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.

[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.

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.

Glycosaminoglycans of tegumental fractions of Hymenolepis diminuta.

The teguments of 6 and 10 day-old Hymenolepis diminuta were removed with Triton X-100 and separated into brush border and vesicular fractions by differential centrifugation. Glycosaminoglycans (GAG) isolated from these tissues and from the denuded carcass were treated with specific GAG-degrading enzymes and other chemical agents and analyzed by sodium dodecyl sulfate-polyacrylamide, agarose gel and cellulose acetate electrophoresis. Both 6 and 10 day-old worm carcasses contained chondroitin sulfate, heparin/heparan sulfate and hyaluronic acid. The 10 day-old worm brush border and vesicle fractions contained chondroitin sulfate but no heparin-like material. Colorimetric analysis showed that the carcasses of both 6 and 10 day-old worms contained uronic acid. About 98% of the detectable uronic acid of 10 day-old worms was found in the carcass, and only 2% in the brush border fraction. No uronic acid was detected in the other tegumental fractions.

Analysis of ecdysteroids in different developmental stages of Hymenolepis diminuta.

Prepatent and patent adult Hymenolepis diminuta from the intestines of rats, H. diminuta eggs recovered from the faeces of rats harbouring patent infections, and infective cysticercoids from the beetle intermediate host were analysed for free and conjugated ecdysteroids. Adult worms and eggs contained both free ecdysteroids and hydrolysable polar conjugated ecdysteroids, with comparatively large amounts of immunoreactive material also being detected following hydrolysis of the possible apolar conjugated ecdysteroid fraction. Free ecdysteroids were not detected in the cysticercoid sample. The concentration of free ecdysteroids in H. diminuta eggs was higher than that detected in the tissues of the adult worms. Ecdysone and 20-hydroxyecdysone were the major identified compounds of the free ecdysteroid fraction, whereas in the hydrolysed polar conjugated ecdysteroid fraction these two compounds were accompanied by 20,26-dihydroxyecdysone. The free ecdysteroid fraction also contained comparatively large amounts of unidentified immunoreactive material.

Developmental and immune regulation of gene expression in Hymenolepis diminuta.

Gene expression and its regulation was studied in the rat tapeworm, Hymenolepis diminuta, during strobilization. RNAs extracted from the different developmental stages of the parasite were translated in vitro in a rabbit reticulocyte lysate system. Two-dimensional patterns of the translational products were compared with the 2-dimensional patterns obtained by metabolic labelling with [35S]methionine. The results indicated a post-transcriptional regulation of gene expression during strobilization in this parasite. Gene expression and its regulation was also studied in H. diminuta obtained from 'non-permissive' hosts (mice) and immune suppressed mice and compared with the parasites of the same age obtained from 'permissive' rats hosts. The 2-dimensional patterns of the in vitro translation products, obtained by translating the RNA of different groups of parasites, were compared with the patterns of gene products obtained from parasites using [35S]methionine metabolic labelling. The results indicated a massive post-transcriptional regulation of gene expression, the latter inhibited in parasites obtained from normal mice, but not in immune suppressed mice.

Alterations in brush border membrane proteins and membrane-bound enzymes of the tapeworm, Hymenolepis diminuta, during development in the definitive host.

During growth and maturation of the tapeworm, Hymenolepis diminuta, significant decreases occur in the brush border membrane-bound alkaline phosphatase, phosphodiesterase, 5'-nucleotidase, adenosine triphosphatase and ribonuclease activities. These decreases are accompanied by qualitative and quantitative changes in the polypeptide profiles of the brush border membrane fraction. Gradients of enzymatic activities and polypeptide profiles are also demonstrable when mature tapeworms are cut into pieces and the brush border membrane of each piece analyzed individually. In fully developed tapeworms the enzymatic activities and polypeptide profiles of membrane preparations reflect mainly the contributions of the more mature proglottids; these proglottids constitute most of the tapeworm biomass. The most anterior sections of these fully developed worms are biochemically similar to young, developing worms.

Intramitochondrial localization of fumarate reductase, NADPH----NAD transhydrogenase, 'malic' enzyme and fumarase in adult Hymenolepis diminuta.

The intramitochondrial localization of the fumarate reductase, NADPH----NAD transhydrogenase, 'malic' enzyme and fumarase was determined in adult Hymenolepis diminuta. The distribution of marker enzymes for the inner membrane, matrix, intermembrane space and outer membrane of H. diminuta mitochondria simulated that of the corresponding ascarid and mammalian organelles. The electron transport-coupled fumarate reductase and the NADPH----NAD transhydrogenase were components of the inner membrane whereas the 'malic' enzyme and fumarase were in the matrix soluble compartment. Assessments of NADH utilization, malate-dependent NADP reduction and NADPH----NAD transhydrogenation by presumedly intact and disrupted mitochondria supported the localization data. The findings presented indicate that in H. diminuta mitochondria (a) NADPH and fumarate are accumulated within the matrix compartment; (b) transhydrogenation between NADPH and NAD is an event associated with the matrix side of the inner membrane; and (c) electron transport-dependent NADH oxidation and fumarate reduction occur at sites on the matrix side of the inner membrane.

Inhibition of growth of a tapeworm Hymenolepis diminuta in its normal host (rat).

The biomass of 8-day-old worms of Hymenolepis diminuta in secondary infections, administered to rats 3-10 days after chemotherapeutically expelling a primary infection, was 70-90% less, and the worms were more posteriorly distributed, than in naive controls. The strong depressive effect on growth waned rapidly over 2-5 weeks, but even in rats not challenged until 17 months later, worm growth was weakly depressed by 30%. The extent to which growth was depressed in a secondary infection was independent of the number of worms in the challenge but increased with number of worms in the immunizing infection up to four to eight worms. Further increase up to 64 worms had little effect. This suggests, as it is known that the biomass of worms in a rat reaches a maximum with infections of between five and 10 worms, that the change in the intestine is proportional to biomass, not number, of worms. It is argued that partially suppressed immuno-inflammatory changes in the intestine, which will affect secondary worms so strongly, will also have depressed growth and fecundity effects on the primary worms, that a dynamic equilibrium is reached between the strength of the intestinal response and the biomass of the tapeworm, and that it is reaching this equilibrium, not a 'crowding effect', which limits H. diminuta to a level compatible with the survival of the rat.

The effects of changes in the definitive host environment on the metabolism of Hymenolepis diminuta during growth and maturation.

Flexibility in the metabolism of Hymenolepis diminuta is associated with changing intrinsic requirements during maturation but is also influenced by extrinsic factors, that is, by the nature of the host environment. End-products of carbohydrate metabolism and enzyme activities in worm extracts were used as indicators of metabolic regulation in H. diminuta recovered at various times postinfection. The predominant end-product from 6-day-old worms is lactate, generated by cytosolic glycolysis. As the cestode matures in the host, lactate production by the whole worm decreases and greater amounts of the mitochondrial end-products, succinate and acetate, are detected. A stable, dichotomous carbon flow to lactate, succinate and acetate is observed from 12 days post-infection. A metabolic gradient along the length of individual strobila is also evident. It extends from glycolysis, in the anterior region, to mitochondrial dismutation in the posterior region. The transition from cytosolic to mitochondrial pathways during maturation and along the strobilus is delayed or suppressed in worms recovered from immunosensitized hosts. Four host environments were compared: unsensitized rats, rats immunosensitized with a primary infection of H. diminuta, rats immunosensitized with a primary infection of Nippostrongylus brasiliensis and mice concurrently infected with Heligmosomoides polygyrus. The specific activities of PK and PEPCK in whole worm extracts were similar in 10-, 21- and 35-day-old worms and did not differ in worms isolated from different host environments. However, the PEPCK/PK ratio is high in worms that utilize mitochondrial pathways and low in worms that produce predominantly lactate. LDH activity is high in lactate producers. It is concluded that the pattern of metabolism in H. diminuta is influenced by many effectors in the host environment.

Immune response to the cestode Hymenolepis nana: cytokine production during infection with eggs or cysts.

Analysis of cytokine production (IFN-gamma, IL-2, IL-3, IL-4, IL-5) by in vitro Con A-stimulated mesenteric lymph node cells measured daily after egg or cyst infection of mice with Hymenolepis nana showed that cytokine production varies during parasite development and between different host strains (BALB/c and C3H/He mice). Egg infection stimulates a rapid increase in IFN-gamma, independent of mouse strain. In addition, in BALB/c mice a Th2-like response (IL-4, IL-5 secretion) was stimulated 4-5 days p.i., when the parasites are thought to begin their lumenal phase. After infection with cysts significant increases in IFN-gamma, IL-2, IL-4 and IL-5 were observed at the time when autoinfection with eggs is thought to occur. The level of IFN-gamma paralleled that seen after a primary egg infection. This suggests that there is a predominantly Th1-type response during the tissue phase of H. nana development and that, in BALB/c mice, a Th2 polarization occurs during the first few days of the lumenal phase. The cytokine patterns observed are discussed in relation to host responses during chronic helminth infection.

Importance of interferon-gamma in protective immunity against Hymenolepis nana cysticercoids derived from challenge infection with eggs in BALB/c mice.

The function of cytokines produced during Hymenolepis nana egg infection in mice in protective immunity against re-infection was examined. Treatment of mice with monoclonal antibody (MAb) against mouse interferon (IFN)-gamma caused suppression of protective immunity against H. nana re-infection when the MAb was injected intraperitoneally at a daily dose of 40.0 mg kg-1 during the effector phase of protective immunity. Although high levels of IFN-gamma, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta were released into the intestinal tracts of the parasitised mice at challenge infection, there was almost no release of these cytokines in mice treated with the MAb. Daily administration of rolipram failed to suppress the protective immunity, even when 400 micrograms kg-1 of the agent was administered into mice during the effector phase of immunity. Treatment of mice with rolipram completely suppressed both TNF-alpha and IL-1 beta production in intestinal tracts, induced by H. nana challenge infection. However, endogenous IFN-gamma production in the intestine was scarcely affected by rolipram. These results strongly suggest that IFN-gamma is the most important (or essential) cytokine in protective immunity to H. nana re-infection, rather than TNF-alpha and IL-1 beta.