various sizes. The early stage of vitellocyte differentiation is completed with the appearance of the first lipid droplet in vitelline cytoplasm (Fig. 2B).

Advanced maturing vitellocytes (Figs. 2C, E, 3C, 4C, D) are elongated cells with the centrally situated nucleus and heterochromatin clumps usually adjacent to the membrane of the nucleus. During the advanced stage of maturation, protein synthesis still continues resulting in the formation of numerous shell globule clusters. Vitelline clusters of S. solidus (ca. 2.5 μm in diameter) and C. namaquensis (ca. 2 μm in diameter) comprise between 5 and 7 loosely packed globules (ca. 0.9 μm in diameter in S. solidus and 0.6 μm in C. namaquensis) embedded in electron-lucent matrix (Figs. 2C, 4D). In contrast, the clusters of D. expansa are bigger (ca. 5 μm in diameter), composed of ∼ 5 tightly packed electron-dense globules. Individual shell globules within the clusters of D. expansa are relatively homogeneous in shape and size (ca. 2 μm) and there is no outer limiting membrane surrounding the clusters (Fig. 3C). In addition, large electron-lucent lipid droplets are abundant in the cytoplasm of D. expansa and C. namaquensis (Figs. 3C, 4C, E). The cytoplasm of S. solidus contains glycogen granules situated around electron-dense lipid droplets (Fig. 4F).

The perinuclear cytoplasm of mature vitellocytes (Figs. 2D, F, G, 3D–F, 4D–H, 5A–D) is almost entirely occupied by four types of vitelline inclusions: shell globule clusters, lipid droplets, glycogen granules, and lamellar bodies. Concentric rows of granular endoplasmic reticulum (GER) are also found close to the cell periphery (Figs. 3D, 4E). The glycogen granules are scattered among lipid droplets and vitelline clusters (Figs. 2G, 3D, 4D). The test of Thiéry was used for revealing the presence of glycogen granules (Fig. 2G, 3D, Inset, Fig. 4F). Mature vitellocytes of D. expansa and S. solidus contain numerous lamellar bodies in the cytoplasm (Figs. 3E, F, 4E, G, H), but these cell structures are missing in C. namaquensis (Figs. 2D, F, 5A). Concentric rows of granular endoplasmic reticulum (GER) occur in the vicinity of the lamellar bodies (Figs. 3E, 4E), which may indicate that the appearance of these structures coincides with the breakdown of granular endoplasmic reticulum. For comparative purposes, a generalized scheme of mature vitellocytes of the three species is provided in Fig. 5(A–D). Table 1 summarizes general characters of the mature vitellocytes in the Diphyllobothriidea.

Of particular interest are the morphological differences of vitelline clusters among the four diphyllobothriidean species studied to date [7,present study]. Vitelline clusters among them differ from each other by the degree of packaging shell globules, the number of shell globules in each cluster, and their size. Shell globule clusters in vitellocytes of S. solidus show a basic ultrastructural organization similar to that reported in D. latum [see 7], both cestodes belonging to the same family and parasitizing homeiothermic hosts. However, the clusters between the two species differ by the number of individual shell globules. The number of globules in clusters of D. latum is almost three times greater than in S. solidus. Surprisingly, C. namaquensis and D. latum share some common features, even though they are biologically and phylogenetically very distant [3,4]. Both exhibit vitelline clusters of approximately the same number of globules and possess individual globules of about the same diameter. Comparing the clusters of D. expansa with the three other species, we found similarities with S. solidus in the number of shell globules in each cluster, although they present different arrangement of shell globules in clusters (Fig. 5). These two taxa may represent phylogenetically closely related groups (R. Kuchta, unpublished data).

The presence of lipid droplets in C. namaquensis, D. expansa, S. solidus and D. latum [7,present study] is consistent with the previous observation in other cestode groups, including the most basal eucestodes. These droplets probably play an important role in supplying nutritive reserves for the developing embryo [6]. In the Diphyllobothriidea, the type and amount of lipids varied across the examined species/families. S. solidus possesses osmiophilic and electron-dense lipid droplets containing a high level of unsaturated fatty acids, whereas lipid droplets in D. expansa are osmiophobic and electron-lucent, thus representing saturated fatty acids. It is interesting to note that the same type of lipids represent the most abundant inclusion in mature vitellocytes of the diphyllobothriid D. latum, but are less numerous in D. expansa than in D. latum (see [7]). In the presumably most basal taxon studied, C. namaquensis, vitelline cells differ in several ultrastructural aspects from those of the other diphyllobothriideans (Fig. 5). One of the characteristic features of mature vitellocytes of C. namaquensis is the presence of both types of lipids; the same character was observed in one of the three spathebothriidean cestodes studied by Bruňanská et al. [12], Cyathocephalus truncatus, a parasite of freshwater teleosts in the Holarctic Region.

Glycogen is the most common vitelline component in the Neodermata as an important source of energy for the future embryo [6]. The glycogen granules usually appear in the final stage of vitellogenesis and occupy almost entirely the cytoplasm of mature vitellocytes. All members of the Diphyllobothriidea contain numerous glycogen granules, usually accumulated around the lipid droplets and shell globule clusters.

Another type of subcellular structures worth discussing, lamellar bodies, were found in mature vitellocytes of three of the four diphyllobothriidean cestodes studied. They appear during the final stage of vitellocyte maturation usually close to concentric rows of granular endoplasmic reticulum (GER) and vitelline clusters. Lamellar bodies are present in mature vitellocytes of S. solidus, D. expansa and D. latum, but they have not been observed in C. namaquensis, i.e. the most basal taxon of the order. Structures with lamellar arrangement are particularly widespread in caryophyllideans and spathebothriideans, i.e. the most basal eucestodes [7,10–13,19,20,24,present study] as well as in some trematodes and monogeneans [25–32].

The formation of lamellar bodies is associated either with the breakdown of granular endoplasmic reticulum [24,29], with the transformation of shell globule clusters during the process of egg formation [13], or with both processes [20]. Based on the present data and on those by Yoneva et al. [7], lamellar bodies in diphyllobothriideans seem to originate from both the granular endoplasmic reticulum and/or by transformation of vitelline clusters. Two distinct types of lamellar bodies were detected in the Caryophyllidea. The first type, referred to as regular lamellar bodies, shows distinct concentric features, and the second type, irregular lamellar bodies, appears to have a non-concentric configuration, as has been observed in D. latum, D. expansa, and S. solidus [7,present study]. The same type is commonly found in spathebothriideans [12,13]. In contrast, caryophyllideans appear to have both types of these lamellar structures, with the irregular type being more common than the regular one [10,11,19,20,24]. However, the function of these bodies in parasitic flatworms including cestodes is unknown.

Some previous studies on vitellogenesis in free-living and parasitic platyhelminths indicated that the ultrastructure of some vitelline cytoplasmic inclusions might differ, even between genera [10,13,33]. In the present study, the process of formation of vitelline cells in representative species of all diphyllobothriidean could be compared and related to their presumed phylogenetic position within the order Diphyllobothriidea. Based on molecular data published by Brabec et al. [4], the most plesiomorphic features would be expected to exist in C. namaquensis, whereas the diphyllobothriids S. solidus and D. latum should rather possess more derived characters; the reptilian parasite D. fimbriata should exhibit intermediate features.

As described above, this assumption is partly supported by the absence of lamellar bodies in C. namaquensis (present in the three more derived taxa) and by the presence of two types of lipid droplets in the former species (in contrast, the derived taxa of the Scyphocephalidae and Diphyllobothriidae possess only one of these types of droplets). Variations in the quantity of lipids in cestodes are supposed to be generally host-related [34]. Thus, such variations in diphyllobothriideans could be related to three different host groups, as described for bothriocephalideans [34]. In addition, the degree of variations in the vitelline inclusions of members of all three families of the order likely depends on their specific life cycles, which include different definitive hosts such as birds and mammals (Diphyllobothriidae), amphibians (Cephalochlamydidae), and reptiles (Scyphocephalidae), and the number of intermediate hosts (one vs. two) or type of egg development (presence or absence of an operculum). Even though data on vitellogenesis of representatives maturing in all major vertebrate groups are now available, it is obvious that more species should be studied, such as representatives of diphyllobothriids from marine hosts and other taxa from reptiles, before a more complex view of the possible evolution of vitellogenesis is presented.

The relationships of the Diphyllobothriidea with other basal groups of the Eucestoda are unclear and analyses inferred from different molecular markers provided different topologies of the phylogenetical tree of the Eucestoda. A close relationship with the Spathebothriidea and all cestode orders from elasmobranchs and remaining acetabulate groups was indicated by the sequences of mitochondrial genomes [1]. A most recent phylogenetic study [2] even placed the Diphyllobothriidea as a sister taxon of the Litobothriidea and all acetabulate (“tetrafossate”) orders, closely related to the Trypanorhyncha.

The ultrastructure of vitellocytes of the spathebothriideans, parasites of freshwater and marine fish that possess operculate eggs, but differ from each other in the presence/absence of polar filaments [35], resemble those in the diphyllobothriideans, which could indicate relatedness of both orders, thus supporting the topology of the cestode tree presented by Waeschenbach et al. [1]. Interestingly, the eggs of the diphyllobothriids (including D. latum, S. solidus and D. expansa) are hard-shelled, operculate and mostly unembryonated when laid. The only exceptions are eggs of the cephalochlamydids that have a membranous shell, are unoperculate, but are embryonated when laid, and whose coracidium is liberated by rupture of the egg membrane [36].

The scarcity of ultrastructural data throughout all major cestode groups at least to the family level limits a more detailed comparison of vitellogenesis in the Cestoda. It is thus impossible to map the ultrastructural characters on the phylogenetic tree of major groups of tapeworms, the phylogenetic relationship of which are not yet well resolved [1,2]. The future ultrastructural research thus should be much more selective, as far as the choice of the species to be studied: instead of incidental selection of taxa based just on their availability, more effort should be given to a more complete taxonomic coverage, i.e. the search for representatives of as many families or even genera as possible, with the focus on phylogenetically important groups, such as the most basal cestodes (“Cestodaria”, i.e. Gyrocotylidea and Amphilinidea), Haplobothriidea, and Litobothriidea.