Search

Background: Tapeworms are parasitic flatworms that independently evolved a segmented body plan, historically confounding comparisons with other animals. Anteroposterior (AP) patterning in free-living flatworms and in tapeworm larvae is associated with canonical Wnt signaling and positional control genes (PCGs) are expressed by their musculature in regionalized domains along the AP axis. Here, we extend investigations of PCG expression to the adult of the mouse bile-duct tapeworm Hymenolepis microstoma, focusing on the growth zone of the neck region and the initial establishment of segmental patterning., Results: We show that the adult musculature includes new, segmental elements that first appear in the neck and that the spatial patterns of Wnt factors are consistent with expression by muscle cells. Wnt factor expression is highly regionalized and becomes AP-polarized in segments, marking them with axes in agreement with the polarity of the main body axis, while the transition between the neck and strobila is specifically demarcated by the expression domain of a Wnt11 paralog., Conclusion: We suggest that segmentation could originate in the muscular system and participate in patterning the AP axis through regional and polarized expression of PCGs, akin to the gene regulatory networks employed by free-living flatworms and other animals., (© 2024 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Introduction: Hymenolepiasis remains among the most common parasitic zoonoses in developing countries. Little information is available about hymenolepiasis in children in Upper Egypt and rodents' contribution to maintaining the disease's epidemiology., Methodology: A cross-sectional study was carried out to investigate the occurrence of Hymenolepis spp. in Rattus rattus and children in Asyut Governorate, Egypt. Rodents (n = 100) were randomly trapped from various localities in Asyut Governorate, and stool samples from 120 children were collected from the same localities. Laboratory examination of the collected samples involved investigation of the small intestine of R. rattus for adult worm detection by morphological examination, followed by examination of stool samples of children using direct smear, formol-ether sedimentation technique, and Sheather's sugar flotation technique. Confirmation of Hymenolepis spp. positive samples were performed using polymerase chain reaction targeting the internal transcribed spacer 1 (ITS-1) and restriction fragment length polymorphism (PCR-RFLP)., Results: This study revealed the occurrence of Hymenolepis spp. in 45% of the examined R. rattus, comprising 43% positivity for H. diminuta and 2% for mixed infection by H. nana and H. diminuta. Hymenolepis nana was detected in 28.3% of the examined children. PCR-RFLP confirmed these findings, showing 100% sensitivity. Collectively, these findings reveal the potential contribution of R. rattus as an important reservoir for Hymenolepis infection in Upper Egypt., Conclusions: This study concluded that personal education, periodical deworming of children, rodent control, and hygienic measures should be implemented by governmental and nongovernmental organizations to reduce the incidence of infection., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Menna-Tala Zakaria Abd-Elrahman, Amal SM Sayed, Doaa Abdelhafez Younes, Alam El-Din Mohamed Abdallah Ahmed, Samia Qasem Alghamdi, Amira A Saleh, Hind Alzaylaee, Manal F El-khadragy, Ehab Kotb Elmahallawy.)

Background: Tapeworm larvae cause important diseases in humans and domestic animals. During infection, the first larval stage undergoes a metamorphosis where tissues are formed de novo from a population of stem cells called germinative cells. This process is difficult to study for human pathogens, as these larvae are infectious and difficult to obtain in the laboratory., Methods: In this work, we analyzed cell proliferation and differentiation during larval metamorphosis in the model tapeworm Hymenolepis microstoma , by in vivo labelling of proliferating cells with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), tracing their differentiation with a suite of specific molecular markers for different cell types., Results: Proliferating cells are very abundant and fast-cycling during early metamorphosis: the total number of cells duplicates every ten hours, and the length of G2 is only 75 minutes. New tegumental, muscle and nerve cells differentiate from this pool of proliferating germinative cells, and these processes are very fast, as differentiation markers for neurons and muscle cells appear within 24 hours after exiting the cell cycle, and fusion of new cells to the tegumental syncytium can be detected after only 4 hours. Tegumental and muscle cells appear from early stages of metamorphosis (24 to 48 hours post-infection); in contrast, most markers for differentiating neurons appear later, and the detection of synapsin and neuropeptides correlates with scolex retraction. Finally, we identified populations of proliferating cells that express conserved genes associated with neuronal progenitors and precursors, suggesting the existence of tissue-specific lineages among germinative cells., Discussion: These results provide for the first time a comprehensive view of the development of new tissues during tapeworm larval metamorphosis, providing a framework for similar studies in human and veterinary pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Montagne, Preza and Koziol.)

Hymenolepis diminuta is a tapeworm commonly found worldwide in small rodents such as rats with occasional reports in other definitive hosts such as primates including chimpanzees and humans. It has not been reported in African green monkey (AGM, Chlorocebus sabaeus), and the parasite's molecular phenotype and phylogeny remain primitively sketchy. The aims of the current study were to determine if H. diminuta infected AGMs, to molecularly characterize H. diminuta and to review its infection in non-human primates. Feces of AGMs were examined visually for adult helminths and microscopically for eggs using centrifugation flotation. Total DNA extracted from eggs was amplified by PCR followed by DNA sequencing of targeted sequences of nuclear rRNA + internal transcribed spacers (ITS) and mitochondrial cox1. Phylogenetic analyses were performed. The DNA sequences of both nuclear rRNA + ITS and mitochondrial cox1 showed more than 98% and 99% identity to the known sequences respectively. Hymenolepis diminuta has been reported in various non-human primates with the highest prevalence of 38.5% in the white-headed capuchin monkey. The study presented here confirms that this tapeworm is capable of infecting various species of non-human primates with the first report of infections in AGM. Phylogenetic analyses of rRNA + ITS and mitochondrial cox1 demonstrated three separated clades I, II and III with the newly described AGM1 isolate belonging to the clade I. Whether these differences are at species level remains to be confirmed., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Spliced leader (SL) trans-splicing is a key process during mRNA maturation of many eukaryotes, in which a short sequence (SL) is transferred from a precursor SL-RNA into the 5' region of an immature mRNA. This mechanism is present in flatworms, in which it is known to participate in the resolution of polycistronic transcripts. However, most trans-spliced transcripts are not part of operons, and it is not clear if this process may participate in additional regulatory mechanisms in this group. In this work, we present a comprehensive analysis of SL trans-splicing in the model cestode Hymenolepis microstoma. We identified four different SL-RNAs which are indiscriminately trans-spliced to 622 gene models. SL trans-splicing is enriched in constitutively expressed genes and does not appear to be regulated throughout the life cycle. Operons represented at least 20% of all detected trans-spliced gene models, showed conservation to those of the cestode Echinococcus multilocularis, and included complex loci such as an alternative operon (processed as either a single gene through cis-splicing or as two genes of a polycistron). Most insertion sites were identified in the 5' untranslated region (UTR) of monocistronic genes. These genes frequently contained introns in the 5' UTR, in which trans-splicing used the same acceptor sites as cis-splicing. These results suggest that, unlike other eukaryotes, trans-splicing is associated with internal intronic promoters in the 5' UTR, resulting in transcripts with strong splicing acceptor sites without competing cis-donor sites, pointing towards a simple mechanism driving the evolution of novel SL insertion sites., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Rodents are hosts of a wide diversity of cestodes. Fifteen genera included in the family Hymenolepididae parasitize rodents, and only four of these genera have been recorded from the Neotropical region. The purpose of this paper is to update species of Hymenolepididae from rodents, describe a new species of Hymenolepis based on morphological and molecular characterization (ITS1 rDNA and cox1 mtDNA), comparing the features among the species from North and South American rodents, and provide phylogenetic inferences of Hymenolepididae from rodents based on sequences available in the GenBank. Rodents were collected in the Parque Provincial Ernesto Tornquist, Buenos Aires, Argentina. Hymenolepis ivanovae n. sp. differs from other Hymenolepis species registered from North and South American rodents by body size, scolex, suckers, cirrus sac, cirrus, testes, and eggs, among others. Comparative morphometric data for Hymenolepis species from North and South American rodents is provided. Molecular analyses place H. ivanovae n. sp. within the genus Hymenolepis with strong support, and show it close to species of zoonotic importance. The new species is the first species of Hymenolepis described from Sigmodontinae rodents., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Chromosome-level assemblies are indispensable for accurate gene prediction, synteny assessment, and understanding higher-order genome architecture. Reference and draft genomes of key helminth species have been published, but little is yet known about the biology of their chromosomes. Here, we present the complete genome of the tapeworm Hymenolepis microstoma, providing a reference quality, end-to-end assembly that represents the first fully assembled genome of a spiralian/lophotrochozoan, revealing new insights into chromosome evolution., Results: Long-read sequencing and optical mapping data were added to previous short-read data enabling complete re-assembly into six chromosomes, consistent with karyology. Small genome size (169 Mb) and lack of haploid variation (1 SNP/3.2 Mb) contributed to exceptionally high contiguity with only 85 gaps remaining in regions of low complexity sequence. Resolution of repeat regions reveals novel gene expansions, micro-exon genes, and spliced leader trans-splicing, and illuminates the landscape of transposable elements, explaining observed length differences in sister chromatids. Syntenic comparison with other parasitic flatworms shows conserved ancestral linkage groups indicating that the H. microstoma karyotype evolved through fusion events. Strikingly, the assembly reveals that the chromosomes terminate in centromeric arrays, indicating that these motifs play a role not only in segregation, but also in protecting the linear integrity and full lengths of chromosomes., Conclusions: Despite strong conservation of canonical telomeres, our results show that they can be substituted by more complex, species-specific sequences, as represented by centromeres. The assembly provides a robust platform for investigations that require complete genome representation.

Background: Reference genome and transcriptome assemblies of helminths have reached a level of completion whereby secondary analyses that rely on accurate gene estimation or syntenic relationships can be now conducted with a high level of confidence. Recent public release of the v.3 assembly of the mouse bile-duct tapeworm, Hymenolepis microstoma, provides chromosome-level characterisation of the genome and a stabilised set of protein coding gene models underpinned by bioinformatic and empirical data. However, interactome data have not been produced. Conserved protein-protein interactions in other organisms, termed interologs, can be used to transfer interactions between species, allowing systems-level analysis in non-model organisms., Results: Here, we describe a probabilistic, integrated network of interologs for the H. microstoma proteome, based on conserved protein interactions found in eukaryote model species. Almost a third of the 10,139 gene models in the v.3 assembly could be assigned interaction data and assessment of the resulting network indicates that topologically-important proteins are related to essential cellular pathways, and that the network clusters into biologically meaningful components. Moreover, network parameters are similar to those of single-species interaction networks that we constructed in the same way for S. cerevisiae, C. elegans and H. sapiens, demonstrating that information-rich, system-level analyses can be conducted even on species separated by a large phylogenetic distance from the major model organisms from which most protein interaction evidence is based. Using the interolog network, we then focused on sub-networks of interactions assigned to discrete suites of genes of interest, including signalling components and transcription factors, germline multipotency genes, and genes differentially-expressed between larval and adult worms. Results show not only an expected bias toward highly-conserved proteins, such as components of intracellular signal transduction, but in some cases predicted interactions with transcription factors that aid in identifying their target genes., Conclusions: With key helminth genomes now complete, systems-level analyses can provide an important predictive framework to guide basic and applied research on helminths and will become increasingly informative as new protein-protein interaction data accumulate.

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.

Tapeworms (cestodes) of the genus Hymenolepis are the causative agents of hymenolepiasis, a neglected zoonotic disease. Hymenolepis nana is the most prevalent human tapeworm, especially affecting children. The genomes of Hymenolepis microstoma and H. nana have been recently sequenced and assembled. MicroRNAs (miRNAs), a class of small non-coding RNAs, are principle regulators of gene expression at the post-transcriptional level and are involved in many different biological processes. In previous work, we experimentally identified miRNA genes in the cestodes Echinococcus, Taenia and Mesocestoides. However, current knowledge about miRNAs in Hymenolepis is limited. In this work we described for the first known time the expression profile of the miRNA complement in H. microstoma, and discovered miRNAs in H. nana. We found a reduced complement of 37 evolutionarily conserved miRNAs, putatively reflecting their low morphological complexity and parasitic lifestyle. We found high expression of a few miRNAs in the larval stage of H. microstoma that are conserved in other cestodes, suggesting that these miRNAs may have important roles in development, survival and for host-parasite interplay. We performed a comparative analysis of the identified miRNAs across the Cestoda and showed that most of the miRNAs in Hymenolepis are located in intergenic regions, implying that they are independently transcribed. We found a Hymenolepis-specific cluster composed of three members of the mir-36 family. Also, we found that one of the neighboring genes of mir-10 was a Hox gene as in most bilaterial species. This study provides a valuable resource for further experimental research in cestode biology that might lead to improved detection and control of these neglected parasites. The comprehensive identification and expression analysis of Hymenolepis miRNAs can help to identify novel biomarkers for diagnosis and/or novel therapeutic targets for the control of hymenolepiasis., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The complete mitochondrial genome of Drepanidotaenia lanceolata was sequenced. The complete mtDNA sequence is 13 573 bp long and contains 12 protein-coding genes (cox1-3, nad1-6, nad4L, atp6, and cytb), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and 2 non-coding regions (NCR). Phylogenetic analysis using three methods (Bayesian inference, maximum parsimony, and maximum likelihood) demonstrates with high statistical support that D. lanceolata is closely related to the genus Hymenolepis. This study reports the first complete mt genome sequence of a representative species of the genus Drepanidotaenia and should provide novel genetic data for further studies of the taxonomy, systematics, and population genetics of these and other related cestodes of socio-economic significance.

Synoptic data and an understanding of helminth parasite diversity among diverse rodent assemblages across temperate latitudes of North America remain remarkably incomplete. Renewed attention to comprehensive survey and inventory to establish the structure of biodiverse faunas is essential in providing indicators and proxies for identifying the outcomes of accelerating change linked to climate warming and anthropogenic forcing. Subsequent to the description of Hymenolepis folkertsi in the oldfield mouse, Peromyscus polionotus, additional specimens of hymenolepidids were collected or discovered in archived museum repositories from multiple species of deer mice (Peromyscus maniculatus, Peromyscus leucopus), the golden mouse (Ochrotomys nuttalli), chipmunks (Tamias striatus, Tamias amoenus), the 13-lined ground squirrel (Ictidomys tridecemlineatus), and tree squirrels (Sciurus carolinensis, Sciurus niger) from disjunct localities in the USA spanning southern Georgia, Virginia, Pennsylvania, Connecticut, the Upper Peninsula of Michigan, Wisconsin, and central Idaho. Specimens were largely consistent morphologically with the original description of H. folkertsi. Initial DNA sequence data, from a portion of the mitochondrial NADH dehydrogenase subunit 1, demonstrated intraspecific variation among three apparently geographically isolated populations attributed to H. folkertsi (uncorrected genetic distances of 2.7 % (Idaho and Michigan), 2.4 % (Virginia + Pennsylvania and Michigan), and 1.89 % (VA + PA and ID). Geography rather than host association explains the distribution and occurrence of H. folkertsi, and host colonization among deer mice, chipmunks, and other sciurids within regional sites is indicated. Genetic divergence revealed across localities for H. folkertsi suggests historically isolated populations, consistent with extended evolutionary and biogeographic trajectories among hymenolepidids and species of Peromyscus and Tamias in North America. Field inventory, that revealed these parasite populations, substantially alters our understanding of the distribution of diversity and provides insights about the nature of the complex relationships that serve to determine cestode faunas in rodents.

Pseudanoplocephala crawfordi is one of the important zoonotic cestodes causing economic significance and public health concern. In the present study, the phylogenetic position of P. crawfordi isolated from pigs was re-inferred using molecular markers of internal transcribed spacer ribosomal DNA (ITS rDNA) and partial NADH dehydrogenase subunit 1 (pnad1) mitochondrial DNA. The lengths of ITS1, ITS2 rDNA and pnad1 were 757 bp, 628 bp and 458 bp, respectively. Sequence differences in the ITS1, ITS2 rDNA and pnad1 between P. crawfordi and Hymenolepis species were smaller than that between cestodes within genus Hymenolepis. Phylogenetic analyses based on three gene fragments showed that P. crawfordi was grouped into cluster of Hymenolepis species. These results suggested that P. crawfordi would be one member of genus Hymenolepis but not in a new genus Pseudanoplocephala.

An adult hymenolepidid tapeworm was recovered from a 52-year-old Tibetan woman during a routine epidemiological survey for human taeniasis/cysticercosis in Sichuan, China. Phylogenetic analyses based on sequences of nuclear 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit 1 showed that the human isolate is distinct from Hymenolepis diminuta and Hymenolepis nana, the common parasites causing human hymenolepiasis. Proglottids of the human isolate were unfortunately unsuitable for morphological identification. However, the resultant phylogeny demonstrated the human isolate to be a sister species to Hymenolepis hibernia from Apodemus mice in Eurasia. The present data clearly indicate that hymenolepidid tapeworms causing human infections are not restricted to only H. diminuta and H. nana., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

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.

We carried out the first survey of Hymenolepis spp. infection in pet rodents in Italy. Fresh fecal samples were collected from 172 pet rodents as follows: guinea pigs (Cavia porcellus; n = 60), squirrels (Callosciurus finlaysonii, Callosciurus prevosti, Tamias striatus, Tamias sibiricus, Sciurus calorinensis; n = 52), hamsters (Phodopus campbelli, Mesocricetus auratus; n = 30), chinchillas (Chinchilla lanigera; n = 13), rats (Rattus norvegicus; n = 10), and mice (Mus minutoides; n = 7). These animals were housed either in pet shops or in private houses. All fecal samples were processed using the FLOTAC pellet technique to assess the number of eggs per gram (EPG) of feces. Eggs of Hymenolepis nana were found in 24 out of 172 (13.9 %; 95 % confidence interval = 9.3-20.2 %) pet rodents. Of those rodents, 41.6 % (10/24) were rats (mean EPG = 55.7; range = 2-200), 29.2 % (7/24) mice (mean EPG = 64.5; range = 32-120), 25.0 % (6/24) were chinchillas (mean EPG = 25.5; range = 10-50), and 4.2 % (1/24) hamsters (P. campbelli) (EPG = 86.0). In addition, Hymenolepis diminuta eggs were found in 2 out of 172 (1.2 %; 95 % confidence interval = 0.2-4.6 %) rodents examined, both of which (100 %; 2/2) were pet squirrels (C. prevosti) (mean EPG = 10; range = 4-16). To the authors' knowledge, this is the first report of a natural infection of H. diminuta in pet squirrels.

A previously unrecognized species of hymenolepidid cestode attributable to Hymenolepis is described based on specimens in Peromyscus polionotus, oldfield mouse, from Georgia near the southeastern coast of continental North America. Specimens of Hymenolepis folkertsi n. sp. differ from those attributed to most other species in the genus by having testes arranged in a triangle and a scolex with a prominent rostrum-like protrusion. The newly recognized species is further distinguished by the relative position and length of the cirrus sac, shape of seminal receptacle, and relative size of external seminal vesicle and seminal receptacle. Hymenolepidid cestodes have sporadically been reported among the highly diverse assemblage of Peromyscus which includes 56 distinct species in the Nearctic. Although the host genus has a great temporal duration and is endemic to the Nearctic, current evidence suggests that tapeworm faunal diversity reflects relatively recent assembly through bouts of host switching among other cricetid, murid, and geomyid rodents in sympatry.

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., (Copyright © 2013. Published by Elsevier Ltd.)

Tapeworms (Cestoda) cause neglected diseases that can be fatal and are difficult to treat, owing to inefficient drugs. Here we present an analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115- to 141-megabase genomes offer insights into the evolution of parasitism. Synteny is maintained with distantly related blood flukes but we find extreme losses of genes and pathways that are ubiquitous in other animals, including 34 homeobox families and several determinants of stem cell fate. Tapeworms have specialized detoxification pathways, metabolism that is finely tuned to rely on nutrients scavenged from their hosts, and species-specific expansions of non-canonical heat shock proteins and families of known antigens. We identify new potential drug targets, including some on which existing pharmaceuticals may act. The genomes provide a rich resource to underpin the development of urgently needed treatments and control.

Wnt genes encode secreted glycoproteins that act in cell-cell signalling to regulate a wide array of developmental processes, ranging from cellular differentiation to axial patterning. Discovery that canonical Wnt/β-catenin signalling is responsible for regulating head/tail specification in planarian regeneration has recently highlighted their importance in flatworm (phylum Platyhelminthes) development, but examination of their roles in the complex development of the diverse parasitic groups has yet to be conducted. Here, we characterise Wnt genes in the model tapeworm Hymenolepis microstoma and mine genomic resources of free-living and parasitic species for the presence of Wnts and downstream signalling components. We identify orthologs through a combination of BLAST and phylogenetic analyses, showing that flatworms have a highly reduced and dispersed complement that includes orthologs of only five subfamilies (Wnt1, Wnt2, Wnt4, Wnt5 and Wnt11) and fewer paralogs in parasitic flatworms (5-6) than in planarians (9). All major signalling components are identified, including antagonists and receptors, and key binding domains are intact, indicating that the canonical (Wnt/β-catenin) and non-canonical (planar cell polarity and Wnt/Ca(2+)) pathways are functional. RNA-Seq data show expression of all Hymenolepis Wnts and most downstream components in adults and larvae with the notable exceptions of wnt1, expressed only in adults, and wnt2 expressed only in larvae. The distribution of Wnt subfamilies in animals corroborates the idea that the last common ancestor of the Cnidaria and Bilateria possessed all contemporary Wnts and highlights the extent of gene loss in flatworms.

Research into the roles played by Hox and related homeotic gene families in the diverse and complex developmental programmes exhibited by parasitic flatworms (Platyhelminthes) can hardly be said to have begun, and thus presents considerable opportunity for new research. Although featured in some of the earliest screens for homeotic genes outside Drosophila and mice, surveys in parasitic flatworms are few in number and almost nothing is yet known of where or when the genes are expressed during ontogeny. This contrasts sharply with a significant body of literature concerning Hox genes in free-living flatworms which have long served as models for the study of regeneration and the maintenance of omnipotent cell lines. Nevertheless, available information suggests that the complement of Hox genes and other classes of homeobox-containing genes in parasitic flatworms is typical of their free-living cousins and of other members of the Lophotrochozoa. Recent work on Schistosoma combined with information on Hox gene expression in planarians indicates that at least some disruption of the clustered genomic arrangement of the genes, as well as of the strict spatial and temporal colinear patterns of expression typical in other groups, may be characteristic of flatworms. However, available data on the genomic arrangement and expression of flatworm Hox genes is so limited at present that such generalities are highly tenuous. Moreover, a basic underlying pattern of colinearity is still observed in their spatial expression patterns making them suitable as cell or region-specific markers. I discuss a number of fundamental developmental questions and some of the challenges to addressing them in relation to each of the major parasitic lineages. In addition, I present newly characterized Hox genes from the model tapeworm Hymenolepis and analyze these by Bayesian inference together with >100 Hox and ParaHox homeodomains of flatworms and select lophotrochozoan taxa, providing a phylogenetic scaffold for their identification.

A piggyBac construct carrying two green fluorescent protein (GFP)-coding sequences one driven by Bombyx mori actin gene promoter and the other by Drosophila melanogaster heat-shock protein 70 (hsp70) promoter were injected together with a nonautonomous helper plasmid containing an active piggyBac transposase gene into the posterior end of mature unfertilized eggs dissected from the ovaries of Athalia rosae (Hymenoptera: Symphyta). These injected eggs, which developed as haploid male embryos upon artificial activation, were cultured to adulthood. Of 278 injected eggs, 61 grew to G(0) haploid adult males. These G(0) haploid males were individually mated to diploid females. The progeny embryos (G(1) generation) were examined for GFP expression. Four GFP-positive embryos (from three independent G(0) matings) were obtained. Two eclosed as diploid adult G(1) females. Mature unfertilized eggs dissected from the GFP-positive G(1) diploid females were activated artificially, and the resultant embryos were examined for GFP expression, separated and cultured to adulthood (G(2) generation). The G(2) haploid embryos segregated to GFP-positive and -negative individuals. By mating the G(2) adult haploid males individually to diploid females, stocks were established in which the piggyBac construct was stably integrated into the genome, as evidenced by GFP expression and Southern blot hybridization. The piggyBac transposition occurred at its canonical target TTAA sequence. These results, which demonstrate the first successful stable transposon-mediated germline transformation in Hymenoptera, will expand the usefulness of the piggyBac vector.

Since isolates of Hymenolepis nana infecting humans and rodents are morphologically indistinguishable, the only way they can be reliably identified is by comparing the parasite in each host using molecular tools. In the current study, isolates of H. nana from rodent and human hosts from a broad geographical range were sequenced at the ribosomal first internal transcribed spacer (ITS1), the mitochondrial cytochrome c oxidase subunit 1 (C01) gene and the nuclear paramyosin gene loci. Twenty-three isolates of H. nana were sequenced at the ITS1 locus and this confirmed the existence of spacers which, although similar in length (approximately 646 bp), differed in their primary sequences which led to the separation of the isolates into 2 clusters when analysed phylogenetically. This sequence variation was not, however, related to the host of origin of the isolate, thus was not a marker of genetic distinction between H. nana from rodents and humans. Sequencing of a 444 bp fragment of the mitochondrial cytochrome c oxidase 1 gene (C01) in 9 isolates of H. nana from rodents and 6 from humans identified a phylogenetically supported genetic divergence of approximately 5% between some mouse and human isolates. This suggests that H. nana is a species complex, or 'cryptic' species (=morphologically identical yet genetically distinct). A small segment of the nuclear gene, paramyosin, (625 bp or 840 bp) was sequenced in 4 mouse and 3 human isolates of H. nana. However, this gene did not provide the level of heterogeneity required to distinguish between isolates from rodent and human hosts. From the results obtained from faster evolving genes, and the epidemiological evidence, we believe that the life-cycle of H. nana that exists in the north-west of Western Australia is likely to involve mainly 'human to human' transmission.

Rodentolepis asymmetrica (Janicki, 1904), is a common hymenolepidid cestode recorded in several vole species (rodents) in the Palearctic. Here, we report a detailed analysis of this species, which includes metrical features and multilocus enzyme electrophoresis. Worms isolated from 4 species of arvicolid hosts in 3 localities in Spain and France from 1994 to 1997 were studied. All the worms used in the morphological study ranged between I and 5 individuals per host. Furthermore, all individuals were analyzed electrophoretically. Statistical analysis of metrical features in scolex, sexual segments, and eggs was carried out, and significant differences were detected only in sexual structures of mature segments. These differences were found in worms from each host species in different localities and in the same host species in 2 localities. Multivariate statistical analysis shows correct classification of worms in all cases. Surprisingly, we observed a lack of genetic variability at the 11 enzymatic loci analyzed, which could be explained by 2 nonexclusive hypotheses: (1) a preferential selfing mode of reproduction for these parasites, and (2) a weak effective size of parasite populations.

The two related species, Rodentolepis straminea (Goeze, 1782) and Rodentolepis microstoma (Dujardin, 1845) (Cestoda, Hymenolepididae), both parasites of rodents, were compared morphologically and electrophoretically. Adult worms were isolated from three wild rodent species of the family Muridae (Apodemus flavicollis, Apodemus sylvaticus, and Mus musculus) from three different sites in Spain and France. Although these two species were strikingly similar in morphological appearance, some of the morphological and metrical features analysed (scolex, mature segments and eggs) can be used for differentiation. Fixed allelic differences were found. Of the ten enzymes detected by starch-gel electrophoresis, six (AAT, AK, GPI, MDH, NP, PGM) showed characteristic isoenzyme profiles in each species. Only in MPI, PEPC, PEPD, and ME enzyme loci were no differences found. The study revealed that the two taxa can be clearly differentiated.

Using "long-PCR," we amplified in overlapping fragments the complete mitochondrial genome of the tapeworm Hymenolepis diminuta (Platyhelminthes: Cestoda) and determined its 13,900-nt sequence. The gene content is the same as that typically found for animal mitochondrial DNA (mtDNA) except that atp8 appears to be lacking, a condition found previously for several other animals. Despite the small size of this mtDNA, there are two large noncoding regions, one of which contains 13 repeats of a 31-nt sequence and a potential stem-loop structure of 25 bp with an 11-member loop. Large potential secondary structures were identified also for the noncoding regions of two other cestode mtDNAS: Comparison of the mitochondrial gene arrangement of H. diminuta with those previously published supports a phylogenetic position of flatworms as members of the Eutrochozoa, rather than placing them basal to either a clade of protostomes or a clade of coelomates.

The karyotype of glirid tapeworm Rodentolepis myoxi (Rudolphi, 1819) (Cestoda: Hymenolepididae) comprises six pairs of small bi-armed chromosomes (2n = 12). All pairs of chromosomes possess uniform morphology, i.e. metacentric, submetacentric or meta-submetacentric types of structures. The formula of the karyotype structure is n = 2m + 1m-sm + 3sm. The absolute chromosome length ranges from 3.78 to 2.00 microm. The mean total length of the haploid complement is 15. 98 microm. The first pair (group A) is the largest, pairs 2 and 3 can be grouped into group B while pairs 4-6 are smaller and can be classified as group C. The number of chromosomes of R. myoxi is the same for the congeneric species, however, karyological characteristics differ from all recently known karyotypes of rodent hymenolepidids.

Three hymenolepidid tapeworms, Hymenolepis diminuta, H. nana and H. microstoma, are commonly maintained in laboratory rodents and used in many experimental model systems of tapeworm infections. We examined partial sequences from the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences to infer phylogenetic relationships of the 3 hymenolepidid species. Parts of the CO1 gene and ITS2 were amplified by PCR and sequenced directly. The CO1 gene sequence obtained was the same in length (391 bp) among all specimens. In the case of ITS2, however, several insertions and deletions were detected (671-741 bp) not only among species but also between an American isolate and a Japanese isolate of H. diminuta. Percentage nucleotide differences between H. diminuta and H. microstoma, or H. diminuta and H. nana were 16.6-18.2% for the CO1 gene and 21.3-22.9% for ITS2. The differences in both sequences between H. microstoma and H. nana were about 14%. Phylogenetic trees inferred from both of the nucleotide sequences showed similar topology, and suggest that H. diminuta may have diverged from the common ancestral line the earliest, and that H. nana is closer to H. microstoma than to H. diminuta.

Three groups of tapeworms obtained 2.5 month after the administration to rats of a dose of 6 cysticercoids proceeding from oncospheres of the 24th generation of Hymenolepis diminuta were compared. Group I came from the oncospheres of only one proglottid of H. diminuta WMS "strain" tapeworm. Group II proceeded from 6 proglottids obtained each from another tapeworm of this "strain". Group III came from 6 proglottids, each from another tapeworm of H. diminuta WMS inbred line 1 (il1). The average number of type 0p3a proglottids in group I and II was similar and amounted to 4.0% and 4.7%, respectively, while in group III it was significantly higher than in the first two groups and reached an average of 8.4% (P < 0.01). There are also other significant differences between the two breeding lines-H. diminuta WMS "strain" and WMS il1 with respect to the number of typical 1p2a proglottids, type 1p3a proglottids, the second after 0p3a most common deviation from the typical 1p2a as well as all deviations taken together. This points to the influence of different breeding condition: non-inbred in case of H. diminuta WMS "strain" and inbred in case of H. diminuta WMS il1 on the investigated features.

The investigations focused on tapeworms obtained as a result of the administration to individual rats a dose of 6 cysticercoids coming from the 39th and 40th generation of one-specimen inbred Hymenolepis diminuta-WMS il1. In 7.5-month old tapeworms the position of genital pores (PGP) was exclusively right-hand side with the genital ducts typical of this feature. Type Op3a proglottids were the most common deviation from the typical number and topography of testes-1p2a (one testis between the ovary and the genital pore marking the poral side and two testes on the aporal side)-in such tapeworms. Their mean incidence amounted to 8.9%, exceeding by at least 2.9% the mean quantitative value of this feature in tapeworms proceeding from non-inbred H. diminuta "strains" which have been subjected to similar studies. 19.5-month old tapeworms, exactly a year older than the previous ones, all had variable PGP. The number of PGP changes was here correlated positively with the number of type 2p1a proglottids and negatively with the number of type 1p2a and 0p3a proglottids.

Variable position of genital ducts and pores (PGP) was found in 23.3% of five months old tapeworms obtained as a result of administering to rats a dose of 6 cysticercoids proceeding from the third generation of inbred Hymenolepis diminuta--WMS il1. In tapeworms coming from generations no 18 and 32 PGP changes were reported only in 7.5 month, with only 5.9% of strobilas in the 32 generation group being characterized by this feature. These results indicate a time shift with respect to the appearance of PGP changes in tapeworms proceeding from latter inbred generations to a further period of tapeworm invasion in rats. The number of PGP changes is positively correlated with the number of type 2p1a proglottids (two testes on the poral and one on the aporal side). The average number of type Op3a proglottids increases with the number of generations. In 5-month old tapeworms of unilateral PGP coming from generation no 3, 18 and 32 it amounts to 5.9%, 6.6% and 8.1%, respectively.

The karyotypes of Vampirolepis nana, Hymenolepis diminuta and Vampirolepis erinacei (Cestoda: Cyclophyllidea) have been described. The number of chromosomes in the diploid cell of the studied taxa was 2n = 12. The absolute and the relative lengths of the chromosomes of V. nana and H. diminuta were measured. The chromosomes had similar metric values. In the karyotypes of V. nana and H. diminuta there were ten one-arm chromosomes and one two-arm chromosome (the third in length in the karyotype of V. nana and the first in length in the karyotype of H. diminuta). The chromosomes in the karyotype of V. erinacei had one arm which was proved by the terminal localization of the constitutive heterochromatin. The examined species differed in the morphology of some chromosomes in the karyotypes.

The technique of allozyme electrophoresis was applied to three strains of Hymenolepis diminuta to distinguish between three hypotheses [(1) multiple species, (2) genetically distinct founder stocks and (3) response to differential selection among similar stocks] proposed to account for metabolic differences among strains. There was no evidence from the 39 enzyme loci established that the three strains represented more than one species. In the absence of knowledge of the population structure of H. diminuta in the wild, electrophoretic data herein could not distinguish between the latter hypotheses. Nevertheless, all three strains were distinguishable on electrophoretic profiles and allelic similarities between strains question the view of their proposed origins.

This study is the first demonstration of individual gene products synthesized by Hymenolepis diminuta. To identify proteins synthesized specifically by H. diminuta, we radiolabeled adult worms with [14C]leucine in vitro. Subsequent one- and two-dimensional gel electrophoretic separation and staining of the protein constituents from lysates of whole worms, brush border denuded worms, and brush border fractions extracted from whole worms demonstrates the diversity of the proteins in each of these lysates. Fluorographic analysis of the radioactivity in the same polyacrylamide gels identified the protein constituents synthesized by H. diminuta. The results from this study disclose and characterize, by molecular weight and isoelectric point, 17 polypeptides which are synthesized by H. diminuta and associated with its surface brush border.

The nucleotide sequence of the 5' ends of the 28S-like rRNA molecules of five species of helminths was determined directly, using a variation on the dideoxynucleotide chain-termination method which requires only 10 micrograms of total cellular RNA for analysis. Nucleotide sequence comparisons over 208 bases allowed the phylogeny of these organisms to be determined. The data show that the rRNA sequence of Nematospiroides dubius, a nematode, is as divergent from that of two platyhelminths, Hymenolepis diminuta and Schistosoma mansoni, as it is from the rRNA sequence of the two nematodes Onchocerca gibsoni and Brugia pahangi. The latter two appear to be very closely related, whereas the two platyhelminths are more distant from each other. The study demonstrates the usefulness and generality of rRNA sequencing for the systematic phylogenetic classification of parasitic organisms whose tissues are only available in relatively small amounts.

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.

Biological characteristics of infectivity, growth rate and fecundity of Hymenolepis diminuta isolated from wild Rattus rattus in Japan were compared with parasites of Texas origin maintained for several generations in this and many other laboratories in laboratory bred Rattus norvegicus. The timing of development and maturation was similar in parasites from both sources, but the mean parasite dry weight was less and the mean egg production lower in Japanese parasites in both single and multiple infections. The differences persisted over 10 weeks in single infections, and were unaffected by rat strain. In all experiments there was much greater variation and heterogeneity in the biological characteristics of the Japanese parasites. It was concluded that the Texas parasites were better adapted to R. norvegicus and in the course of adaptation had become more homogeneous, and that the difference between the parasite strains had a complex genetic basis. Some Japanese parasites of the early isolated generations failed to produce normal eggs, but the proportion of these decreased in later generations. However, selected individual Japanese parasites whose egg production was equal to that of parasites of Texas origin did not produce offspring of larger mean size or higher mean fecundity, and heterogeneity of these characteristics was maintained. In the intermediate host, the mean number of cysticercoids of the Japanese isolate per beetle was higher than that of the Texas strain in Tribolium confusum but lower in Tenebrio molitor. The greater heterogeneity exhibited by Japanese parasites and the genetic basis of the strain difference are discussed with particular reference to geographical isolation, differences in definitive hosts and co-evolution of wild and laboratory host and parasite populations.