Larisa G. Poddubnaya

On the Position of Archigetes and Its Bearing on the Early Evolution of the Tapeworms

The tapeworm Archigetes sieboldi Leuckart, 1878 (Platyhelminthes: Cestoda: Caryophyllidea) has been cited as a likely representative of the “protocestode” condition, owing to its lack of segmentation and ability to attain sexual maturity in the invertebrate host (aquatic oligochaetes). The idea has been variously amplified or rejected in the literature, although the actual phylogenetic position of the species has not been investigated until now. New collections of Archigetes sp. from both its vertebrate and invertebrate hosts provided the opportunity to estimate its phylogenetic position with the use of molecular systematics, while prompting new analyses aimed at assessing the early diversification of the Cestoda. Additional collections representing the Amphilinidea, Caryophyllidea, and Gyrocotylidea were combined with published gene sequences to construct data sets of complete 18S (110 taxa) and partial (D1-D3) 28S (107 taxa) rDNA sequences, including 8 neodermatan outgroup taxa. Estimates resulting from Bayesian inference, maximum likelihood, and maximum parsimony analyses of the separate and combined data sets supported a derived position of the genus within the Caryophyllidea, and thus reject the idea that Archigetes sp. may exemplify a “primitive” condition. Topological constraint analyses rejected the hypothesis that Archigetes represents the most basal lineage of the Eucestoda, but did not rule out that it could represent the earliest branching taxon of the Caryophyllidea. In all analyses, the Eucestoda were monophyletic and supported basal positions of the nonsegmented Caryophyllidea and Spathebothriidea relative to other major lineages of the Eucestoda, implying that segmentation is a derived feature of the common ancestor of the di- and tetrafossate eucestodes. However, constraint analyses could not provide unequivocal evidence as to the precise branching patterns of the cestodarian, spathebothriidean, and caryophyllidean lineages. Phylogenetic analyses favor the interpretation that sexual maturity of Archigetes sp. in the invertebrate host, and similar examples in members of the Spathebothriidea, are the result of progenesis and have little if any bearing on understanding the protocestode condition.

SPERMIOGENESIS AND SPERM ULTRASTRUCTURE OF CYATHOCEPHALUS TRUNCATUS (PALLAS, 1781) KESSLER, 1868 (CESTODA: SPATHEBOTHRIIDEA)

Spermiogenesis and sperm ultrastructure of adult Cyathocephalus truncatus, a member of presumably basal group of “true” cestodes (Eucestoda), have been examined for the first time by using transmission electron microscopy. The process of sperm formation corresponds in basic pattern to that of the Pseudophyllidea. In addition, the 2 pairs of electron-dense attachment zones are present in median cytoplasmic process of C. truncatus. However, mature spermatozoa of C. truncatus differ significantly from those of the pseudophyllideans, especially in the morphology of the proximal and distal spermatozoon extremities. The proximal extremity of the mature spermatozoon lacks a crested body, which is present in more derived cestodes and some pseudophyllideans. The distal end of the mature spermatozoa exhibits different morphology in the gametes from testes and those from receptaculum seminis. New for the Eucestoda is a finding that a lateral cytoplasmic extension creates the distal end of the spermatozoa from testes, resembling sperm of some Monogenea and Digenea. In contrast, the distal extremity of the spermatozoa from receptaculum seminis contains only a nucleus. Despite the above-mentioned peculiarities, the ultrastructural data on sperm/spermiogenesis suggest close relationships of the Spathebothriidea and Pseudophyllidea.

Vitellogenesis in two spathebothriidean cestodes

Vitellogenesis in two spathebothriidean tapeworms, dixenous adult Cyathocephalus truncatus and monoxenous progenetic Diplocotyle olrikii, has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. Each vitelline follicle consists of vitellocytes at various stages of development and one irregularly shaped interstitial cell. Projections of the interstitial cell enclose the vitellocytes and extend as a cytoplasmic sheath on the follicular periphery. An outer thin fibrous layer (= extracellular lamina) covers the cytoplasmic sheath in C. truncatus, but lacks in D. olrikii. Maturing and mature vitellocytes contain vitelline material in the form of single small shell globules that gradually fuse and give rise to the large shell globule clusters. Morphology of shell globule clusters differs slightly in both species. In addition, single “lamellar” granules are present in the cytoplasm of vitellocytes of C. truncatus, but not in D. olrikii. Both electron lucent and electron dense lipid droplets are present in the vitellocytes of C. truncatus, whereas only electron dense lipids occur in D. olrikii. A single lipid droplet turns up occasionally in the nuclei of some of the vitellocytes of C. truncatus. The ultrastructural features of vitellogenesis in spathebothriideans resemble those reported previously in “lower” cestodes, especially in pseudophyllideans.

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.

Spermiogenesis in the caryophyllidean cestode Khawia armeniaca (Cholodkovski, 1915).

Spermiogenesis of the monozoic cestode Khawia armeniaca (Cholodkovski, 1915), a parasite of Capoeta capoeta sevangi (De Filippi, 1865) (Pisces: Cyprinidae), has been investigated by means of transmission electron microscopy for the first time. The present results show that the type of spermiogenesis in K. armeniaca shares the main features described previously in the pseudophyllideans and/or spathebothriideans: apical dense material in the zone of differentiation in the early stages of spermiogenesis, rotation of flagellum and flagellar bud, proximodistal fusion, and penetration of the nucleus into the spermatid body after the fusion of the free flagellum with the MCP has started. However, only one axoneme and a simple intercentriolar body represent presumably more evolved characters, typical for “higher” tapeworms. Ultrastructural data of spermiogenesis in K. armeniaca may support the hypothesis that the Caryophyllidea are secondarily derived from an ancestor closely allied with the Pseudophyllidea.

Spermatological characters of the spathebothriidean tapeworm Didymobothrium rudolphii (Monticelli, 1890)

Spermiogenesis and the spermatozoon ultrastructure of the adult Didymobothrium rudolphii (Spathebothriidea) have been examined using transmission electron microscopy for the first time. The dense material is present in the apical region of the differentiation zone in the early stage of spermiogenesis, similarly as in other basal tapeworms examined to date. The orthogonal development of the two flagella is followed by a flagellar rotation and their proximodistal fusion with the median cytoplasmic process (MCP). The two pairs of electron-dense attachment zones in the MCP mark the lines where the proximodistal fusion of MCP with two axonemes takes place. D. rudolphii exhibits polymorphism of the intercentriolar body during spermiogenesis. The mature spermatozoon possesses the two axonemes of 9 + “1” trepaxonematan pattern, nucleus, cortical microtubules (CMs), and electron-dense granules. The anterior extremity of the gamete lacks a crested body and exhibits a centriole surrounded by a semiarc of electron-dense tubular structures. The two parallel rows of the CMs have been found in the proximal part of the two-axoneme region of the spermatozoon for the first time in the Eucestoda. The posterior extremity of the gamete exhibits pattern of the disorganized axoneme. The ultrastructural features of the sperm/spermiogenesis support a view about the close relationships of the Spathebothriidea and Diphyllobothriidea and the basal position of the Spathebothriidea within the Eucestoda.

Ultrastructural features of the spermatozoon and its differentiation in Brandesia turgida (Brandes, 1888) (Digenea, Microphalloidea, Pleurogenidae)

Spermatological characters of the digenean Brandesia turgida (Brandes, 1888), an intestinal parasite of the frog Pelophylax ridibundus (Pallas, 1771), have been investigated by means of transmission electron microscopy for the first time. The process of the spermatozoon formation begins with the appearance of the differentiation zone bordered by cortical microtubules and containing two centrioles associated with striated rootlets and with an intercentriolar body. The intercentriolar body is made up of seven distinct electron-dense plates, two less electron-dense, and four electron-lucent zones. The orthogonal development of the two flagella is followed by a flagellar rotation and their proximodistal fusion with the median cytoplasmic process. This process is accompanied by an extension of both the mitochondrion and nucleus into the median cytoplasmic process. The mature spermatozoon of B. turgida contains two parallel axonemes of unequal lengths with the 9 + “1” trepaxonematan pattern, mitochondrion, nucleus, parallel cortical microtubules, four electron-dense attachment zones, an external ornamentation of the plasma membrane, and electron-dense glycogen granules. The anterior extremity of the male gamete contains one complete centriole, a small component of the central element of the second centriole, and peripheral cortical microtubules (up to 45). The posterior extremity of the mature spematozoon exhibits tubular elements of the disorganized axoneme. The present study provides the first data on spermiogenesis within the family Pleurogenidae. Variations of the spermatozoa ultrastructural characters within Digenea, in particular, between different families of the superfamily Microphalloidea, are discussed.

FIRST EVIDENCE OF THE PRESENCE OF MICROTRICHES IN THE GYROCOTYLIDEA

The Gyrocotylidea, a small and enigmatic group of intestinal parasites of chimaeras, has been considered to be related either to the Monogenea, or, more frequently, to the most primitive monozoic tapeworms (Cestoda), i.e., the Amphilinidea and Caryophyllidea. The present study, based on transmission electron microscopical observations of a species of Gyrocotyle from the rabbit fish, Chimaera monstrosa, in the North Atlantic, demonstrates for the first time the presence of microtriches as surface structures of gyrocotylideans. Because microtriches are considered to be an autapomorphy of tapeworms (Cestoda), in which they differ from other Neodermata (Monogenea and Trematoda), the present data represent another source of evidence in support of a close relationship between the gyrocotylideans and the tapeworms sensu stricto (Eucestoda). Simple morphology, small size, and shape uniformity of the microtriches of Gyrocotyle sp. may indicate they represent an original (plesiomorphic) form that then evolved in more derived cestode groups into a variety of types present mainly on the scolex. The microtriches of Gyrocotyle sp. resemble those found in caryophyllidean, spathebothriidean, pseudophyllidean, and trypanorhynch cestodes, which are considered to represent the most basal groups of the Eucestoda.

Ultrastructure of the ovary, ovicapt and oviduct of the spathebothriidean tapeworm Didymobothrium rudolphii (Monticelli, 1890)

The ultrastructural details are presented of the ovary, ovicapt and oviduct of the spathebothriidean tapeworm Didymobothrium rudolphii (Monticelli, 1890) from the intestine of the sand sole Solea lascaris. Oogonia, maturing oocytes and mature oocytes are surrounded by a syncytial interstitial cytoplasm, one of the distinctive traits of which is the presence of numerous myelin-like bodies. Oocyte inclusions comprise cortical granules and a small number of lipid droplets. The thickened, nucleated epithelium of the ovicapt lacks any apical structure and is a prolongation of the narrow ovarian epithelium. The muscular sphincter of the ovicapt is formed by a band of longitudinal muscles and bands of radial muscles at right angles to the longitudinal layer, and numerous myocytes surround the ovicapt wall. The oviduct of D. rudolphii is subdivided into three regions: (1) the proximal oviduct; (2) the fertilization chamber — the region distal to the point of entry of the duct from the seminal receptacle; and (3) the ovovitelline duct — the region distal to the point of entry of the duct from the vitelline reservoir. A comparative analysis is made between the structures of the ovary, ovicapt and oviduct of D. rudolphii and those of two other spathebothriideans, Cyathocephalus truncatus and Diplocotyle olrikii, with a discussion of ultrastructural traits that might be used as taxonomic criteria within the order Spathebothriidea.

Vitellogenesis of basal trematode Aspidogaster limacoides (Aspidogastrea: Aspidogastridae)

The vitellogenesis of the trematode Aspidogaster limacoides (Aspidogastrea: Aspidogastridae), a parasite of cyprinid fishes, is described here using transmission electron microscopy. Four different stages of vitellocytes are differentiated: immature vitellocytes, early maturing vitellocytes, advanced maturing vitellocytes and mature vitellocytes. The process follows the same general pattern already described in other free-living neoophorans and parasitic flatworms (i.e. Trematoda, Monogenea and Cestoda): differentiation into mature vitelline cells involves the development of mitochondria, granular endoplasmic reticulum, Golgi complexes, lipid droplets and shell-globules. Mature vitellocytes of A. limacoides are composed of numerous shell-globule clusters, few lipid droplets and glycogen granules. They differ from those of another aspidogastrean Rugogaster hydrolagi in that they possess numerous globules tightly packed and by the presence of only one type of vitelline material. The interstitial tissue of vitelline follicles of A. limacoides contains a peripheral nucleus and long cytoplasmic projections extending between vitelline cells. Since aspidogastreans are considered as an archaic group of parasitic flatworms and thus have a strategic phylogenetic position, future works needs to pay special attention to the ultrastructural and chemical composition of mature vitellocytes within this basal group of trematodes.