Ultrastructural and cytochemical study on vitellogenesis in the diphyllidean cestode Echinobothrium euterpes (Echinobothriidae) and its phylogenetical implications

The first description of vitellogenesis in the Diphyllidea is presented in this paper. Though the type of vitellogenesis and mature vitellocyte in Echinobothrium euterpes appear to be unique among the Eucestoda, however, they somewhat resemble that observed in the two orders of the lower cestodes, Tetraphyllidea and Proteocephalidea. Vitellocyte maturation is characterized by: (1) an increase in cell volume; (2) extensive development of short, parallel, frequently concentric cisternae of GER that produce dense proteinaceous granules; (3) development of Golgi complexes engaged in packaging this material; (4) progressive formation of saturated lipid droplets; their continuous enlargement and fusion; (5) formation of small accumulations of glycogen particles scattered between and among lipid droplets in the cytoplasm of maturing vitellocytes; (6) concentration of dense proteinaceous granules in the peripheral layer of cytoplasm, around the cell plasma membrane; and (7) vacuolization of cytoplasm of mature vitellocytes accompanied by a rapid increase in its volume. A new, unreported type of dense proteinaceous granules, situated around the limiting plasma membranes of mature vitellocytes, is described. Vitellogenesis evidently differs from that with typical shell-globules and shell-globule clusters previously reported in other taxa of lower cestodes. Cytochemical staining with periodic acidthiosemicarbazide-silver proteinate for glycogen indicates a strongly positive reaction for glycogen particles between and around large unsaturated lipid droplets of the maturing and mature vitellocytes. Some hypotheses concerning the interrelationship between this pattern of vitellogenesis, possible mode of egg formation, embryonic development and diphyllidean life cycle, and their phylogenetic implications are drawn and discussed.

Ultrastructure and cytochemistry of vitellogenesis in Wenyonia virilis Woodland, 1923 (Cestoda, Caryophyllidea)

Vitellogenesis in Wenyonia virilis was examined by transmission electron microscopy (TEM), including the cytochemical detection of glycogen at the ultrastructural level with the periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) technique. Mature vitelline follicles have cells in various stages of development, progressing from immature cells of gonial type near the periphery of the follicle to maturing and mature vitellocytes towards the centre. Maturation is characterized by: (1) increase in cell volume; (2) increase in nuclear surface area restoring the N/C (nucleo-cytoplasmic) ratio; (3) nucleolar transformation; (4) extensive development of parallel cisternae of GER, the shell-protein producing units; (5) development of Golgi complexes, engaged in shell-granule/shell-globule formation and packaging; (6) synthesis and storage of glycogen in the cytoplasm; (7) simultaneous, independent formation and storage of intranuclear glycogen; (8) continuous fusion of small shell-granules into larger shell-globules and fusion of these into large shell-globule clusters with a progressive increase in the number and size of the latter; and (9) disintegration of GER in the medial layer of vitellocyte cytoplasm, degenerative changes and accumulation of glycogen and shell-globule clusters within the cytoplasm. The functional significance of numerous shell-globule clusters and the relatively small amount of nuclear and cytoplasmic glycogen is analysed. Unlike vitellogenesis of other caryophyllids, the nuclear glycogen of mature vitellocytes in W. virilis is randomly dispersed in the nucleoplasm and never forms a high central accumulation, the so-called “nuclear vacuole”. The nutritive function of vitellocytes appears greatly reduced in W. virilis, a fact perhaps related to the intrauterine development of the early embryos. The ultrastructure of vitellogenesis in W. virilis is compared with that in other lower cestodes, both monozoic and polyzoic. Conclusions concerning interrelationships of the vitellogenesis pattern of the ultrastructural cytochemistry of mature vitellocytes of W. virilis to intrauterine embryonation, absence of uterine glands and an extensive uterus characteristic for this species, are drawn and discussed.

Ultrastructural and cytochemical studies on vitellogenesis in the trypanorhynch cestode Parachristianella trygonis Dollfus, 1946 (Eutetrarhynchidae)

During vitellogenesis in Parachristianella trygonis Trypanorhyncha, Eutetrarhynchidae) we distinguished four stages: (1) gonial or stem cell stage; (2) early differentiation stage concentrated on protein synthetic activity and shell-globule formation; (3) advanced differentiation stage with main cell activity concentrated on carbohydrate synthesis (glycogenesis) and massive glycogen storage in the form of α-glycogen rosettes and β-glycogen particles; and finally (4) mature vitellocyte stage. Early vitellocyte maturation is characterised by: (1) an increase in cell volume; (2) extensive development of large, parallel cisternae of GER that produce proteinaceous granules; (3) development of Golgi complexes engaged in packaging this material; (4) continuous enlargement of proteinaceous granules within vacuoles and their transformation into shell-globule clusters composed of heterogeneous material. Cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for polysaccharides indicated a strongly positive reaction for the presence of α-glycogen rosettes and β-glycogen particles in the advanced stage of vitellocyte maturation. Both protein synthesis for shell-globule formation and carbohydrate synthesis or glycogenesis, important storage of nutritive reserves for the developing embryos, observed during cytodifferentiation of P. trygonis vitellocytes overlap in time to some extent. Mature vitelline cells are very rich in three types of cell inclusions accumulated in large amounts in their cytoplasm: (1) shell-globule clusters, playing an important role in egg-shell formation; (2) numerous large lipid droplets, as well as a high accumulation of lipid and α-glycogen rosettes and β-glycogen particles that undoubtedly represent important nutritive reserves for the developing embryos. Despite the fact that the type of vitellogenesis and ultrastructure of the mature vitellocyte in P. trygonis appears to differ to some extent from those of three other trypanorhynch species, its general pattern and ultrastructure greatly resembles those observed in other lower cestodes. Factors that may have contributed to the qualitative and quantitative variation in lipids during vitellogenesis among the four species of Trypanorhyncha, are identified and discussed.

Ultrastructural and cytochemical studies on vitellogenesis in trypanorhynch cestode Dollfusiella spinulifera Beveridge, Neifar et Euzet, 2004 (Eutetrarhynchidae)

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 acid-thiosemicarbazide-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 vitellogensis, possible mode of egg formation in D. spinulifera, its embryonic development and trypanorhynchean life cycle, are drawn and discussed.

Vitellocyte ultrastructure in the cestode Didymobothrium rudolphii (Monticelli, 1890): possible evidence for the recognition of divergent taxa within the Spathebothriidea

In the spathebothriidean tapeworm Didymobothrium rudolphii (Monticelli, 1890) the fine structure of the vitellocytes at different stages of their development within the vitelline follicles, vitelline ducts and uterus was studied for the first time using transmission electron microscopy. The vitellocyte inclusions of D. rudolphii are shell globule clusters containing tightly packed shell globules associated with a matrix of moderate electron density, glycogen granules, large electron-lucent lipid droplets (up to 3 μm in diameter), and, occasionally, a lipid droplet may occur in the nucleus of the vitellocytes. The diameter of the clusters ranges from 0.4 to 2.5 μm, the number of shell globules in the clusters varies from 8 to 45, and the size of the globules ranges from 0.12 to 0.25 μm and they are of approximately homogeneous sizes within a cluster. Most vitellocyte lipid droplets have a heterogeneous configuration with a ‘cavity’ inside them when they are within vitelline ducts and intrauterine eggs. Vitellocytes of the eggs contain dark concentric bodies and lipid droplets. The interstitial tissue has a syncytial structure. The morphological parameters of the diameter and shape of shell globule clusters, arrangement of shell globules in clusters, number and diameter of globules within clusters, types of lipid droplets and presence of dark concentric bodies are compared with those of two other spathebothriidean genera, Cyathocephalus and Diplocotyle. The comparative data demonstrate that vitelline material morphology has unique features in three spathenothriidean genera and may be used as evidence for the recognition of separate taxa.

Ultrastructure of vitellocytes in the cestode Progrillotia pastinacae Dollfus, 1946 (Trypanorhyncha, Progrillotiidae)

The present study describes the ultrastructure of mature vitellocytes of the trypanorhynch cestode Progrillotia pastinacae Dollfus, 1946 (Progrillotiidae), a parasite of the common stingray Dasyatis pastinaca (Linnaeus, 1758) (Dasyatidae). The vitelline cells of this species measure about 24 μm in length and about 20 μm in width. They have small, elongated, slightly lobulated nuclei, about 4–5 μm in length, with large dense elongated nucleoli and numerous irregularly-shaped dense clumps of heterochromatin. The extensive cytoplasm is rich in numerous cell organelles and cell inclusions. While the perinuclear cytoplasm contains numerous long parallel cisternae of GER, ribo-and polyribosomes, several Golgi complexes and mitochondria, the peripheral cytoplasm contains predominantly three types of cell inclusions: a great number of large lipid droplets, several shell globule clusters, and a very small amount of glycogen-like particles. The most characteristic features of vitellocytes in P. pastinacae are having almost no traces of glycogen and the great number of large, highly osmiophobic lipid droplets representing saturated fatty acids. The presence of large amounts of lipids also in two other trypanorhynchs, Grillotia erinaceus (Beneden, 1858) Guiart, 1927 and Dollfusiella spinulifera (Beveridge et Jones, 2000) Beveridge, Neifar et Euzet, 2004, is in strong contrast to the condition in the most evolved cestodes, Cyclophyllidea, that usually show no trace of lipids.

Development of the vitellaria of the liver fluke, Fasciola hepatica in the rat host

The development of the vitellaria of Fasciola hepatica within the liver of its rat host was studied by means of whole-mount stained preparations and transmission electron microscopy, together with light and electron immunocytochemistry using an antibody to vitelline protein B, an eggshell precursor protein synthesized by F. hepatica. No vitelline cells could be identified in flukes recovered from the liver parenchyma, by any of the methods used. In contrast, follicles were present in flukes at the earliest time of recovery from the bile duct, namely, 5 weeks 3 days post-infection. The vitellaria in these flukes formed a row of small follicles on either side of the body. Development of the follicles was rapid: by 6 weeks 3 days, the vitellaria resembled those in the adult fluke and eggs were present in the uterus. Immunolabelling was confined to the shell protein globules in the vitelline cells, confirming the packaging of the eggshell protein within the shell globule clusters.

Fine structure of the vitteline cells in the cestode Proteocephalus longicollis (proteocephalidea)

The mature vitelline cells of Proteocephalus longicollis (Zeder, 1800) are ovoid or spherical and measure about 30μm in diameter. They represent the holocrine type of secretory cells.The spherical nuclei, about 10μm in diameter, are localized in the central part of the cell. They contain more or less prominent nucleoli enclosed in the moderately electrondense nucleoplasm (Fig. 1).The cytoplasm (Fig. 2, 3, 4) is packed with numerous shell globules of heterogenous type, large lipid droplets, and a few glycogen islands composed mainly of α-glycogen rosettes.The remaining granular cytoplasmic matrix (Fig. 3, 4) contains: (a) numerous polysomes, (b) cysternae of granular endoplasmic reticulum, (c) several mitochondria, and (d) extended Golgi regions, composed of vesicles of different sizes and density.The extensive development of granular endoplasmic reticulum (GER) and Golgi complexes indicates high secretory activity of these cells. Both Golgi and GER are evidently engaged in shell globule formation and are considered therefore to function as the shell-protein producing units

Electron microscopy of embryonic envelope formation by the cestode proteocephalus longicollis (zeder, 1800)(proteocephalidea)

Four main embryonic envelopes, the capsule, outer envelope, inner envelope and oncospheral membrane, are formed around the developing embryos of Proteocephalus longicollis, a parasite of fishes.The capsule, formed from shell-globule material of vitelline cells, is the first embryonic envelope which encloses the fertilized oocyte and a single vitelline cell (Fig. 1) when they pass through the ootype and enter the uterus. At this initial stage of embryogenesis, the capsule is formed of two closely apposed membranes (Fig. 1, inset). In more advanced stages, the capsule slightly increases its volume, and a fine-fibrillar material accumulates in the space between its two limiting membranes (Fig. 2).