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.

Description of embryonic development and ultrastructure in miracidia of Cardiocephaloides longicollis (Digenea, Strigeidae) in relation to active host finding strategy in a marine environment.

The functional ultrastructure and embryonic development of miracidia in naturally released eggs of the trematode Cardiocephaloides longicollis were studied using light and transmission electron microscopy. This species has operculated eggs and embryogenesis occurs in the marine environment before an actively infecting ciliated miracidium hatches. Six different developmental stages were identified. The lack of pores in the eggshell indicates its impermeability and the miracidium's dependency on glycogen nutritive reserves, contained in numerous vitellocytes in early embryos. As the development advances, these merge into larger vitelline vacuoles that encircle the miracidium and may aid its hatching. Tissue and primary organ differentiation were observed in advanced stages, i.e., terebratorium, glands, cerebral ganglion, peripheral sensory endings, and eyespots. The anterior part of the body contains a single apical and paired lateral glands, as well as two types of sensory endings, which permit location, adhesion, and penetration of the host. No previous studies describe the embryonic development and ultrastructure of miracidia in strigeids, however, some of the structural features shared with other, well described species with unknown life cycles are emphasised. This study highlights that ultrastructural data have to be interpreted in relation to parasite biology to understand the structural requirements of specific parasite strategies.

CHANGES IN SPINATION PATTERNS OVER THE COURSE OF METACERCARIAL DEVELOPMENT OF DIPLOSTOMUM PSEUDOSPATHACEUM NIEWIADOMSKA, 1984 (TREMATODA, DIPLOSTOMIDAE).

Metacercarial development of most Diplostomum species including D. pseudospathaceum occurs in the eye lenses of their fish hosts and is accompanied by radical morphological changes often referred to as metamorphosis. One of the structures undergoing substantial changes in D. pseudospathacewn are tegumental spines. The present study used phalloidin staining and confocal microscopy to examine these changes in D. pseudospathaceum over the course of development from 3-day-old to infective metacercariae. Although the general pattern of spination remained essentially unchanged, most larval spines continued growing in size until late in metacercarial development. From day 10 of development, larval spination was gradually replaced by small incipient definitive spines and the infective metacercariae had only definitive spination. The possible adaptive role of spines in developing metacercariae is discussed.

Active chi-like sequences are present in the ITS1 region of polyembryonic adult Collyriclum faba trematodes encysted in pairs.

Collyriclum faba (Plagiochiida: Collyriclidae) adults occur in pairs within subcutaneous cysts. Here, we tested the extensive C. faba infrapopulation for five DNA loci known to display variability among Central European C. faba individuals. The infrapopulation tested shared 100% similarity in four of the five mitochondrial and nuclear DNA loci tested. Contrariwise, the internal transcribed spacer 1 (ITS1) loci in all but one individual differed from each other. We found only 0.0-1.5 base substitutions per 1,000 sites within the cysts, while we found 0.7-9.0 substitutions between the cysts of the single host and 3.0-9.0 substitutions when comparing C. faba individuals isolated from different host individuals. We observed the most of the ITS1 variability within 48 bp repetitive sequences featured by the chi-like sequence 5'-GCTTGTCTGCC-3' at their beginning. Similarly to the extensive C. faba infrapopulation examined, we determined the presence of highly variable number of repetitive sequences within the ITS1 locus of C. faba isolated from multiple host species and from various geographic locations. While similar variability was observed earlier in mutually unrelated specimens of several Schistosomatidae and Microphallidae species, here, we for the first time document it among multiple individuals of a single infracommunity possessing single mitochondrial haplotype. Lower ITS1 evolutionary divergence rates observed between individuals within the cysts when compared to those between the cysts suggest that the recombination occurs at multiple stages of the life cycle. We propose DNA recombination involving chi-like sequences to serve as a general feature shared by multiple families of digenetic trematodes to increase genetic diversity of their polyembryonic populations infecting their definitive hosts.

Ultrastructure of the intrauterine eggs of the microphallid trematode Maritrema feliui: evidence of early embryonic development.

Intrauterine embryonic development in the microphallid trematode Maritrema feliui is examined by means of transmission electron microscopy. Both fertilization and eggshell formation take place in the ootype. The eggshell is formed from a shell globule material derived from the vitelline cells combined with secretions of Mehlis' gland. The proximal uterus is packed with unembryonated eggs of the oligolecithal type, each composed of a fertilized oocyte and several vitelline cells, all surrounded by the shell. Intrauterine embryonic development of the egg is followed to the early stage of outer embryonic envelope formation, resulting in an embryo of ~20 blastomeres of three different types: macromeres, mesomeres and micromeres. The first equal cleavage division of the zygote produces two macromeres. The outer envelope is of cellular origin and formed by the cytoplasmic fusion of two macromeres, which become situated at opposite poles in the peripheral layer of the embryo just beneath the eggshell. Simultaneously, other blastomeres multiply and differentiate, whereas several micromeres exhibit clear signs of degeneration or apoptosis. These results show that the embryonic development of M. feliui starts in utero and represents an example of early stage ovoviviparity. A reduction in the number of blastomeres results from a continued degeneration of micromeres, which after autolysis and re-absorption, appear to represent an important source of nutritive reserves for the embryo. The embryonic development of this digenean is discussed in relation to its life cycle.

Advanced stages of embryonic development and cotylocidial morphogenesis in the intrauterine eggs of Aspidogaster limacoides Diesing, 1835 (Aspidogastrea), with comments on their phylogenetic implications.

Ultrastructural aspects of the advanced embryonic development and cotylocidial morphogenesis of the aspidogastrean Aspidogaster limacoides are described. The posterior or distal regions of the uterus are filled with eggs containing larvae at advanced stages of morphogenesis and fully-formed cotylocidia. Various stages and organs of this larva are described in detail, including the aspects of the developing and fully-differentiated cotylocidium, the body wall (tegument and musculature), glandular regions and the protonephridial excretory system. Blastomere multiplication by means of mitotic divisions takes place simultaneously with the degeneration or apoptosis of some micromeres; this frequently observed characteristic is compared and discussed in relation to corresponding reports for other neodermatans. During the advanced stages of the embryonic development of A. limacoides, the vitelline syncytium disappears and the size of the embryo increases rapidly. Evident polarization of the differentiating larva was observed; towards one pole of the egg, cytodifferentiation of the mouth, surrounded by the oral sucker and cephalic glands, takes place, whereas, towards the opposite pole, differentiation of the posterior sucker (incipient ventral disc) occurs. The oral and posterior suckers are formed from numerous embryonic cells which have differentiated into myocytes. The central part of the oral sucker undergoes invagination and forms the future pharynx and intestine. Fully-developed cotylocidia of A. limacoides have a neodermatan type of tegument, flame cells and two types of glandular structures. These results suggest a sister relationship between the Aspidogastrea and the Digenea, although the systematic position of aspidogastreans in relation to other platyhelminth taxa remains somewhat equivocal.

Egg formation and the early embryonic development of Aspidogaster limacoides Diesing, 1835 (Aspidogastrea: Aspidogastridae), with comments on their phylogenetic significance.

Ultrastructural aspects of the early embryonic development of the aspidogastrean Aspidogaster limacoides are described and their phylogenetic implications discussed. Whereas the proximal regions of the uterine lumen usually contain unembryonated eggs or eggs with early embryos, the posterior or distal regions of the uterus are filled with eggs containing a fully-developed cotylocidium. The eggs of A. limacoides can be classified as polylecithal due to the presence of numerous vitellocytes which accompany each fertilized oocyte or ovum during egg formation. The results of the study are described in details under six headings: (1) general characteristics of the intrauterine eggs; (2) eggshell and operculum formation; (3) unembryonated eggs; (4) zygote formation and early cleavage divisions; (5) embryonic envelope formation; and (6) early degeneration or apoptosis of some blastomeres. The late differentiation of the operculum, possible functions of GER-bodies, and the early degeneration of vitellocytes and some blastomeres in this species are compared, drawn and discussed with corresponding observations reported for other parasitic Platyhelminthes. The most important differences are apparent in the number of egg envelopes and their mode of formation in A. limacoides compared with previous reports for both digeneans and cestodes. The results of the present TEM study indicate that the three macromeres, resulting from two cleavage divisions, take part in the formation of a single embryonic outer envelope in A. limacoides, and that this takes place at a very early stage of embryogenesis. Their fusion results in the formation of a single continuous cytoplasmic layer surrounding the early embryo, which is composed of only a small number of undifferentiated blastomeres. The early separation of the macromeres may indicate an equal cleavage pattern. These results suggest that the systematic position of the Aspidogastrea among the Platyhelminthes still remains somewhat equivocal, and indicate the need for more studies on the embryonic development, larval morphogenesis and molecular phylogeny for the elucidation of the relationships between this enigmatic group and related taxa.

Trematode embryology: a new method for whole-egg analysis by confocal microscopy.

Trematode worms have the neoophoran mode of development in which several specialized vitelline cells surround the zygote. This vitelline cell mass appears just before the zygote passes through the ootype, a thickening of the oviduct, where the egg shell is formed. The great amount of vitelline material blurs the visualization of embryo development in whole egg seen by brightfield microscopy. The eggshell is difficult to cut into thin or ultrathin sections and acts as a barrier to fixation and infiltration with embedding media. The egg shell is also brightly fluorescent when analyzed by fluorescence microscopy. To overcome these technical disadvantages a simple staining protocol widely used in adult helminth morphological analysis was adapted for the study of the embryonic development of two different trematode species. The effects of potassium hydroxide as bleach and ethylene glycol as mounting medium were also evaluated. Confocal microscopy allowed virtual sectioning of whole-mounted eggs and made possible internal morphological detailed analysis of different embryonic stages. This method could contribute to the study of helminth egg embryology.

Survival, feeding and embryo development of Gyrodactylus gasterostei (Monogenea: Gyrodactylidae).

Viviparous gyrodactylid monogeneans which are detached from their host may form an important source of infection in fish communities. This is particularly significant for these ectoparasites which have no specific free-living stage and where transmission usually occurs through transfer of adult parasites when fish come into close contact. In this study, in vitro survival of Gyrodactylus gasterostei was correlated with changes in tissue ultrastructure of parents and their embryos during detachment and following return to a host. At 10 degrees C, detached worms survived for up to 89 h (50%) survival = 20 h) with mortality influenced by both age-independent and age-dependent factors. The gastrodermis of attached, fed parasites is syncytial and contains a variety of vesicles and feeding vacuoles. However, following detachment and starvation, degenerative changes occur within the gastrodermis, eventually leading to marked damage to the embryo/s. When starved worms reattached to a fish, they began browsing on host epithelial cells within 1 min and ultrastructural evidence for phagocytic activity in the gastrodermis was detected after 5 min. Putative waste vacuoles increased in abundance after 5-30 min on the new host, coinciding with the reappearance of electron-dense vesicles and possibly indicating completion of the first intracellular cycle of digestion. Parental feeding directly influenced the normal cyclical maturation and regression of the uterine lining between births. In extreme cases, starvation led to detached parasites (6.1%) aborting their embryos. However, even short periods off the host influenced development and survival of embryos, suggesting that temporary interruption of nutrient flow to the embryo can significantly affect gyrodactylid reproductive rates.

Biology of Crassicutis cichlasomae, a parasite of cichlid fishes in Mexico and Central America.

Field study on the biology of Crassicutis cichlasomae Manter, 1936 (Digenea: Homalometridae) was carried out in a small swamp in a limestone factory near Mérida, Yucatán, Mexico. Aquatic snails, Littorina (Littoridinopis) angulifera, harbouring C. cichlasomae rediae, cercariae and metacercariae, served both as the first and second intermediate hosts. Feeding experiments confirmed the conspecificity of metacercariae from naturally infected snails with adults from naturally infected fish. Gravid C. cichlasomae worms were obtained from experimentally infected fish 19 days post exposure at 22-24 degrees C. Examination of fish from the swamp in Mitza and other localities in the Yucatan Peninsula showed that the cichlids Cichlasoma urophthalmus and C. meeki were definitive hosts of C. cichlasomae. There was no pronounced preference of C. cichlasomae adults for the site of their location in the intestine of the definitive host; a slightly higher proportion (41%) of worms was only found in the anterior third of the gut. The time of miracidium development varied from 18.5 to 27.5 days; different temperature (20.1-35.7 degrees C) or light/darkness regimes influenced only slightly the rate of embryonic development, with shorter development times at higher temperature (34.8-35.7 degrees C) and constant darkness and/or light. With the exception of the sporocyst, all developmental stages are described and figured.

Ovoviviparity in platyhelminth life-cycles.

The encapsulated embryos of platyhelminths may be retained and complete their development in utero in a range of circumstances. However, hatching within the parent (the criterion of ovoviviparity) is relatively rare and larvae generally emerge only after deposition. Viviparity is characterized by the nutritional dependency of the unencapsulated larva upon the parent, but in several cases larvae retained within a shell also receive parental nutrients during intra-uterine development. Uptake of exogenous nutrients via shell pores occurs in Schistosoma mansoni but the eggs, which gain all the advantages of intra-uterine retention, are supported by host nutrients. Intra-uterine larval development avoids the hazards of development in the external environment and eliminates the time delay between oviposition and infection. Deposition of immediately infective offspring may be concentrated in time and space to exploit periods of host vulnerability. The control and precision of transmission is illustrated by examples in which the opportunity for invasion is restricted because of either host behaviour or environmental instability. This strategy has been an important factor in the evolution of polystomatid monogeneans, and its effectiveness is demonstrated by comparison of the life-cycles of Polystoma integerrimum and Pseudodiplorchis americanus. Ovoviviparity also increases reproductive potential in some polystomatids by extending the period of multiplication and by increasing established populations through internal re-infection. In Eupolystoma alluaudi, the capacity for ovoviviparity is programmed into larval development and this regulates population growth within individual hosts.

[Seasonal dynamics of the development and reproduction of parthenogenetic generations of Xiphidiocercaria sp. VII Odening 1962 (Trematoda: Plagiorchiidae) in the mollusk, Coretus corneus]. / Sezonnaia dinamika razvitiia i razmnozheniia partenogeneticheskikh pokolenii Xiphidiocercaria sp. VII Odening 1962 (Trematoda: Plagiorchiidae) v molliuske Coretus corneus.

Intensity of reproduction of parthenogenetic generations of Xiphidiocercaria sp. VII Odening 1962 was shown to exhibit a seasonal dependence. Comparative studies of generative and somatic cell nuclei size, amount of divisional cells, quantity of cercariae and embryos were conducted in summer and winter.

Larval and post-larval development of Ergenstrema mugilis Paperna, 1964 (monogenea, ancyrocephalidae) parasite of Liza ramada (Risso, 1826) (teleostel, mugilidae).

The ciliated cells, chaetotaxy and excretory system of the oncomiracidium are fully described in Ergenstrema mugilis Paperna, 1964. The study of the post larval development shows a phase of cutaneous infestation in the fish host and the mode of transmission is discussed among Dactylogyroidea. The morphogenesis of the parasite haptor is described. The origin and migration of the hamuli in Dactylogyroidea are discussed.

[Experimental study of the biology of the miracidium of Philophthalmus rhionica Tichomirov, 1976 (Trematoda, Philophthalmidae)]. / Eksperimental'noe izuchenie biologii miratsidiia Philophthalmus rhionica Tichomirov, 1976 (Trematoda, Philophthalmidae)

Some peculiarities of the biology of miracidia of Philophthalmus rhionica have been studied: penetration of the parasite's eggs into the environment, hatching of larvae, the miracidia motion, their life span and changes in the invasion ability character in time.

[The eyespots of the oncomiracidium of Euzetrema knoepffleri (Monogenea): ultrastructure and evolution during the life-cycle (author's transl)]. / Les taches pigmentaires de l'oncomiracidium d'Euzetrema knoepffleri (Monogenea): ultrastructure et évolution au cours du cycle biologique

Euzetrema knoepffleri (Monogenea, Monopisthocotylea) is a parasite of the urinary bladder of Euproctus montanus (Amphibia, Urodela). The eyespots of the oncomiracidium of E. knoepffleri are rhabdomeric; each anterior pigmented cell has one rhabdomere, each posterior pigmented cell has two rhabdomeres. This pattern is quite similar to that of the eyespot of another Monopisthocotylea, Entobdella soleae but the lack of cristalline lens in E. knoepffleri appears as a new feature of the Monopisthocotylea. The symmetry of the pigmented structures seems to be effectively connected with the swimming mode of the larva. Moreover, the ultrastructural differences between the two species Euzetrema knoepffleri and Entobdella soleae may be in relation with their different behaviour concerning light. After the fixation of the larva on its host, the comparative study of the evolution of the eyespots, shows the disappearance of the cristalline lens in Entobdella, and the loss of rhabdomeric structures in Euzetrema. These differences seem related with the nature of the microbiotope of the adult: Entobdella soleae is a skin parasite, Euzetrema knoepffleri a reno-vesical one.