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.

First ultrastructural and cytochemical data on the spermatozoon and its differentiation in progenetic and adult Archigetes sieboldi Leuckart, 1878 (Cestoda, Caryophyllidea, Caryophyllaeidae).

Spermiogenesis in progenetic and adult stages of Archigetes sieboldi Leuckart, 1878, a tapeworm parasitic in oligochaetes and fish respectively, has been examined using transmission electron microscopy and cytochemical staining for glycogen. General pattern of spermiogenesis is essentially like that of other caryophyllideans, i.e., apical dense material in the zone of differentiation in the early stages of spermiogenesis, rotation of free flagellum and a flagellar bud, and proximo-distal fusion. Interestingly, rotation of a free flagellum and flagellar bud to the median cytoplasmic process (MCP) has been observed unconventionally at > 90° only in progenetic stages. Typical striated roots associated with the centrioles occur rarely in A. sieboldi, and only in form of faint structures in advanced stages of spermiogenesis. In contrast to most caryophyllideans studied to date, penetration of the nucleus into the spermatid body has started before the fusion of the free flagellum with the MCP. This feature has been reported rarely but exclusively in the family Caryophyllaeidae. The unipartite mature spermatozoon of A. sieboldi is composed of one axoneme of the 9 + '1' trepaxonematan pattern with its centriole, parallel nucleus, and parallel cortical microtubules which are situated in a moderately electron-dense cytoplasm with glycogen particles. An unusual arrangement of cortical microtubules in the two parallel rows in region I of the spermatozoon is described here for the first time in the Caryophyllidea. Ultrastructural data on spermiogenesis and the spermatozoon in A. sieboldi from tubuficids and carp are compared and discussed with those in other caryophyllideans and/or Neodermata.

Ultrastructural, cytochemistry and electron tomography analysis of Caryophyllaeides fennica (Schneider, 1902) (Cestoda: Lytocestidae) reveals novel spermatology characteristics in the Eucestoda.

Spermatozoon formation in Caryophyllaeides fennica (Schneider, 1902) is characterised by the following: (1) apical electron-dense material in the zone of differentiation, (2) typical striated roots situated unconventionally in opposite directions in early stages of spermiogenesis, (3) intercentriolar body composed of three electron-dense and two electron-lucent plates, (4) free flagellum and a flagellar bud that correspond to a greatly reduced flagellum and (5) rotation of free flagellum and a flagellar bud to the median cytoplasmic process at 90°. The development of two flagella of significantly unequal length clearly supports a derived form of spermiogenesis in the Caryophyllidea. New for cestodes is a finding of two additional striated roots situated opposite each other, in conjunction with both the flagellar bud and free flagellum. Mutual position of additional striated roots and typical striated roots is parallel in early stages and perpendicular in advanced stages of spermiogenesis. A complete proximodistal fusion gives rise to a mature spermatozoon consisting of one axoneme, parallel cortical microtubules, a nucleus and a moderately electron-dense cytoplasm with glycogen particles, detected by a technique of Thiéry (J Microsc 6:987-1018, 1967), in the principal regions (II, III, IV). Electron tomography analysis of the free flagellum and one axoneme of a mature spermatozoon of C. fennica provides clear evidence, for the first time, that two tubular structures are present in the central axonemal electron-dense core. Phylogenetically important aspects of spermiogenesis of the Caryophyllidea with one axoneme, and other cestodes with one or two axonemes, are briefly reviewed and discussed.

Cytocomposition of the vitellarium in Khawia sinensis Hsü, 1935 (Cestoda, Caryophyllidea, Lytocestidae): another caryophyllidean species with lamellar bodies and lipids.

The vitellarium of the invasive caryophyllidean tapeworm Khawia sinensis Hsü, 1935 from carp Cyprinus carpio L. was examined by means of transmission electron microscopy and cytochemical staining for glycogen with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP). A vitellarium consists of numerous follicles of irregular size that are interconnected by a net of vitelline ducts. Vitelline follicles are composed of vitelline cells at various stages of development that are interconnected by interstitial tissue. Vitelline follicles are surrounded by a cytoplasmic sheath associated with an intercellular matrix. Extensive development of the granular endoplasmic reticulum and Golgi complexes are both involved in the production of shell globules/shell globule clusters and characterise cytodifferentiation of vitellocytes. Nuclear and nucleolar transformation lead to the formation and storage of intranuclear glycogen, a feature specific for the Caryophyllidea. Newly observed within the mature vitellocytes of Khawia sp. is the presence of lamellar bodies and a few lipid droplets. These cytoplasmic inclusions first occur in the mature cells within the follicles and persist in the vitelline cells within vitelloducts and intrauterine eggs. Two types of lamellar bodies are detected: regular lamellar-structured body and irregular lamellar-structured body. None of the lamellar bodies are membrane bound. Results of the present study indicate that the formation of lamellar bodies may be closely related to the endoplasmic reticulum or shell globule clusters. Some of the shell globule clusters are transformed into lamellar body clusters. Ultrastructural features of vitellocytes in K. sinensis are compared with those of other monopleuroid, polypleuroid, and strobilated cestodes.

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.

Scolex morphology of monozoic cestodes (Caryophyllidea) from the Palaearctic Region: a useful tool for species identification.

A comparative study of the scoleces of caryophyllidean tapeworms (Cestoda: Caryophyllidea), parasitic in cypriniform fishes in the Palaearctic Region, was carried out using light and scanning electron microscopy. Three-dimensional pictures of the scoleces of 18 species of caryophyllidean cestodes of the Capingentidae (1 species), Caryophyllaeidae (7) and Lytocestidae (10), and outlines of the scoleces and anterior extent of the testes and vitelline follicles of 19 Palaearctic taxa were documented. Both species of Atractolytocestus Anthony, 1957 possess a bulboacuminate scolex, whereas species of Archigetes Leuckart, 1876 have fossate scoleces of the bothrioloculodiscate type, with loculi, bothrium-like depressions and an apical disc. Breviscolex orientalis Kulakovskaya, 1962, the only member of the Capingentidae, has a cuneiform scolex, as do both taxa of the lytocestid genus Caryophyllaeides Nybelin, 1922. The scoleces of two species of Caryophyllaeus Gmelin, 1790 are flabellate, whereas that of the congeneric C. fimbriceps Annenkova-Chlopina, 1919 is cuneicrispitate. Khawia Hsü, 1935, the most specious Palaearctic genus, with seven taxa that we consider to be valid, has the highest diversity in scolex morphology: semi-bulbate, flabellate, cuneiform, cuneifimbriate, truncated cuneiform-flabellate and festoon-like. Species of Monobothrium Nybelin, 1922 have either a digitiform scolex with widened posterior part or cuneiform, with lateral auricular extensions. Paracaryophyllaeus gotoi (Motomura, 1927) is characteristic in its possessing a bulbate scolex, whereas Paraglaridacris limnodrili (Yamaguti, 1934) has a fossate scolex of the bulboloculate type with bothrium-like depressions and feebly developed lateral loculi. Anterior extent of the testes and vitelline follicles and their mutual position show a somewhat higher variability than scolex shape, with intraspecific variation in some taxa, such as Atractolytocestus sagittatus (Kulakovskaya et Akhmerov, 1965), B. orientalis, Khawia armeniaca (Cholodkovsky, 1915) and K. sinensis Hsii, 1935. Based on scolex morphology and relative position of the anterior testes and vitelline follicles, a key is provided to facilitate the routine identification of 20 Palaearctic caryophyllidean taxa.

Scolex morphology of monozoic tapeworms (Caryophyllidea) from the Nearctic Region: taxonomic and evolutionary implications.

A comparative study of the scoleces of monozoic tapeworms (Cestoda: Caryophyllidea), parasites of catostomid and cyprinid fishes (Teleostei: Cypriniformes) in the Nearctic Region, was carried out using light and scanning electron microscopy. Scoleces of 22 genera of North American caryophyllideans were characterised and their importance for taxonomy, classification and phylogenetic studies was critically reviewed. Nearctic genera exhibit a much higher variation in the shape and form of scoleces compared with taxa in other biogeographical regions. The following basic scolex types can be recognised in Nearctic caryophyllideans: monobothriate (Promonobothrium Mackiewicz, 1968), loculotruncate (Promonobothrium, Dieffluvium Williams, 1978), bothrioloculodiscate (Archigetes Leuckart, 1878, Janiszewskella Mackiewicz et Deutsch, 1976, Penarchigetes Mackiewicz, 1969, Pseudoglaridacris Oros, Uhrovic et Scholz, 2018), fixomegabothriate (Capingens Hunter, 1927), bulbate and bulboacuminate (Atractolytocestus Anthony, 1958), cuneiloculate (Hypocaryophyllaeus Hunter, 1927, Rowardleus Mackiewicz et Deutsch, 1976, Spartoides Hunter, 1929), biacetabulate, bulboloculate, bothrioloculodiscate (Biacetabulum Hunter, 1927), tholate (Hunterella Mackiewicz et McCrae, 1962), cuneifimbriate (Khawia Hsü, 1935), cuneiform (Calentinella Mackiewicz, 1974, Caryophyllaeides Nybelin, 1922, Edlintonia Mackiewicz, 1970), hastate (Pseudolytocestus Hunter, 1929), loculotholate (Bialovarium Fischthal, 1953, Pliovitellaria Fischthal, 1951), and cuneiformoloculate (Glaridacris Cooper, 1920, Isoglaridacris Mackiewicz, 1965). The same type of scolex may be shared by species of different genera or families and species of the same genus can have a scolex of conspicuously different morphology, e.g. in Promonobothrium. Scolex morphology may be therefore of limited use in generic designation.

Spermatological characteristics of the enigmatic monogenean Dictyocotyle coeliaca Nybelin, 1941 (Monopisthocotylea: Monocotylidae) reveal possible adaptation to endoparasitism.

Spermatological characters of Dictyocotyle coeliaca Nybelin, a unique endoparasitic monogenean from the body cavity of the ray Amblyraja radiata (Elasmobranchii: Rajidae), was investigated by means of scanning and transmission electron microscopy for the first time. The process of the spermatozoon formation begins with the appearance of the differentiation zone which contains two centrioles. Subsequently developed two free flagella rotate to lie parallel before their fusing. After fusion, both the mitochondrion and nucleus migrate alongside the axonemes. Spermatids are formed within parallel cytoplasmic canals. During late spermiogenesis, an electron-dense spiral end-piece is formed proximally to the centriolar region of some spermatids. At the end of spermiogenesis, this end-piece is separated by a plasma membrane from the late spermatid and remains in the residual cytoplasm when spermatozoa are released into the testis lumen. The mature spermatozoon of D. coeliaca contains two parallel axonemes with the 9 + "1" trepaxonematan pattern, mitochondrion, nucleus, parallel cortical microtubules, and electron-dense granules. Especially the presence of electron-dense granules, occurring frequenly in the spermatozoa of endoparasitic flatworms, may indicate an adaptation to endoparasitic lifestyle in D. coeliaca. The anterior extremity of the male gamete contains one centriolar derivative, a small component of the second centriolar derivative and anterior extremity of the mitochondrion. The posterior extremity of the mature spematozoon exhibits tubular elements of the disorganized axoneme. Variations of the spermatozoon ultrastructural characters within monocotylid monogeneans and possible evolutionary significance of glycogen in sperm to the endoparasitic habit are discussed.

Fine structure of the male reproductive ducts, vagina and seminal receptacle of Cyathocephalus truncatus (Cestoda: Spathebothriidea).

Fine structure of the vas efferens, vas deferens, ejaculatory duct with accessory glands and vagina with seminal receptacle is described in the spathebothriidean tapeworm, Cyathocephalus truncatus (Pallas, 1781) Kessler, 1868. The numerous well-developed prostate glands are characterised by having secretory granules with an electron-dense core surrounded by a matrix of lower electron density. Coalescence of the outer part of the granules with each other takes place in the terminal end of the secretory ducts. The position of prostate glands around the proximal part of the cirrus pouch and terminating in the ejaculatory duct is a characteristic feature of the Spathebothriidea. Up to 20 closely arranged muscle layers make up the muscular cirrus pouch wall with 4 well-developed muscular layers in the ejaculatory duct and cirrus. Both the cirrus and the vagina are covered with the same uniform cone-shaped microtriches. The vagina has an extensive seminal receptacle. All of these structures are well-adapted to insure successful sperm transfer involving ejaculation and storage, probably for both self- and cross-insemination. Cyathocephalus truncatus has a cirrus similar to that of the monozoic, progenetic caryophyllidean, Archigetes sieboldi and well-developed prostate glands like those of the polyzoic pseudophyllidean, Diphyllobothrium latum. The ultrastructural aspects of the male and female reproductive system of C. truncatus are compared with those of other tapeworms.

Fine structure of the female reproductive ducts of Cyathocephalus truncatus (Cestoda: Spathebothriidea), from salmonid fish.

The fine structure of the ovary, ovicapt, oviduct, fertilisation canal, vitelline ducts, vitelline reservoir, ovovitelline duct, ootype and Mehlis' gland, and proximal, middle and distal parts of the uterus of the spathebothriidean cestode, Cyathocephalus truncatus (Pallas, 1781), from salmoniform fish, has been studied for the first time by transmission electron microscopy (TEM). Emphasis was given to characteristics which might shed light on the unclarified phylogenetic position of spathebothriideans, belonging among the most basal tapeworms (Eucestoda). New for cestodes is the finding of a multinucleate cell that plugs the ovicapt lumen. The morphology of the proximal part of the oviduct resembles that of the pseudophyllidean tapeworm Diphyllobothrium latum. After fertilisation in the fertilisation canal, vitellocytes of C. truncatus become associated with fertilized oocytes in the ovovitelline duct. Only one type of Mehlis' gland secretory cell is present. The eggs with electron-dense eggshells containing large pores first appear in the proximal part of the uterus. The middle portion of the uterus has well-developed uterine glands. The distal portion of the uterus has apical microtriches. Ultrastructural data on the female genital system of C. truncatus are compared and discussed with those for other cestodes. However, on the basis of available ultrastructural data it is not possible to conclude whether the Spathebothriidea are phylogenetically closer to the Caryophyllidea or to the Pseudophyllidea.

Ultrastructure of Archigetes sieboldi (Cestoda: Caryophyllidea): relationship between progenesis, development and evolution.

Ultrastructural characteristics of progenetic and monoxenic Archigetes sieboldi Leuckart, 1878 from the oligochaete Limnodrilus hoffmeisteri Claparède are described. Our observations demonstrate that progenetic Archigetes sieboldi shares characteristics of both larval (progenetic) and adult stages. The primary larval characteristics are: the presence of a cercomer; a surface filamentous coat covering the whole worm; the presence of the penetration glands and the absence of tegumental ones; wide sarcoplasmic processes connecting the circular and longitudinal external tegumental muscles; the absence of the dense homogenous zone of the basal lamina beneath the epithelial cytoplasm of all reproductive organs and ducts; non-functional gonopores; and an orthogonal plan of nervous system with three pairs of longitudinal nerve trunks. The principle adult characteristics are: oogenesis, spermiogenesis and vitellogenesis that produce fertilized eggs; the uterine glands; a well-developed longitudinal tegumental muscle layer between tegumental cytons; and the presence of different microtriches. As a result of this progenetic development there has been a secondary reduction in the life cycle of A. sieboldi. It is postulated that a similar process of progenesis may have played a major role in the early evolution of the Caryophyllidea by first appearing in a plerocercoid stage of an ancestral strobilate cestode from fish.

Cytogenetics and chromosomes of tapeworms (Platyhelminthes, Cestoda).

Tapeworms (Cestoda, Platyhelminthes) are a highly diversified group of parasites that can have significant veterinary importance as well as medical impact as disease agents of human alveococcosis, hydatidosis, taeniosis/cysticercosis/neurocysticercosis, hymenolepidosis or diphyllobothriasis. Because of their great diversity, there has been keen interest in their phylogenetic relationships to other obligate parasitic platyhelminthes, as well as within the group itself. Recent phylogenetic analyses of cestodes, however, have focused on morphological, molecular, life cycle, embryology and host-specificity features and conspicuously omitted inclusion of karyological data. Here we review the literature from 1907 to 2010 and the current status of knowledge of the chromosomes and cytogenetics within all of the cestode orders and place it within an evolutionary perspective. Karyological data are discussed and tabulated for 115 species from nine eucestode orders with ideograms of 46 species, and a comparison of cytogenetic patterns between acetabulate and bothriate cestode lineages is made. Attention is drawn to gaps in our knowledge for seven remaining orders and cestodarian groups Gyrocotylidea and Amphilinidea. Among the cytogenetic aspects covered are: chromosome number, triploidy, classical karyotype cytogenetics (banding patterns, karyotype asymmetry, secondary constrictions), as well as advanced karyotype techniques allowing location of genes on chromosomes by fluorescence in situ hybridization. We demonstrate that further progress in cestode karyosystematics rests with new molecular approaches and the application of advanced cytogenetic markers facilitating intimate karyotype analysis.

Phylogenetic relationships of the monozoic tapeworms (Eucestoda: Caryophyllidea) inferred from morphological characters.

Phylogenetic relationships of all genera of the order Caryophyllidea, possibly the earliest branching group of true tapeworms (Platyhelminthes: Eucestoda) and the only one that is monozoic, have been assessed for the first time. Results of this cladistic analysis, inferred from 30 unweighted morphological characters, are only partly congruent with the existing classification, which consists of four families based on the position of the inner longitudinal muscles in relation to the internal genital organs. Whereas all but five genera of the Caryophyllaeidae form a monophyletic clade, members of the Capingentidae are split, occurring within six unrelated groups. The Lytocestidae is also paraphyletic, as some genera appear in four unrelated clades. Archigetes appears in a derived clade, indicating that its direct (monoxenous) life-cycle involving only tubificid oligochaetes is secondarily derived and not plesiomorphic among the Eucestoda, as postulated by some authors.