Early morphogenesis in the platyhelminthes with special reference to egg development and development of cestode larvae

Early development of fertilized eggs is reviewed for the main groups within the phylum Platyhelminthes (Turbellaria, Temnocephaloidea, Monogenea, Digenea, Cestodaria and Cestoda). The Turbellaria show two different patterns of embryological development consequent on whether yolk material is contained within the ovum membrane (endolecithal) or whether yolk is added to the outside of the ovum (ectolecithal). In the parasitic groups, those that are oviparous or ovoviviparous produce shelled eggs of the ectolecithal type and, in addition, there are viviparous forms in which egg development shows variations and the amount of yolk, where present, is reduced. In every case examined, however, cleavage is of the spiral type. Within the Cestoda, clear distinction is made between those groups (orders) of worms which are oviparous as compared to those which are viviparous. It is suggested that oviparity is more primitive (ancestral) than viviparity based on adult morphology and on the evidence of life-cycle studies.The continued, post-oncospheral development of cestodes is described as is the ultrastructure of various representative cercoid larval forms. These include: the procercoid of Pseudophyllidea and Haplobothrioidea; the cercoid of Tetraphyllidea; and, within the Cyclophyllidea, the cercoscolex of the Dilepididae, the cysticercoid of the Hymenolepidae, and the cysticercus of the Taeniidae. During post-oncospheral development, in every case examined the posterior part of the developing larva, the cercomer, can be characterized by a microvillar tegument (at least during early development) whereas the more anterior larval body itself, destined to develop in the definitive host, develops microtriches. Modifications to the cercomer, and its microvilli, in the later stages of post-oncospheral development, occur particularly in the more advanced Cyclophyllidea such as the Taeniidae and in some Hymenolepididae.     

Changes in the monocercus cercomer in the cysticercoid cavity and the hemocoel of the nonspecific host

In order to elucidate the functional role of cercomer in larvae of the monocercus type their transplantation from the specific host Chironomus obtusidens to Gammarus lacustris was conducted. At early stages after the transplantation proceeds an increase in the functional activity of the tegument of follicles of the cercomer followed by their complete destruction in 3 or 4 days. On the surface of the exocyst membrane an adhesion of the host's haemocytes occurs, which becomes more distinct in 3-4 days when the process acquires a character of local encapsulation. Within the same period, in the places of haemocytes aggregation, a local resorption of the exocyst external membrane takes place. Later intensification of the host response to transplant is associated with the destruction of follicles of the cercomer. In one case the occurrence of follicles of the cercomer in the cavity of cysticercoid was observed that is caused by the microbe affection of the latter. In the zone of contact of the tegument of scolex and neck with follicles of the cercomer an increased secretion (the microapocrine type) of the tegument, disturbance of the microvillous tegument of the cercomer's follicles and their destruction are observed. Incompatibility of the tegument of definitive departments and cercomer, which arises during differentiation of larvae, is supposed to affect the formation of scolex invagination in the evolution of larvae of Hymenolepis.     

Life cycle and postembryonic development of Oochoristica anolis (Cyclophyllidea: Linstowiidae)

Gravid proglottids of Oochoristica anolis from naturally infected anole lizards, Anolis carolinensis, were placed in covered Petri dishes with laboratory-reared beetles, Tribolium confusum and Tenebrio molitor. After maintenance at 25 C, metacestodes developed in 29 of 61 T. confusum (48%), but in none of 5 T. molitor. Beetles contained from 1 to 22 metacestodes (means = 3.3), which were fully developed by day 40 postexposure. A primary lacuna was never observed, but the possibility of its presence could not be ruled out without histological study. No cercomer was formed and metacestodes retained larval hooks throughout development. Scolices were invaginated at removal from the hemocoel, but usually evaginated quickly in Ringer's. On day 60 postexposure, metacestodes were fed by stomach tube to 5 anoles, 2 lacertid lizards (Podarcis muralis) and 2 mice. Worms developed only in anoles, 3 of which were infected upon examination. Oncospheral hooks were present in worms after 7 days development in the lizard; a median excretory pore was present at the posterior tip of all stages examined, including the terminal mature proglottid of a worm after 105 days in a lizard. Scolex growth rate was linear throughout metacestode and adult development, but growth rate in body length was diphasic, punctuated by change of hosts, associated with strobilization. Attempts to establish parenteral infections in anoles were unsuccessful. Present data constitute the most complete life history study thus far for any species of Oochoristica.     

The terminology of larval cestodes or metacestodes

The terminology associated with the nomenclature of larval or metacestodes is reviewed as well as the various morphological and developmental characters used to define different types of larval cestodes. Based on a review of the literature, the key characters differentiating the types of larval cestodes are the presence of a primary lacuna and the invagination/retraction of the scolex. The presence of a cercomer and of a bladder-like enlargement of the larval cestode were considered to be useful secondary characteristics. Using these characters, six basic types of larval cestodes were identified: the procercoid, an alacunate form which cannot develop further until ingested by a second intermediate host; the plerocercus, an alacunate form with a retracted scolex; the plerocercoid, an alacunate form with an everted scolex; the merocercoid, an alacunate form with an invaginated scolex; the cysticercoid, a lacunate form with a retracted scolex; and the cysticercus, a lacunate form with an invaginated scolex. The diversity of larval types within the broad classifications of cysticercoid and cysticercus can be differentiated by the use of appropriate prefixes. Deficiencies in knowledge of specific types of larval cestodes are identified and further avenues of research are indicated.Pseudonym for the contributors of a workshop on metacestode terminology, chaired by I. Beveridge, which took place during the Third International Workshop for Tapeworm Systematics,Sofia, Bulgaria, 20-24 July, 1999 (Organiser: B.B. Georgiev).     

Prey sex pheromone as kairomone for a new group of predators (Coleoptera: Dasytidae, Aplocnemus spp.) of pine bast scales

During the last decades, an increasing number of predators were found to use specific prey pheromones as chemical cues. Beyond its ecological relevance, this knowledge has practical applications on insect conservation and pest control. In this study, we present first evidence that two species of the family Dasytidae (Coleoptera) Aplocnemus brevis Rosenhauer and A. raymondi Sainte-Claire Deville use the sex pheromone of the pine bast scale Matsucoccus feytaudi Ducasse (Hemiptera: Matsucoccidae) as kairomone to locate this prey. The feeding habits and biology of Aplocnemus species are practically unknown. In the laboratory, the adults of Aplocnemus sp. accepted M. feytaudi egg masses as food source as well as other diets. Females represented more than 90% of Aplocnemus sp. attracted to the pheromone lures. We believe that females use this olfactory cue to locate suitable places for oviposition and that larvae are the predators of Matsucoccus. This study further demonstrates that the response to the kairomone elicited short prey searching times, about 23% of the individuals appeared less than 12 min after lure exposure, being consistent with the hypothesis of prey specialization. Habitat and geographical distribution predict an ancestral association of A. brevis with M. feytaudi and of A. raymondi with M. pini. Nevertheless, a recent prey shift of A. raymondi to the invasive M. feytaudi in Corsica is in progress.     

Phylogenetic analysis among the families of the Cyclophyllidea (Eucestoda) based on comparative morphology,with new hypotheses for co-evolution in vertebrates

Phylogenetic analysis of the families of the Cyclophyllidea based on comparative morphology revealed support for monophyly of the order. Four equal length trees (CI = 0.71) resulted from analysis of 42 binary and multistate characters. Major conclusions are the following: (1) a basal position for the arostellate groups, with Mesocestoididae + Nematotaeniidae representing the basal subclade; (2) polyphyly for the Anoplocephalidae with sister-group associations postulated respectively for Anoplocephalinae + Thysanosomatinae and Linstowiinae + Inermicapsiferinae; (3) recognition within the rostellate cyclophyllideans of taeniid, dilepidid and davaineid subclades and the Dipylidiidae; (4) designation of Metadilepididae + Paruterinidae as sister taxa; (5) monophyly for the Davaineidae with all inclusive subfamilies; and (6) a close relationship for the Hymenolepididae and acoleate cyclophyllideans. Monophyly for such classically defined groups as the Mesocestoididae, Taeniidae and Davaineidae is corroborated. Polyphyly of the Dilepididae sensu lato, with independence of the Dipylidiidae, Dilepididae sensu Bona (1994), the Metadilepididae + Paruterinidae, and the Gryporhynchidae is confirmed. As presented these constitute testable hypotheses for monophyly and relationships among the families of the Cyclophyllidea. Initial diversification of the cyclophyllideans occurred in mammalian hosts and three independent events of colonisation of avian taxa are postulated. Origins of the cyclophyllideans extend into the Mesozoic or earlier, with extant taxa representing lineages that were persistent across the extinction event that defines the Cretaceous-Tertiary boundary.     

Histogenesis in the metacestode of Echinococcus vogeli and mechanism of pathogenesis in polycystic hydatid disease.

Histogenesis of the metacestode of Echinococcus vogeli was traced mainly in rodents inoculated intraperitoneally with finely minced infective vesicles. The fragments aggregated in the peritoneal cavity and coalesced, forming structures (plaques) from which primary vesicles arose. From primordia in their germinal tissue, exogenous vesicles developed, enlarged, and migrated outward to the surface of the laminated membrane, where they remained attached and proliferated. Each unit of vesicles so formed retained discrete identity and, within 6-8 mo, acquired an adventitia; thereafter, exogenous multiplication ceased and endogenous proliferation supervened. Large numbers of daughter cysts arose in the germinal tissue lining chambers within the units; endogenous proliferation also finally ceased, and the daughter cysts produced brood capsules containing protoscoleces. Primordia of exogenous vesicles were not observed in the walls of daughter cysts. Production of protoscoleces involved 3 processes: they developed in typical brood capsules, singly in minute brood capsules, or directly from germinal tissue. Exogenous proliferation is not characteristic in the natural intermediate host of E. vogeli, the paca. Evidently in primates, the initial proliferation in the liver is followed by extension of the metacestode into the peritoneal cavity and eventual invasion of abdominal and thoracic organs. Exogenous proliferation by a process unique to E. vogeli accounts for the clinical course of polycystic hydatid disease.     

Amyloidogenic peptides of yeast cell wall glucantransferase Bgl2p as a model for the investigation of its pH-dependent fibril formation

The pH-dependence of the ability of Bgl2p to form fibrils was studied using synthetic peptides with potential amyloidogenic determinants (PADs) predicted in the Bgl2p sequence. Three PADs, FTIFVGV, SWNVLVA and NAFS, were selected on the basis of combination of computational algorithms. Peptides AEGFTIFVGV, VDSWNVLVAG and VMANAFSYWQ, containing these PADs, were synthesized. It was demonstrated that these peptides had an ability to fibrillate at pH values from 3.2 to 5.0. The PAD-containing peptides, except for VDSWNVLVAG, could fibrillate also at pH values from pH 5.0 to 7.6. We supposed that the ability of Bgl2p to form fibrils most likely depended on the coordination of fibrillation activity of the PAD-containing areas and Bgl2p could fibrillate at mild acid and neutral pH values and lose the ability to fibrillate with the increasing of pH values. It was demonstrated that Bgl2p was able to fibrillate at pH value 5.0, to form fibrils of various morphology at neutral pH values and lost the fibrillation ability at pH value 7.6. The results obtained allowed us to suggest a new simple approach for the isolation of Bgl2p from Saccharomyces cerevisiae cell wall.     

Echinococcus multilocularis: the parasite-host interplay

Alveolar echinococcosis (AE) is a severe chronic helminthic disease caused by the intrahepatic tumor-like growth of the metacestode of Echinococcus multilocularis. Metacestodes are fluid-filled, asexually proliferating vesicles, which are entirely covered by the laminated layer, an acellular carbohydrate-rich surface structure that protects the parasite from immunological and physiological reactions on part of the host. The E. multilocularis metacestode has acquired specific means of manipulating and using the immunological host response to its own advantage. These include the expression of distinct immunoregulatory parasite molecules that manipulate and interfere in the functional activity of macrophages and T cells. Recent research findings have led to a better understanding of the protein- and glycoprotein composition of the laminated layer and the E/S fraction of the metacestode, including Em2- and Em492-antigens, two metacestode antigen fractions that exhibit immunosuppressive or -modulatory properties. Understanding of the events taking place at the host-parasite interface is the key for development of novel immuno-therapeutical and/or chemotherapeutical tools.     

A hydrophobic ligand-binding protein of the Taenia solium metacestode mediates uptake of the host lipid: implication for the maintenance of parasitic cellular homeostasis

Parasitic organisms are incapable of de novo fatty acid synthesis due to a down-regulated expression of enzymes involved in the oxygen-dependent pathway. We investigated the uptake of host lipids by a 150-kDa hydrophobic ligand-binding protein (HLBP) of Taenia solium metacestode, an agent causative of neurocysticercosis. The protein was found to be a hetero-oligomeric complex consisting of multiple subunits (M(r) 7, 10, and 15 kDa within pH 8.0-9.7), which may originate from four unique genes of 7- and 10-kDa gene families with 2-3 polymorphic alleles/paralogs. The 15-kDa protein represented glycosylated forms of the 10-kDa. With high binding affinity to lipid analogs, these subunits evidenced high-level sequence identity with other cestode HLBPs and form a novel clade associated with excretory-secretory type HLBP. In vitro experiments with viable worms suggested that the excreted 150-kDa protein might bind to lipids, and participate in the translocation of host lipids across the syncytial membrane. This process was substantially inhibited by the specific anti-150 kDa antibodies. The protein was localized in the parasite syncytium and in the lipid droplets within host granuloma wall, where significant lipase activity was expressed. HLBP-mediated uptake of the host lipid may be critical for the parasite survival and thus could be targeted by chemotherapeutics and/or vaccine.     

Phenotypic 'variant' forms of Trichomonas vaginalis trophozoites from cervical neoplasia patients

With the present study, a culture system for successive life-cycle stages of the tapeworm Schistocephalus solidus was developed and this report documents for the first time, cultivation of the procercoid stage of S. solidus from eggs. Additionally we have transformed procercoids dissected from experimentally infected copepods and cultured procercoids into the early plerocercoid stage in vitro. Observations in the culture suggest that the coracidia can interact with their external environment and need no host specific stimuli, except for the components in the culture medium, for activation and hatching from the embryophore. Increasing the culture medium pH from 7.3 to 8.0 improved escape rates and frequencies of hook contractions, suggesting that the oncosphere may recognize and respond to environmental conditions along the host intestine. Procercoids in the culture did not stop growing indicating that conditions within the copepod may be important to limit growth and to induce transformation to plerocercoids. When procercoids are dissected from copepods and transferred to the culture, the outer tegument layers and cercomer starts to loosen. Comparison of the lectin staining of the loosened outer tegument layers and cercomer in procercoids dissected from copepods confirms that transitions of both, the oncosphere to procercoid and procercoid to plerocercoids, has taken place in the in vitro cultures.     

Identity of Cysticercoides menidiae Chandler, 1935 (Cestoda: Dilepididae)

Cysticercoides menidiae Chandler, 1935, described as a metacestode of an unknown dilepidid cestode from the intestinal wall and mesenteries of the silversides Menidia menidia (Pisces: Atherinidae), from Galveston Bay, Texas, was found to be conspecific with Ascodilepis transfuga (Krabbe, 1869), a tapeworm described from the spoonbill. Platalea ajaja Linnaeus (syn. Ajaja ajaja), from Brazil. Consequently. C. menidiae becomes a junior synonym of A. transfuga.     

EmTIP,a T-Cell Immunomodulatory Protein Secreted by the Tapeworm Echinococcus multilocularis Is Important for Early Metacestode Development

Background

Alveolar echinococcosis (AE), caused by the metacestode of the tapeworm Echinococcus multilocularis, is a lethal zoonosis associated with host immunomodulation. T helper cells are instrumental to control the disease in the host. Whereas Th1 cells can restrict parasite proliferation, Th2 immune responses are associated with parasite proliferation. Although the early phase of host colonization by E. multilocularis is dominated by a potentially parasitocidal Th1 immune response, the molecular basis of this response is unknown.

Principal Findings

We describe EmTIP, an E. multilocularis homologue of the human T-cell immunomodulatory protein, TIP. By immunohistochemistry we show EmTIP localization to the intercellular space within parasite larvae. Immunoprecipitation and Western blot experiments revealed the presence of EmTIP in the excretory/secretory (E/S) products of parasite primary cell cultures, representing the early developing metacestode, but not in those of mature metacestode vesicles. Using an in vitro T-cell stimulation assay, we found that primary cell E/S products promoted interferon (IFN)-γ release by murine CD4+ T-cells, whereas metacestode E/S products did not. IFN-γ release by T-cells exposed to parasite products was abrogated by an anti-EmTIP antibody. When recombinantly expressed, EmTIP promoted IFN-γ release by CD4+ T-cells in vitro. After incubation with anti-EmTIP antibody, primary cells showed an impaired ability to proliferate and to form metacestode vesicles in vitro.

Conclusions

We provide for the first time a possible explanation for the early Th1 response observed during E. multilocularis infections. Our data indicate that parasite primary cells release a T-cell immunomodulatory protein, EmTIP, capable of promoting IFN-γ release by CD4+ T-cells, which is probably driving or supporting the onset of the early Th1 response during AE. The impairment of primary cell proliferation and the inhibition of metacestode vesicle formation by anti-EmTIP antibodies suggest that this factor fulfills an important role in early E. multilocularis development within the intermediate host.     

The Echinococcus granulosus antigen B shows a high degree of genetic variability

Echinococcus granulosus larvae secret a polymeric lipoprotein known as antigen B (AgB) into the metacestode hydatid fluid. Three similar AgB subunits have been previously identified (AgB1, AgB2, and AgB3), and their respective genes isolated, but the actual number of genes encoding AgB subunits remains uncertain. In this study, we characterize the variability of genes encoding the AgB2 subunit, using PCR and RT-PCR followed by cloning and sequencing. We have analyzed 32 cDNA and 34 genomic sequences from a single metacestode, showing a high degree of sequence polymorphism. In addition, we have identified a possibly new AgB subunit, which we call AgB4. Additionally, we describe an AgB2 genomic clone lacking (i) a segment corresponding to the intron and (ii) a short, 45 bp sequence within exon II. The 45 bp segment encompasses the conserved splicing signals and corresponds to a highly conserved insect promoter motif.     

Taenia solium: identification and preliminary characterization of a lipid binding protein with homology to the SEC14 catalytic domain

The objective of this work is to identify proteins of the human and porcine parasite, Taenia solium, which may be exploited for control of the parasite. Through screening a cDNA library of T. solium metacestodes, we have identified a novel Sec-14-like Taenia lipid-binding protein that may play an important role in membrane trafficking. The Sec14-like sequence is a single copy gene, encoding a putative polypeptide of 320 amino acids and 36.1 kDa (sec14Tsol protein). Secondary amino acid structural analysis suggested that the sec14Tsol protein might contain two distinct structural domains, an amino-terminal alpha-helix rich domain and a mixed alpha-helix/beta-stand carboxy-terminal zone, showing homology with the conserved SEC14 domain found in a great number of proteins that bind lipids, as the regulators of membrane trafficking between Golgi membrane bilayers. Significantly, therefore, in a phosphoinositide-binding assay, sec14Tsol purified recombinant protein specifically interacted with important lipid regulators of membrane trafficking, with a preference for PI(3)P(2), PI(3,4)P(2), PI(4,5)P(2) and phosphatidic acid. Moreover, the sec14Tsol protein was localized in the Golgi apparatus of transfected cells and in the spiral canal region of T. solium metacestode tegument. As sec14Tsol protein may play an important role in membrane trafficking, its demonstrated localisation in the intact parasite tegument suggests its involvement in the function of the tegument and thus perhaps interaction with the host.     

Amyloid Aggregation on Lipid Bilayers and Its Impact on Membrane Permeability

Fibrillar protein aggregates (amyloids) are involved in several common pathologies, e.g., Alzheimer's disease and type II diabetes. Accumulating evidence suggests that toxicity in amyloid-related diseases originates from the deposition of protein aggregates on the cell membrane, which results in bilayer disruption and cell leakage. The molecular mechanism of damage to the membrane, however, is still obscure. To shed light on it we have performed coarse-grained molecular dynamics simulations of fibril-forming amphipathic peptides in the presence of lipid vesicles. The simulation results show that highly amyloidogenic peptides fibrillate on the surface of the vesicle, damaging the bilayer and promoting leakage. In contrast, the ordered aggregation of peptides with low amyloidogenicity is hindered by the vesicles. Remarkably, leakage from the vesicle is caused by growing aggregates, but not mature fibrils. The simulation results provide a basis for understanding the range of aggregation behavior that is observed in experiments with fibril-forming (poly)peptides.     

Echinococcus granulosus: ultrastructure of epithelial changes during the first 8 days of metacestode development in vitro

The epithelium of artificially hatched and activated oncospheres of E. granulosus was studied ultrastructurally over the first 8 days of metacestode development in vitro. Within 4 h of activation, the epithelium was transformed from a thin cytoplasmic layer into a much wider layer packed with penetration gland granules and containing mitochondria and Golgi apparatus. Microvilli were extended from the outer plasma membrane and the basal lamina on the inner epithelial surface virtually disappeared. Microvilli increased in number and length over the first 24 h of development while granules in both the epithelium and penetration gland decreased in number. The granules appear to be involved in microvilli formation. After 3 days of development, the first lamination resolved ultrastructurally as shortened microvilli and some microtriches extending from the epithelium surrounded by an electron-dense microfibrillate material containing sloughed microvilli. By 6 days post-activation, no microvilli remained and only double-walled truncated microtriches extended from the epithelium. The microfibrillate material had become more electron-dense and was closer to the epithelium than at day 1. Within 8 days of metacestode development, a second lamination had developed. Both microfibrillate and particulate material of a greater electron density than the first lamination was added to the microthrix side of the first lamination.     

Shape variation of cysticercoids of Hymenolepis diminuta (Cyclophyllidea) from fed, partially fed, and fasted Tribolium confusum (Coleoptera)

Quantitative studies of a crowding effect on cysticercoids of Hymenolepis diminuta in the intermediate host are few and limited in scope. In this study, we developed a technique to rapidly collect morphological information on large numbers of parasites, and verified the utility of geometric models for simple and accurate estimation of cysticercoid size for quantitative studies. These models were tested using measurements from 4,899 H. diminuta obtained from 666 Tribolium confusum exposed 1-4 wk previously. Length, width, and depth of the body and cercomer (when present) can be used in conjunction with these models to provide the most accurate estimation of parasite size. However, parasite body length alone can be used, with adjustment for effects of host diet and infection intensity, to predict the remaining measurements in incomplete specimens. Parasites that developed in higher intensity infections, or in hosts with reduced food intake, were narrower and had a proportionately shorter cercomer. Host age, sex, and mating status, and parasite age also had statistically significant, but small-magnitude, effects on parasite shape.     

Phylogenetic systematics of the genus Echinococcus (Cestoda: Taeniidae)

Echinococcosis is a serious helminthic zoonosis in humans, livestock and wildlife. The pathogenic organisms are members of the genus Echinococcus (Cestoda: Taeniidae). Life cycles of Echinococcus spp. are consistently dependent on predator–prey association between two obligate mammalian hosts. Carnivores (canids and felids) serve as definitive hosts for adult tapeworms and their herbivore prey (ungulates, rodents and lagomorphs) as intermediate hosts for metacestode larvae. Humans are involved as an accidental host for metacestode infections. The metacestodes develop in various internal organs, particularly in liver and lungs. Each metacestode of Echinococcus spp. has an organotropism and a characteristic form known as an unilocular (cystic), alveolar or polycystic hydatid. Recent molecular phylogenetic studies have demonstrated that the type species, Echinococcus granulosus, causing cystic echinococcosis is a cryptic species complex. Therefore, the orthodox taxonomy of Echinococcus established from morphological criteria has been revised from the standpoint of phylogenetic systematics. Nine valid species including newly resurrected taxa are recognised as a result of the revision. This review summarises the recent advances in the phylogenetic systematics of Echinococcus, together with the historical backgrounds and molecular epidemiological aspects of each species. A new phylogenetic tree inferred from the mitochondrial genomes of all valid Echinococcus spp. is also presented. The taxonomic nomenclature for Echinococcus oligarthrus is shown to be incorrect and this name should be replaced with Echinococcus oligarthra.