Stage-specific transcriptomic analysis of the model cestode Hymenolepis microstoma.

Most parasitic flatworms go through different life stages with important physiological and morphological changes. In this work, we used a transcriptomic approach to analyze the main life-stages of the model tapeworm Hymenolepis microstoma (eggs, cysticercoids, and adults). Our results showed massive transcriptomic changes in this life cycle, including key gene families that contribute substantially to the expression load in each stage. In particular, different members of the cestode-specific hydrophobic ligand-binding protein (HLBP) family are among the most highly expressed genes in each life stage. We also found the transcriptomic signature of major metabolic changes during the transition from cysticercoids to adult worms. Thus, this work contributes to uncovering the gene expression changes that accompany the development of this important cestode model species, and to the best of our knowledge represents the first transcriptomic study with robust replicates spanning all of the main life stages of a tapeworm.

Divergent Axin and GSK-3 paralogs in the beta-catenin destruction complexes of tapeworms.

The Wnt/beta-catenin pathway has many key roles in the development of animals, including a conserved and central role in the specification of the primary (antero-posterior) body axis. The posterior expression of Wnt ligands and the anterior expression of secreted Wnt inhibitors are known to be conserved during the larval metamorphosis of tapeworms. However, their downstream signaling components for Wnt/beta-catenin signaling have not been characterized. In this work, we have studied the core components of the beta-catenin destruction complex of the human pathogen Echinococcus multilocularis, the causative agent of alveolar echinococcosis. We focused on two Axin paralogs that are conserved in tapeworms and other flatworm parasites. Despite their divergent sequences, both Axins could robustly interact with one E. multilocularis beta-catenin paralog and limited its accumulation in a heterologous mammalian expression system. Similarly to what has been described in planarians (free-living flatworms), other beta-catenin paralogs showed limited or no interaction with either Axin and are unlikely to function as effectors in Wnt signaling. Additionally, both Axins interacted with three divergent GSK-3 paralogs that are conserved in free-living and parasitic flatworms. Axin paralogs have highly segregated expression patterns along the antero-posterior axis in the tapeworms E. multilocularis and Hymenolepis microstoma, indicating that different beta-catenin destruction complexes may operate in different regions during their larval metamorphosis.

Comparative analysis of Wnt expression identifies a highly conserved developmental transition in flatworms.

BACKGROUND: Early developmental patterns of flatworms are extremely diverse and difficult to compare between distant groups. In parasitic flatworms, such as tapeworms, this is confounded by highly derived life cycles involving indirect development, and even the true orientation of the tapeworm antero-posterior (AP) axis has been a matter of controversy. In planarians, and metazoans generally, the AP axis is specified by the canonical Wnt pathway, and we hypothesized that it could also underpin axial formation during larval metamorphosis in tapeworms. RESULTS: By comparative gene expression analysis of Wnt components and conserved AP markers in the tapeworms Echinococcus multilocularis and Hymenolepis microstoma, we found remarkable similarities between the early stages of larval metamorphosis in tapeworms and late embryonic and adult development in planarians. We demonstrate posterior expression of specific Wnt factors during larval metamorphosis and show that scolex formation is preceded by localized expression of Wnt inhibitors. In the highly derived larval form of E. multilocularis, which proliferates asexually within the mammalian host, we found ubiquitous expression of posterior Wnt factors combined with localized expression of Wnt inhibitors that correlates with the asexual budding of scoleces. As in planarians, muscle cells are shown to be a source of secreted Wnt ligands, providing an explanation for the retention of a muscle layer in the immotile E. multilocularis larva. CONCLUSIONS: The strong conservation of gene expression between larval metamorphosis in tapeworms and late embryonic development in planarians suggests, for the first time, a homologous developmental period across this diverse phylum. We postulate these to represent the phylotypic stages of these flatworm groups. Our results support the classical notion that the scolex is the true anterior end of tapeworms. Furthermore, the up-regulation of Wnt inhibitors during the specification of multiple anterior poles suggests a mechanism for the unique asexual reproduction of E. multilocularis larvae.

Basic and applied problems in developmental biology and immunobiology of cestode infections: Hymenolepis, Taenia and Echinococcus.

Differentiation and development of parasites, including longevity in host animals, are thought to be governed by host-parasite interactions. In this review, several topics on the developmental biology of cestode infections are discussed from immunobiological perspective with a focus on Hymenolepis, Taenia and Echinococcus infections. The basic premise of this review is that 'differentiation and development of cestodes' are somehow affected by host immune responses with an evolutionary history.

RNA-mediated gene suppression and in vitro culture in Hymenolepis microstoma.

Hymenolepis microstoma, the mouse bile-duct tapeworm, is a classical rodent-hosted model that provides easy laboratory access to all stages of the life cycle. Recent characterisation of its genome has greatly advanced its utility for molecular research, albeit contemporary techniques such as those for assaying gene function have yet to be developed in the system. Here we present research on the development of RNA-mediated gene suppression via RNA interference (RNAi), and on in vitro culture of the enteric, adult phase of the life cycle to support this work. We demonstrate up to 80% quantitative suppression of a Hox transcript via soaking activated juvenile worms with double-stranded RNAs. However, we were unable to achieve segmentation of the worms in culture despite extensive manipulations of the culture media and supplements, preventing functional interpretation. An alternative, in vivo approach to RNAi was also tested by exposing cysticercoids prior to inoculation in mice, but fluorescent labelling showed that the RNAs did not sufficiently penetrate the cyst body and no difference in expression was found between exposed and control groups grown in vivo. Genomic and transcriptomic data revealed that H. microstoma has two orthologs each of Dicer, Drosha and Ago-1-like genes and that expression of one of the Ago-1 genes appears exclusive to germline development, suggesting that two or more independent RNA-mediated pathways are in operation. These studies demonstrate the viability of RNAi in H. microstoma and extend the utility of the model for research in the genomic era.

Effect of some immunomodulators on the host-parasite system in experimental Hymenolepiasis nana.

Mice experimentally infected with H. nana and injected with immunosuppressant {cyclophosphamide (Cp) and lead nitrate (Ln)} showed significant increase in infection intensity, significant decrease in intestinal mast cell count, dissemination of larvae to the liver, toxic hepatitis and absence of specific serum IgG. Cyclophosphamide caused morphological abnormallities in adult worms, prolongation of patent period and more severe villous changes. Immuno-stimulants represented by Levamisol (Lv) and gamma interferon (IFN-alpha) caused significant decrease in infection intensity, significant shortening of patent period and early improvement of histopathological changes. Immunostimulants, particularly IFN-alpha, were highly effective in counteracting hyperinfection seen with immuno-suppression. The study confirmed the deleterious effects of immunosuppression on hymenolepiasis and suggested a beneficial role for immunotherapy for immunosuppressed patients.

[The evolution of forms of hermaphroditism in Cyclophyllidea (Cestoda). 1. Morpho-functional causes of the formation of tapeworms with the protandrous type of genital apparatus development].

Morpho-fuctional causes of the formation of protandrous Cyclophyllidea (tapeworms) have been studied. Two forms of protandry are described. The protandry type I is typical for polymeric (polysegmental) Hymenolepididae. It appears independently in different taxa of this family (Aploparaksis, Echinatrium, Wardium, Diorchis and others) while the narrow-strobila forms with a low prolificacy of proglottids are formed. The development of this living form of hymenolepidids is ecologically caused by the high density of their aggregation in intestines of hosts. The primordium results in the development of genitals in the juvenile strobila proglottids with the limited internal space. Due to this process, parallel morphogenesis of male and female gonads is proved to be impossible. A selection leading to the overtaking development of testicles and copulative apparatus regarding morphogenesis of ovary and vitellarium is based an earlier maturation of testicles and group copulation of proglottids with and underdeveloped ovary that is typical in original euandrogyne forms. The group insemination of proglottids from a polysegmented strobila reduces the number of copulation and improves an efficiency of cross-copulation of tapeworms and. As a result, morpho-functional zones of male proglottids characterized by an immature ovary and those of fertile female segments losing their testicles are differentiated in the strobila. The protandry type II is typical of mesomeric tapeworms (Dilepididae, Schistotaeniidae, Anoplocephalidae). It is also resulted from a limited space of proglottids for developing the hermaphroditic genital apparatus. This is caused by the shift of genital morphogenesis process into juvenile proglottids and also by the enlargement of gonad sizes as the result of a selection for a higher prolificacy of proglottids. The dissociation of the development of male and female gonads takes place because of the retardation of ovary morphogenesis.

Hymenolepis pseudodiminuta Tenora et al. 1994 from Apodemus speciosus and H. diminuta: a comparison of experimental infections in rats.

The successful maintenance of Hymenolepis pseudodiminuta, isolated from Apodemus speciosus, is described for the first time. In the laboratory, the flour beetle, Tribolium confusum, and F344 rats could serve as intermediate and definitive hosts, respectively. In single worm infections with H. pseudodiminuta, which were carried in two groups of rats, adult worms were recovered from eight and seven out of ten rats, respectively, while Hymenolepis diminuta was found in all of ten rats 6 weeks after inoculation. The worm weight of H. pseudodiminuta in rats was significantly lower than that of H. diminuta. The egg output of H. pseudodiminuta occurred significantly earlier than that of H. diminuta. The number of eggs in the faeces of H. diminuta-infected rats was approximately twofold higher than the number in the faeces of H. pseudodiminuta-infected rats throughout the course of the infection. Mucosal mast cells in rats infected with H. pseudodiminuta were significantly more common than in rats infected with H. diminuta. No detectable IgE antibodies were found in the uninfected and H. diminuta-infected rat groups; however total IgE was detected in H. pseudodiminuta-infected rats but the concentrations were variable between individuals.

Cestode parasites: application of in vivo and in vitro models for studies on the host-parasite relationship.

Cestode worms, commonly also known as 'flat' worms or tapeworms, are an important class of endoparasitic organisms. In order to complete their life cycle, they infect intermediate and definitive hosts in succession, through oral ingestion of eggs or larvae, respectively. Serious disease in humans or other mammalian hosts is mostly caused by the larval stages. Echinococcus spp. and Taenia spp. have been extensively investigated in the laboratory due to the fact that they represent important veterinary medical challenges and also cause grave diseases in humans. In contrast, Hymenolepis spp. and Mesocestoides spp. infections are relatively rare in humans, but these parasites have been extensively studied because their life cycle stages can be easily cultured in vitro, and can also be conveniently maintained in laboratory animal hosts. Thus they are more easily experimentally accessible, and represent important models for investigating the various aspects of cestode biology. This review will focus on in vitro and in vivo models which have been developed for studies on the host-parasite relationship during infection with Echinococcus, Taenia, Hymenolepis, Mesocestoides and Spirometra, and will cover the use of these models to investigate the morphology and ultrastructure of respective genera, the immunological relationship with the host and the development of vaccination approaches, as well as applications of these models for studies on parasite metabolism, physiology and gene expression. In addition, the use of these models in the development of chemotherapeutic measures against cestode infections is reviewed.

Failure to infect laboratory rodent hosts with human isolates of Rodentolepis (= Hymenolepis) nana.

Confusion exists over the species status and host-specificity of the tapeworm Rodentolepis (= Hymenolepis) nana. It has been described as one species, R. nana, found in both humans and rodents. Others have identified a subspecies; R. nana var. fraterna, describing it as morphologically identical to the human form but only found in rodents. The species present in Australian communities has never been identified with certainty. Fifty one human isolates of Rodentolepis (= Hymenolepis) nana were orally inoculated into Swiss Q, BALB/c, A/J, CBA/ CAH and nude (hypothymic) BALB/c mice, Fischer 344 and Wistar rats and specific pathogen free (SPF) hamsters. Twenty four human isolates of R. nana were cross-tested in flour beetles, Tribolium confusum. No adult worms were obtained from mice, rats or hamsters, even when immunosuppressed with cortisone acetate. Only one of the 24 samples developed to the cysticercoid stage in T. confusum; however, when inoculated into laboratory mice the cysticercoids failed to develop into adult worms. The large sample size used in this study, and the range of techniques employed for extraction and preparation of eggs provide a comprehensive test of the hypothesis that the human strain of R. nana is essentially non-infective to rodents.

Ileal mucosal mast cell, eosinophil, and goblet cell populations during Hymenolepis diminuta infection of the rat.

The population dynamics in the enteric connective tissues of eosinophils, mucosal mast cells (MMC), and in the mucosal epithelium of goblet cells were examined morphometrically in fixed ileal tissue of outbred Sprague Dawley rats during the first 32 days of infection with the tapeworm Hymenolepis diminuta. MMC and eosinophils were present in the lamina propria and submucosa; however, only eosinophils were also present in the muscularis externa. Eosinophilic infiltrate was first observed in the lamina propria at 15 days postinfection (dpi) and the numbers of eosinophils remained elevated through 32 dpi. Initial mucosal mastocytosis was detected on 6 dpi and MMC numbers continued to rise over the study period without reaching a plateau. Goblet cell hyperplasia occurred only at 32 dpi. In contrast to some intestinal nematode infections where these same 3 cell types are associated with the host's expulsion responses, H. diminuta is not lost by a rapid host response in the outbred Sprague Dawley rat strain used in these experiments. We suggest that either the induction of hyperplasia of these host effector cells in ileum tissue during H. diminuta infection is not capable of triggering parasite rejection mechanisms, or the function of the induced hyperplasia is necessary for some as yet unassociated physiological or tissue architecture change in the host's intestine.

Effect of surgical alteration of the rat gastrointestinal tract on the growth and development of Hymenolepis diminuta.

Eight groups of rats were used to study the involvement of the enteric (ENS) and central (CNS) nervous systems in the development of Hymenolepis diminuta using surgical intestinal transection, or CNS denervation, or both procedures. The transection procedure was used to isolate the ENS of the small intestine from either orad and/or caudal portions of the alimentary system, while the CNS denervation was used to eliminate direct visceral efferent inputs from the CNS. Nine days after the surgical procedures, all rats were infected with 35 cysticercoids of H. diminuta. On 20 days postinfection, the infection intensity, tapeworm dry weight, tapeworm morphology, intestine length, and intestinal wet weight were recorded. Only the combination of the duodenal and ileal transections with a CNS denervation reduced infection intensity and prevented the increased intestinal length normally observed in infected rats. In contrast, none of the various intestinal transection procedures alone or CNS denervation alone had any effect on the survival, ability to produce oncospheres or morphology of the tapeworms. In conclusion, tapeworm survival is decreased when both CNS and ENS inputs into the small intestine are altered or absent.

Immunoglobulin E and mast cell responses are related to worm biomass but not expulsion of Hymenolepis diminuta during low dose infection in rats.

It is well known that the destrobilation and later expulsion are characteristics of multiple Hymenolepis diminuta infections in rats. This process is suggested to be mediated by a variety of host cellular responses. It has also been suggested that immunoglobulin (Ig) E may have a beneficial role for some cestodes including H. diminuta. We examined the intestinal mast cell and serum IgE responses to a 10-H. diminuta infection in three different rat strains. Tapeworm infection induced no increased mast cell and IgE responses in F344 rats in which neither worm biomass nor worm burden decreased during 6 weeks of observation. The number of mast cells and amounts of serum rat mast cell protease (RMCP) II and IgE markedly increased from 3 weeks postinfection (p.i.) in BN rats. The worm biomass in BN rats was significantly lower than that in F344 rats, but worm burden was not different from that in F344 rats at 3 or 6 weeks p.i. In DA rats, the number of mast cells and levels of serum RMCP II and IgE increased at 6 weeks but not at 3 weeks p.i. Although numbers of mast cells and serum RMCP II and IgE levels were lower in DA rats than in BN rats, smaller and fewer worms were recovered in DA rats than in F344 and BN rats at from 3 and 6 weeks p.i. Worms were recovered from all of F344 and BN rats, while only 40% of DA rats harboured worms at 6 weeks p.i. These results suggested that the worm biomass was related to mast cell and IgE responses, but these responses were not required for worm expulsion during low dose H. diminuta infection in rats.

Allometric growth of the proglottids and strobila of the tapeworm, Hymenolepis diminuta.

HYMENOLEPIS DIMINUTA: adults were killed and fixed in fully extended positions, and the total lengths of the strobilae, numbers of proglottids, and distances of proglottids along the strobilae were determined. The relationship of proglottid number to distance along the strobila was exponential. Beginning at proglottid 100 (P100), the lengths and widths of proglottids at 100 proglottid intervals were determined, and the surfaces areas were calculated. The relationships of proglottid length and width to proglottid number were linear, but the relationship of proglottid number to surface area (SA) was exponential. The volumes of proglottids were calculated, and the relationship of volume (V) to proglottid number was exponential. The relationship of surface area to volume ratio (SAVR) to proglottid number was exponential; at the anterior end of the worm (P100), the SAVR was 14.6, while at the posterior end of the worm (P1300) the ratio was 4.2. A single exponential equation describing the relationships among proglottid number, SA, and V was derived.

Metabolic transition in the development of Hymenolepis diminuta (Cestoda).

Cysticercoids as well as 6-, 10-, and 14-day Hymenolepis diminuta were evaluated in terms of enzymatic activities related to phosphoenolpyruvate (PEP) utilization and mitochondrial succinate accumulation. The data obtained support a transition toward anaerobic electron-transport-dependent succinate accumulation, characteristic of adult H. diminuta, with development from cysticercoid to adult. This transition was reflected most prominently in the increasing activities of PEP carboxykinase (PEPCK), malate dehydrogenase, NADPH-->NAD+ transhydrogenase, and fumarate reductase. Developmental increases in PEPCK/pyruvate kinase (PK), fumarate reductase (FR)/NADH oxidase (NO), and FR/succinate dehydrogenase (SDH) activity ratios were also apparent. Evaluations of "egg-free" immature, mature, and pregravid-gravid segments of adult H. diminuta revealed that in general the greater levels of activity were associated with the immature and mature segments. Whereas FR/NO and FR/SDH ratios remained relatively constant in segment comparisons, the greatest PEPCK/PK ratio was associated with the pregravid-gravid segment.