ULTRASTRUCTURE OF EGG ENVELOPES AND EARLY EMBRYOS OF ROHDELLA AMAZONICA (TREMATODA: ASPIDOGASTREA) PARASITIC IN BANDED PUFFER FISH, COLOMESUS PSITTACUS.

Egg structure and early embryonic development of the aspidogastrean, Rohdella amazonica, a basal trematode, were studied by transmission electron microscopy (TEM) to gain insight into functional, developmental, and phylogenetic characteristics. Gravid worms were removed from the intestine of naturally infected banded puffer fish Colomesus psittacus, collected from the Bay of Marajó, Paracauari River (Pará, Brazil) and processed by standard TEM methods. By the time of pronuclear fusion, the fertilized zygote was already enclosed in a thick, electron-dense pre-operculate eggshell and an underlying layer of vitellocytes that fused into a vitelline syncytium as they were still secreting their shell granules. When cleavage commenced, a small number of macromeres moved to the area just underneath the eggshell, where they fused to form a single syncytial embryonic envelope. Simultaneously, the smaller blastomeres continued to divide as they maintained contact with each other, but remained separate from the vitelline syncytium. Concurrent with these cellular changes, a thickened knob expanded at one pole of the eggshell and began to form an opercular suture. By the time the operculum was fully formed, the vitelline syncytium had mostly degenerated, while the smaller blastomeres had become cohesive as a single mass that preceded the differentiation and morphogenesis of the cotylocidium larva. The general pattern of cleavage and eggshell formation resembles that of other trematodes and polylecithal cestodes, but the single embryonic envelope has been reported only in a few basal taxa. The only other aspidogastrean studied in detail to date is very similar, indicating close phylogenetic affinity and conservatism within this basal neodermatan and neoophoran group.

ULTRASTRUCTURE AND CYTOCHEMISTRY OF LATE EMBRYOS AND COTYLOCIDIUM LARVAE OF ROHDELLA AMAZONICA (TREMATODA: ASPIDOGASTREA), FROM THE TROPICAL ESTUARINE FISH, COLOMESUS PSITTACUS.

Developmental ultrastructure of late embryos and cotylocidium larval morphogenesis of Rohdella amazonica, an aspidogastrean parasite of fish, were studied to reveal the functional aspects of larvigenesis within the egg as well as phylogenetically relevant characteristics of the embryos and larvae in this basal trematode group. Gravid worms were removed from the intestine of naturally infected banded puffer fish Colomesus psittacus, collected from the Bay of Marajó, Paracauari River (Pará, Brazil) and processed by standard methods of transmission electron microscopy (TEM) and cytochemistry. During late cleavage and rearrangement of the blastomeres, the vitelline syncytium that plays a role in eggshell formation and nutrient provision to the embryo completes its apoptotic degeneration as the embryonic mass grows substantially. Early larval morphogenesis involves cellular positioning that defines the anteroposterior polarity of the differentiating larva. Progressing through larvigenesis, the anterior end forms a muscular oral sucker surrounding the mouth, which leads inward into the pharynx and expanding digestive cavity. At the posterior end, a large disc forms as a precursor to the eventual ventral disc. The fully formed cotylocidium, still within the eggshell, is flexed ventrally, bringing the 2 poles into near juxtaposition. The neodermatan tegument with outwardly projecting small microvilli becomes fully formed, as myocytons, a protonephridial system, and 2 glandular regions occupy the body's interior. The ultrastructural features described here are very similar to those reported for Aspidogaster limacoides from fish and somewhat similar to those reported for Cotylogaster occidentalis from molluscs, but differ from the more diverse larvae of neodermatan taxa that have been studied more extensively.

Ultrastructure and cytochemistry of intrauterine embryonic and larval stages of Ityogonimus lorum (Digenea: Brachylaimidae) involving transitory development of ciliated miracidia.

Results of the present study provide ultrastructural evidence that miracidial morphogenesis is fully completed within the intrauterine eggs while in the most posterior uterine regions of Ityogonimus lorum, a digenean parasite of an Iberian mole, Talpa occidentalis (Eulipotyphla, Talpidae). Using transmission electron microscopy (TEM), the ultrastructural characteristics of diverse cell types and their organelles of these developing embryos and fully formed miracidia within the eggshell were examined. The eggshell and embryonic envelopes are similar to those described previously by many authors for other digeneans. However, the developing miracidia are unique among previously described digeneans in possessing transitory cilia during larvigenesis, but completely lacking cilia in fully formed miracidium larvae. The evidence for completion of miracidial maturation in intrauterine eggs is based on the presence of the following structures: (1) transitional stage of ciliated differentiating miracidial epithelium; (2) apical and lateral glands, characteristic for digenean miracidia; and (3) fully developed germinative cells grouped together in the germinative sac localized in the posterior region of the miracidium. The protonephridial system with its characteristic flame cells and the nervous system with diverse types of neurons and nerve centers, which are characteristic for other digenean species reported until now, are absent from all these developmental stages of I. lorum. Based on these observations, we hypothesize that the life cycle of I. lorum is entirely terrestrial, involving passive transmission by ingestion of eggs containing unciliated miracidia to the first intermediate host.

Sperm characters of the aspidogastrean Rohdella amazonica (Aspidogastridae, Rohdellinae), a parasite of the banded puffer fish Colomesus psittacus.

The ultrastructural characteristics of the mature spermatozoon of the aspidogastrean Rohdella amazonica (Aspidogastridae, Rohdellinae) were studied by means of transmission electron microscopy. The sperm cell shows two axonemes of the 9 + '1' trepaxonematan pattern of Platyhelminthes, parallel cortical microtubules, a well-developed lateral expansion, external ornamentation of the plasma membrane, one mitochondrion, an electron-dense ring, a nucleus and granules of glycogen. The present results were compared with those observed in the aspidogastreans studied to date and in other Platyhelminthes. The lateral expansion and the electron-dense ring are typical characters for aspidogastreans. Although a lateral expansion has been described in other Platyhelminthes such as monogeneans and digeneans, the Aspidogastrea shows a much larger lateral expansion with both peripheral and internal microtubules. The dense ring is observed as a cylinder in a longitudinal view and shows a more granular appearance in sperm cells from the seminal vesicle in comparison to a more electron-dense appearance in sperm cells from the seminal uterine receptacle.

Functional ultrastructure and cytochemistry of vitellogenesis and mature vitellocytes of the digenean Cainocreadium labracis (Dujardin, 1845), parasite of Dicentrarchus labrax (L., 1758).

Vitellogenesis and vitellocytes of Cainocreadium labracis were studied by transmission electron microscopy (TEM) and TEM cytochemistry. Four developmental stages were distinguished during vitellogenesis: (I) stem cell of high nucleo-cytoplasmic ratio; (II) early differentiation with chief activity focused on the beginning of protein synthesis and shell globule formation; (III) advanced differentiation with rapid intensification of protein synthesis, progressive fusion of single shell globules into large globule clusters, and formation of unsaturated lipid droplets surrounded by ß-glycogen particles; and (IV) mature vitellocyte. Early vitellogenesis with vitellocyte maturation consists of: (1) increase in cell volume; (2) increased development of large, parallel cisternae of GER with production of proteinaceous granules; (3) development of small Golgi complexes that package granules; and (4) within vacuoles, progressive enlargement of proteinaceous granules into shell globule clusters formed during vitellogenesis. Three types of inclusions accumulate in large amounts in mature vitelline cells: (1) shell globule clusters, important component in the formation of egg shell; (2) numerous unsaturated lipid droplets. Though fewer, there are also diphasic droplets consisting of saturated and unsaturated lipids in the same droplet, and (3) a relatively small amount of ß-glycogen particles, usually surround a few groups of lipid droplets. The ß-glycogen and lipid droplets are nutritive reserves for embryogenesis. General pattern and functional ultrastructure of vitellogenesis greatly resemble those observed in some lower cestodes, such as bothriocephalideans and diphyllobothrideans. Variations and differences in the amount of lipids and of glycogen during vitellogenesis in lower cestodes and other trematodes are compared and discussed.

Echinococcus multilocularis (Cestoda, Cyclophyllidea, Taeniidae): functional ultrastructure of the penetration glands and nerve cells within the oncosphere.

The fine structure of the infective hexacanths of Echinococcus multilocularis was examined with particular emphasis on the functional ultrastructure of penetration glands and nerve cells directly involved in the mechanism of initial host infection. The oncosphere contains two types of penetration glands, PG1 and PG2, that differ slightly in size and form a large U-shaped bi-nucleated syncytial structure. The arms of each gland at each end of the U, directed towards the hook region, exit into the tegument peripheral layer between the median and lateral hook pairs. The lobate nuclei of PG1 and PG2 contain prominent spherical nucleoli surrounded by several large electron-dense islands of heterochromatin. The syncytial cytoplasm of both types of glands is rich in free ribosomes, polysomes, several mitochondria, and heavy accumulations of discoid secretory granules of moderate to high electron density. The secretory granules, sg1 and sg2, differ in their ultrastructure and electron density; the sg2 are much smaller and more flattened in shape. A common characteristic for sg1 and sg2, evident under high magnification, is their high electron density and discoidal shape, with two bi-concave surfaces. Both sg1 and sg2 are frequently grouped in characteristic parallel stacks, the "rouleau"-shaped assemblages with sometimes six to ten granules. Two nerve cells of neurosecretory type are situated in the central part of the hexacanth, each one in a deep U-shaped invagination between the two penetration glands. The nuclei of nerve cells contain several large heterochromatin islands closely adjacent to their nuclear membranes. Their cytoplasm is characterized by having membrane-bound, dense-cored neurosecretory-like granules not only in nerve cell perikarya but also in the elongated nerve processes frequently adjacent to gland arms and to both somatic or body musculature, including the complex system of hook muscles. The results of the present study, when supported with literature data on oncospheres of other cestode species, allow for a better understanding of the important role and coordinated functions of three structural components, i.e., oncospheral hooks, penetration glands, and nerve cells, in the mechanism of intermediate host infection. Presence or absence of nerve cells in oncospheres of various cestodes is reviewed, and perspectives on the value and application of research on functional morphology of oncospheres are discussed.

Echinococcus multilocularis (Cestoda, Cyclophyllidea, Taeniidae): origin, differentiation and functional ultrastructure of the oncospheral tegument and hook region membrane.

Both the oncospheral tegument and the hook region membrane (HRM) of Echinococcus multilocularis hexacanths originate from a syncytial binucleate complex that appears in the early stage of morphogenesis and organogenesis of the hexacanth larva. The primordium of this binucleate complex forms a binucleate syncytial cap or "calotte" situated beneath the inner envelope at one pole of the developing embryo. During oncospheral differentiation, the binucleate perikaryon of the syncytial cap is sunk progressively deeper into the central part of the embryo, but remains always connected with the distal cytoplasm by a tendrillar cytoplasmic connection or bridge. Following migration or sinking of the binucleate perikaryon, numerous cytoplasmic vesicles appear in the distal cytoplasm. These vesicles fuse progressively together and form a single large cavity or lacuna. The walls of this cavity are becoming at this point the walls of two delaminated layers: (1) the distal anucleated cytoplasmic layer is transformed into the oncospheral tegument and (2) the proximal thin cytoplasmic layer is transformed into the "hook region membrane". This delamination of the initially compact layer of distal cytoplasm into two layers seems to be closely associated with differentiation of oncospheral hooks, the elongating blades of which protrude progressively into a newly formed cavity. The pressure of hook blades on the hook region membrane appears to facilitate its further separation from the basal layer of distal cytoplasm which is transformed into the peripheral layer of oncospheral tegument. In the mature oncosphere, the surface of this peripheral layer forms a regular brush border of cytoplasmic processes or microvilli and represents the true body covering of the hexacanth. The very thin cytoplasmic connection between the peripheral layer of tegument and binucleate perikaryon appears only very seldom in the ultrathin sections as a narrow cytoplasmic strand and has a plasma membrane that is reinforced by a single row of cortical microtubules. The HRM covers only one pole of the oncosphere and is attached to the oncosphere surface. The HRM is clearly visible in the mature oncosphere and is draped over the hook blades, the sharp points of which are protected by moderately electron-dense caps. Comparison of the above morphology with that of TEM study of the tegument of adult cestodes shows a great similarity as well as homology in the body covering of both larval and adult cestodes.

Ultrastructure of digenean trematode eggs (Platyhelminthes: Neoophora): A review emphasizing new comparative data on four European Microphalloidea.

Despite their tremendous diversity and their medical and veterinary importance, details of egg ultrastructure among the digenean trematodes has been studied rather little. The available literature is spread over several decades and several species, but has not been adequately reviewed to reveal patterns of similarity and divergence. We present this review to synthesize and analyse what is known from the available literature reporting studies using both transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To support our general review of existing literature, we also have synthesized our own previously published descriptions, and present herein our new previously unpublished data. From these new electron micrographs, we provide a comparative analysis of the intrauterine eggs of four digenean species, representing four genera and three families of the superfamily Microphalloidea, collected from four different host wildlife species in four European countries: 1) Mediogonimus jourdanei (Prosthogonimidae) from Myodes glareolus (Mammalia: Rodentia), collected in France; 2) Maritrema feliui (Microphallidae) from Crocidura russula (Mammalia: Soricimorpha), collected in Spain; 3) Brandesia turgida (Pleurogenidae) from Pelophylax ridibundus (Amphibia: Anura: Ranidae), collected in Russia; and 4) Prosotocus confusus (Pleurogenidae) from Rana lessonae (Amphibia: Anura: Ranidae), collected in Belarus. All were studied by preparing whole worms by various techniques for TEM, so that eggs could be studied in situ within the uterus of the parent worm. Based on the literature review and the new data presented here, we describe basic similarities in patterns of embryogenesis and egg formation among all trematode species, but substantial variations in timing of larvigenesis, sculpturing of egg shell surfaces, and some other features, especially including accessory cocoon coverings outside the egg shells of B. turgida and P. confusus. In the future, many more studies are needed to explore egg ultrastructure in other digenean taxa, to explore potential phylogenetic patterns in egg development and structure, and to correlate structure with function in the life cycle.

Ultrastructural characters of the spermatozoon of the liver fluke Opisthorchis viverrini (Poirier, 1886) (Opisthorchiidae).

The present study records the ultrastructural organization of the mature spermatozoon of Opisthorchis viverrini by means of transmission electron microscopy. The spermatozoon of O. viverrini is a filiform cell, tapered at both extremities. It exhibits the characteristics of type IV spermatozoon of digeneans, namely with two axonemes of the 9+'1' trepaxonematan pattern, external ornamentation of the plasma membrane associated with cortical microtubules that are in the posterior part of the anterior region of the sperm cell, and with two mitochondria. The maximal number of cortical microtubules is in the anterior part of the spermatozoon and arranged into two bundles. Other characteristics are spine-like bodies and a posterior extremity with only the second axoneme. Ultrastructural characters of the spermatozoon of O. viverrini are compared with those of other known digeneans belonging to the Opisthorchioidea, with particular emphasis on representatives of the family Opisthorchiidae. The main differences between O. viverrini and its congener Opisthorchis felineus are the spine-like bodies (present and absent, respectively) and the posterior spermatozoon character (axoneme and nucleus, respectively).

Origin, differentiation and functional ultrastructure of egg envelopes in the cestode Echinococcus multilocularis Leuckart, 1863 (Cyclophyllidea: Taeniidae).

The origin, differentiation and functional ultrastructure of oncospheral or egg envelopes in Echinococcus multilocularis Leuckart, 1863 were studied by transmission electron microscopy (TEM) and cytochemistry. The purpose of our study is to describe the formation of the four primary embryonic envelopes, namely vitelline capsule, outer envelope, inner envelope and oncospheral membrane, and their transformation into the oncospheral or egg envelopes surrounding the mature hexacanth. This transformation takes place in the preoncospheral phase of embryonic development. The vitelline capsule and oncospheral membrane are thin membranes, while the outer and inner envelopes are thick cytoplasmic layers formed by two specific types of blastomeres: the outer envelope by cytoplasmic fusion of two macromeres and the inner envelope by cytoplasmic fusion of three mesomeres. Both outer and inner envelopes are therefore cellular in origin and syncytial in nature. During the advanced phase of embryonic development, the outer and inner envelopes undergo great modifications. The outer envelope remains as a metabolically active layer involved in the storage of glycogen and lipids for the final stages of egg development and survival. The inner envelope is the most important protective layer because of its thick layer of embryophoric blocks that assures oncospheral protection and survival. This embryophore is the principal layer of mature eggs, affording physical and physiological protection for the differentiated embryo or oncosphere, since the outer envelope is stripped from the egg before it is liberated. The embryophore is very thick and impermeable, consisting of polygonal blocks of an inert keratin-like protein held together by a cementing substance. The embryophore therefore assures extreme resistance of eggs, enabling them to withstand a wide range of environmental temperatures and physicochemical conditions.

Echinococcus multilocularis (Cestoda, Cyclophyllidea, Taeniidae): oncospheral hook morphogenesis.

Ultrastructural characteristics of the oncospheral hook morphogenesis in the taeniid cestode Echinococcus multilocularis Leuckart, 1863, a parasite of medical and veterinary importance, are described. Oncospheral hook primordia appear at the preoncospheral phase of the embryonic development. Within six specialised cells of the so-called oncoblasts, high concentration of mitochondria, numerous ribosomes and extended Golgi regions are involved in hook development. During hook growth, the blade and base gradually protrude outside the oncoblast plasma membrane. The nucleated oncoblast persists around the handles of fully formed hooks. Simultaneously with the hook primordium elongation and transformation into a blade, handle and base, the hook material differentiates into an electron-dense cortex and a less dense inner core. The exit of the blade of each mature hook, protruding from the oncosphere, is surrounded by a circular, septate desmosome and two rigid, dense rings on either side. The pattern of oncospheral hook morphogenesis in E. multilocularis is compared with that of other previously examined cyclophyllidean cestodes. Though oncoblasts have never been observed around the mature hooks, their remnants are often still visible in the fully developed infective oncospheres in particular in some taeniid species so far examined in this respect. The origin and formation of oncospheral hooks in E. multilocularis, evidently differs from that of the rostellar hooks. Thus, although the hooks may have slight similarity at the gross level, they are neither analogous nor homologous structures.

Spermatozoon ultrastructure of Thysanotaenia congolensis (Cyclophyllidea, Anoplocephalidae, Inermicapsiferinae): phylogenetic implications.

The mature spermatozoon of Thysanotaenia congolensis, an intestinal parasite of black rat Rattus rattus from Cape Verde, is described by means of transmission electron microscopy. The ultrastructural organization of the sperm cell of T. congolensis follows Levron et al.'s type VII of the Eucestoda. It corresponds to a uniflagellate spermatozoon that presents crested bodies, periaxonemal sheath and intracytoplasmic walls, spiralled cortical microtubules and nucleus spiralled around the axoneme. These characteristics are also present in the spermatozoa of other inermicapsiferines and differ from the characters found in species belonging to the remaining subfamilies of anoplocephalids, namely Anoplocephalinae, Linstowiinae and Thysanosomatinae. Several authors consider the family Anoplocephalidae as a polyphyletic group, and its relationships with the Davaineidae are a matter of controversy. The phylogenetic implications of spermatological ultrastructural features present in inermicapsiferines and in the remaining anoplocephalids are discussed, and the available data on anoplocephalids are compared to similar results in davaineids in order to contribute to a better knowledge of relationships between these cyclophyllidean families.

Echinococcus multilocularis Leuckart, 1863 (Taeniidae): new data on sperm ultrastructure.

The present study establishes the ultrastructural organisation of the mature spermatozoon of Echinococcus multilocularis, which is essential for future research on the location of specific proteins involved in the sperm development in this species and also in Echinococcus granulosus. Thus, the ultrastructural characteristics of the sperm cell are described by means of transmission electron microscopy. The spermatozoon of E. multilocularis is a filiform cell, which is tapered at both extremities and lacks mitochondria. It exhibits all the characteristics of type VII spermatozoon of tapeworms, namely a single axoneme, crested bodies, spiralled cortical microtubules and nucleus, a periaxonemal sheath and intracytoplasmic walls. Other characteristics observed in the male gamete are the presence of a >900-nm long apical cone in its anterior extremity and only the axoneme in its posterior extremity. The ultrastructural characters of the spermatozoon of E. multilocularis are compared with those of other cestodes studied to date, with particular emphasis on representatives of the genus Taenia. The most interesting finding concerns the presence of two helical crested bodies in E. multilocularis while in the studied species of Taenia, there is only one crested body. Future ultrastructural studies of other species of the genus Echinococcus would be of particular interest in order to confirm whether or not the presence of two crested bodies is a characteristic of this genus.

Fertilization in the cestode Echinococcus multilocularis (Cyclophyllidea, Taeniidae).

Fertilization in the taeniid cestode Echinococcus multilocularis with uniflagellate spermatozoa was examined by means of transmission electron microscopy (TEM). Fertilization in this species occurs in the oviduct lumen or in the fertilization canal proximal to the ootype, where the formation of the embryonic capsule precludes sperm contact with the oocytes. Cortical granules are not present in the cytoplasm of the oocytes of this species, however, several large bodies containing granular material where frequently observed. Spermatozoa coil spirally around the oocytes and syngamy occurs by lateral fusion of oocyte and sperm plasma membranes. In the ootype, one vitellocyte associates with fertilized oocyte, forming a membranous capsule which encloses both cell types. In this stage, the spirally coiled sperm body adheres partly to the external oocyte surface, and partially enters into the perinuclear cytoplasm. The electron-dense sperm nucleus becomes progressively electron-lucent within the oocyte cytoplasm after penetration. Simultaneously with chromatin decondensation, the elongated sperm pronucleus changes shape, forming a spherical male pronucleus, which attains the size of the female pronucleus. Cleavage begins immediately after pronuclear fusion.

Functional ultrastructure of eggs and cellular organization of hexacanths of the cyclophyllidean cestode Thysanotaenia congolensis: a phylogenetic implication of obtained results.

The functional ultrastructure of eggs and cellular organization of hexacanths from gravid proglottids of Thysanotaenia congolensis, from black rats from Cape Verde, were examined by transmission electron microscopy. Mature eggs with fully formed hexacanths are grouped within parenchymatous capsules of gravid proglottids. Oncospheral envelopes surrounding mature hexacanths are reduced to a very thin membranous embryophore as their protective function is taken over by the parenchymatous capsules originating from the medullary parenchyma of immature proglottids and composed of three layers. Six major cell types are present: a bi-nucleate medullary centre; a six-nucleate U-shaped penetration gland; a second type of penetration gland; two neurosecretory-type nerve cells; about 30 somatic cells; and about 12 germinative cells. Present results on the functional ultrastructure of eggs and cellular organization of hexacanths support the phylogenetic distinction between T. congolensis and cestodes of the subfamily Anoplocephalinae.

Ultrastructural study of the egg wall surrounding the developing miracidia of the digenean Prosotocus confusus (Looss, 1894) (Plagiorchiida: Pleurogenidae), with the description of a unique cocoon-like envelope.

Helminth eggs play a critical role in movement of the parasite from definitive to intermediate host. Eggs of the pleurogenid digenean trematode Prosotocus confusus (Looss, 1894), a parasite of naturally infected frogs Pelophylax lessonae (Amphibia: Ranidae) in Europe, are described here for the first time. Particular emphasis is placed on the ultrastructure on the egg wall and on the detailed description of a unique cocoon-like envelope. Each embryonating egg is composed of an early embryo surrounded by a four-layered egg wall: (1) an outer, anucleate layer external to the eggshell, which forms a thick cocoon; (2) the operculate eggshell; (3) not fully formed, a differentiating outer embryonic envelope containing large nuclei of macromeres; and (4) situated below, an undifferentiated layer of the future inner embryonic envelope containing mesomere nuclei. Layers enveloping the egg apparently play an important role in the protection, metabolism, and storage of nutritive reserves for the developing miracidium. The outer anucleate layer, or cocoon, is situated externally to the eggshell and composed of an electron-lucent substance with numerous electron-dense islands attached to its peripheral membrane. A cocoon envelope such as this has never been seen in previous TEM studies of the eggs of parasitic platyhelminths, with the exception of another pleurogenid Brandesia turgida. The origin, formation, functional ultrastructure, and chemical composition of this peculiar layer remain enigmatic, although its function appears to be protective. The thick, electron-dense eggshell resembles that of other trematodes, exhibiting a characteristic fissure zone around the operculum. Numerous lysosome-like structures observed in some eggs may be involved in the autolysis of both the embryonic envelopes (particularly the early degeneration of macromere nuclei of the outer envelope, characteristic for this species) and in the disintegration of several early micromeres. The inner envelope, which forms later from mesomeres, persists longer during embryogenesis.

Functional ultrastructure of the parenchymatic capsules of the cestode Thysanotaenia congolensis (Cyclophyllidea, Anoplocephalidae, Inermicapsiferinae).

Proglottids from adult Thysanotaenia congolensis from naturally infected black rats Rattus rattus from Santiago Island, Cape Verde, were examined by transmission electron microscopy (TEM). The uterus in mature proglottids is composed of an ephemeral transverse tube or sac that breaks down, releasing eggs into the parenchyma where they are sequestered in groups and encapsulated by parenchymatous layers. In gravid proglottids, eggs accumulate in groups of 6-12 at the distal end of sac-like uterine ducts. As eggs accumulate, the end of the uterine ducts expands until it pinches off, releasing groups of eggs into the parenchyma surrounded by remnants of uterine epithelium. These epithelium-bound groups of eggs remain in the parenchyma until they are encapsulated with several parenchymatous layers, forming parenchymatic egg capsules, typical for mature and gravid proglottids of Inermicapsiferinae. The parenchymatic capsules originate from the medullary parenchyma of immature proglottids, which undergoes differentiation into the three layers of gravid proglottids: (1) an outer connective tissue layer composed of long delicate filaments of unknown chemical nature embedded in a granular extracellular matrix; (2) a middle layer appearing as an accumulation of large closely packed PAS-positive mucous goblets that are intensely metachromatic after toluidine blue staining and (3) an inner compact layer composed of lipid-containing cells, muscle cells with elongated muscle fibres and cells of various sizes and shapes forming or containing calcareous corpuscles. The mature hexacanths of T. congolensis are surrounded by reduced oncospheral envelopes consisting of remnants of a very thin membranous layer of degenerating embryophore with long, irregularly shaped cytoplasmic processes and by remnants of uterine epithelium extending as numerous apical microlamellae into the parenchymatic capsule lumen.

Ultrastructural evidence for completion of the entire miracidial maturation in intrauterine eggs of the digenean Brandesia turgida (Brandes, 1888) (Plagiorchiida: Pleurogenidae).

Results of this TEM study provide ultrastructural evidence that miracidial morphogenesis is fully completed within the intrauterine eggs situated in the most posterior uterine regions of the pleurogenid trematode Brandesia turgida (Brandes, 1888). The ultrastructural characteristic of different larval organelles and cell types of these eggshell-enclosed, but fully formed, cilated miracidia is described. The body wall of the pyriform mature miracidium of B. turgida is composed of ciliated epidermis and underlying peripheral body musculature. Two miracidial flame cells of the protonephridial excretory system are localized in the central region of the ciliated larvae. Three types of miracidial glands were observed: a single apical gland, two lateral glands, and several small vesiculated glands; each gland type contains characteristic, but different types of secretory granules. The anterior end of each miracidium consists of an apical papilla on which are situated the exits of the three main larval glands: an exit of a single apical gland as well as the individual exits of two lateral glands. The exits of vesiculated glands, containing characteristic spherical membrane-bound and highly electron-dense granules, evidently different from the two other types of secretory granules of apical and lateral glands, were not identified. Germinative cells, grouped together in a sac-like germinative follicle, are situated in the medioposterior part of the larva, the germatophore. The germinative cells contain numerous electron-dense heterochromatin islands arranged in the form of a network or chain-like pattern and distributed mainly in the karyoplasm adjacent to the nuclear membrane. The thin layer of granular cytoplasm is rich in free ribosomes and contains a few small mitochondria. Both nuclear and cytoplasmic features if these cells indicate their great developmental potential for further growth and multiplication in postembryonic stages of the life cycle. In the mature eggs, the areas of focal cytoplasmic degradation were frequently observed and may be involved in the autolysis of some embryonic structures. Obtained results are compared with available literature data on the functional ultrastructure of the miracidia of other digeneans.

A transmission electron microscopical study of the tegument of Maritrema feliui (Digenea: Microphallidae).

The tegument of the microphallid digenean Maritrema feliui, examined by means of TEM, is described as a syncytial epithelium organised into two layers. The outer layer is an external anucleate, cytoplasmic region connected to a second region composed of nucleate perikarya (cytons) deeply embedded in the surrounding cortical parenchyma. The surface layer of the tegument is covered by a plasma membrane with many deep invaginations, which are apparently pinocytotic. This layer also bears numerous large, electron-dense spines of two types, which are intracellular and attached to the basal plasma membrane. Its cytoplasm is rich in free ribosomes, contains numerous mitochondria, disc-shaped granules frequently arranged in a rouleau, and several large, moderately electron-dense, membranous bodies. The subtegumentary perikarya and their nuclei, which are both flattened, are described in detail, as are their connections with the surface tegument. These perikarya appear to be the source of the disc-shaped granules and some of the other inclusions present in the surface layer. The main characteristics of the tegumental structure of M. feliui are commented upon in relation to the findings of previous publications and their suggested functions.