Daniel Młocicki

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.

An ultrastructural study of embryonic envelope formation in the anoplocephalid cestode Mosgovoyia ctenoides (Railliet, 1890) Beveridge, 1978

In this study the ultrastructural aspects of egg envelope formation in the anoplocephalid cestode Mosgovoyia ctenoides are described. In the early stage of oncospheral morphogenesis, formation of three following primary embryonic envelopes takes place: (1) the capsule, (2) the outer envelope, and (3) the inner envelope. The capsule is formed from the vitellocyte material. Two macromeres contribute to the formation of the outer envelope and three mesomeres take part in the formation of the inner envelope. The three primary envelopes undergo further differentiation and transformation into the secondary envelopes, the so-called oncospheral or egg envelopes. In the advanced preoncospheral phase, the inner envelope undergoes differentiation into three sublayers: (1) a thick extra-embryophoral cytoplasmic layer; (2) an electron-dense embryophore, as a stiff pyriform apparatus; and (3) a thin intra-embryophoral cytoplasmic layer containing mesomere nuclei. The oncosphere is located in the extended cupule-like part of the pyriform apparatus. The two embryophoral horns elongate and fuse, thus forming a rigid cone. Four egg envelopes surround the mature infective oncosphere of M. ctenoides: (1) a thick capsule; (2) the outer envelope; (3) the inner envelope with a characteristic embryophore, in the form of the pyriform apparatus; and (4) the oncospheral membrane. The differentiation and ultrastructure of the egg envelopes of M. ctenoides are compared, in particular to those described in other anoplocephalids, and in general to the oncospheres of other cestode species.

CELLULAR ORGANIZATION OF THE ONCOSPHERE OF MOSGOVOYIA CTENOIDES (CESTODA: ANOPLOCEPHALIDAE)

The ultrastructure of the infective oncosphere of the cestode Mosgovoyia ctenoides (Anoplocephalidae) is described. The surface of the infective oncosphere is covered by a thin cytoplasmic layer of tegument connected by a narrow cytoplasmic process with the binucleate subtegumental cell, situated deeper in the body. Below the basal matrix of the cytoplasmic layer of the tegument are situated wide bands of the peripheral, somatic musculature responsible for body movements. The 3 pairs of hooks and their muscles form a complex hook muscle system, responsible for coordinated hook action. Five major types of cells have been distinguished: (1) a binucleate subtegumental cell, (2) a binucleate penetration gland, (3) 2 nerve cells, (4) numerous somatic cells, and (5) about 6 germinative cells. The approximate number of cells is 24 (26 nuclei, including 2 syncytial structures). The results of this study, when compared with other published reports from other cestode taxa, support previous hypotheses that the progressive reduction of oncosphere cells is an adaptive feature in cestode evolution.

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.

The ultrastructure of taeniid cestode oncospheres and localization of host-protective antigens

Taeniid eggs contain an infective larval form of the parasite, known as the oncosphere, which has been found to be highly susceptible to attack by the hosts immune system and this fact has been exploited in the development of highly effective vaccines. Relatively little is known about the structure of taeniid oncospheres and the localization of host-protective antigens within or on the oncosphere. Here, we briefly review the current state of knowledge of the structure of the oncosphere and present preliminary data on the localization of a host-protective antigen within the oncospheres of Taenia ovis. The precise localization of the antigens, in the context of a detailed knowledge of the ultrastructure of the parasite, may reveal the immune mechanisms by which the taeniid parasites are killed by vaccine-induced immune responses, which, in turn, may provide clues about how vaccines could be developed against other parasitic helminths.

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.

Ultrastructural and cytochemical studies of GER-bodies in the intrauterine eggs of Wenyonia virilis Woodland, 1923 (Cestoda, Caryophyllidea)

Ultrastructural and cytochemical characteristics of GER-bodies observed in the vitellocyte cytoplasm of the intrauterine eggs of the caryophyllidean cestode Wenyonia virilis are described. In this species GER-bodies were observed only in the cytoplasm of vitellocytes, surrounded by a newly formed egg-shell. They are composed of spherical areas of condensed, electron-dense cytoplasm which contains concentrically arranged parallel lamellae of granular endoplasmic reticulum (GER), forming characteristic balls of different sizes. Each GER-body is surrounded by numerous free ribosomes, polyribosomes, α-glycogen rosettes and large mitochondria. Results of cytochemical analysis by means of PATSC-SP test for polysaccharides indicated that glycogen is absent within the GER-bodies, however, a strongly positive reaction was observed only in large aggregations of α-glycogen rosettes and β-glycogen particles, localised usually near GER-bodies.

Ultrastructural reconstruction of Taenia ovis oncospheres from serial sections

The cellular organisation of Taenia ovis oncospheres is interpreted from ultrathin serial sections and transmission electron microscopy following high pressure freezing and freeze-substitution. The surface of a hatched, non-activated T. ovis oncosphere is covered by an oncospheral membrane below which is the tegument bearing microvilli. The basal lamina of the tegument is underlain by broad bands of peripheral somatic musculature. Three pairs of hooks and associated muscles are present in the somatophoric third of the oncosphere. Approximately 19 cells of seven different types were identified which include: (i) a quadri-nucleated syncytium of penetration gland type 1 containing two lateral pairs of cell bodies interconnected by narrow cytoplasmic bridges (PG1); (ii) a quadri-nucleated syncytium of penetration gland type 2 (PG2); (iii) a single-nucleated median mesophoric gland cell; (iv) 10 somatic cells; (v) two germinative cells; (vi) two nerve cells; and (vii) a pair of median somatophoric cells. This study provides a clear understanding of the morphology of T. ovis oncospheres and forms the basis for further investigations into the biology of taeniid oncospheres.

Post-embryonic development and ultrastructural characteristics of the polycephalic larva of Taenia parva Baer, 1926 (Cyclophyllidea, Taeniidae)

Post-embryonic development and fully-formed polycephalic larvae of Taenia parva Baer, 1926 were examined by light (LM) and transmission electron microscopy (TEM). Three developmental stages were recognised: (1) an early stage of exogenous budding at the surface of the central vesicle; (2) a stage of polycephalic cyst development accompanied by segmentation of the growing larval strobile and an obvious decrease in the size of the central vesicle; (3) fully-formed larval strobile and invaginated scoleces. In fully-developed encysted polycephalic larvae, there are usually 14–24 segmented larval strobilae, each terminating with an invaginated scolex; larval strobilae arise from a common central vesicle and remain attached posterior to it during the entire development. The number of segments varies between 109 and 120 per larval strobila. The polycephalic larvae examined closely resemble the strobilocercus type of taeniid larvae. The structure of developing and fully-formed larvae was examined by TEM. The tegument, scolex, subtegumental musculature of the strobilar segments, protonephridial system, calcareous corpuscles and medullary parenchyma of larvae exhibit general similarity with the same structures in adults at both LM and TEM levels. The morphogenesis of the larva of T. parva is compared with that of the polycephalic larvae of other Taenia spp. (T. krepkogorski, T. twitchelli and T. endothoracica) and with other asexually-multiplying cestode larvae (mesocestoidids, hymenolepidids and dilepidids).

Early intrauterine embryonic development in Khawia sinensis Hsü, 1935 (Cestoda, Caryophyllidea, Lytocestidae), an invasive tapeworm of carp (Cyprinus carpio): an ultrastructural study

Intrauterine embryonic development in the caryophyllidean tapeworm Khawia sinensis has been investigated using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. Contrary to previous light microscopy findings that reported the release of non-embryonated eggs of K. sinenesis to the external environment, the present study documents various stages of embryonation (ovoviviparity) within the intrauterine eggs of this cestode. At the initial stage of embryonic development, each fertilised oocyte is accompanied by several vitellocytes that become enclosed within the operculate, electrondense shell. Cleavage divisions result in formation of blastomeres (up to about 24 cells) of various sizes. Mitotic divisions and apparent rosette arrangment of the blastomeres, the latter atypical within the Eucestoda, are observed for the first time in the intrauterine eggs of K. sinenesis. The early embryo enclosed within the electrondense shell is surrounded by a thin membraneous layer which in some enlarged regions shows presence of nuclei. Simultaneously to multiplication and differentiation, some of the blastomeres undergo deterioration. A progressive degeneration of the vitellocytes within eggs provides nutritive reserves, including lipids, for the developing embryo. The possible significance of this atypical timing of the intrauterine embryonic development to (1) the ecology of K. sinensis and that of a recent introduction of another invasive tapeworm, the caryophyllidean Atractolytocestus huronensis Anthony, 1958 to Europe; and (2) the affiliation of caryophyllideans with other lower cestodes, are discussed.