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The phylum Platyhelminthes shares a unique population of undifferentiated cells responsible for the proliferation capacity needed for cell renewal, growth, tissue repair and regeneration. These cells have been extensively studied in free-living flatworms, whereas in cestodes the presence of a set of undifferentiated cells, known as germinative cells, has been demonstrated in classical morphology studies, but poorly characterized with molecular biology approaches. Furthermore, several genes have been identified as neoblast markers in free-living flatworms that deserve study in cestode models. Here, different cell types of the model cestode

Many anthelmintics target the neuromuscular system, in particular by interfering with signaling mediated by classical neurotransmitters. Although peptidergic signaling has been proposed as a novel target for anthelmintics, current knowledge of the neuropeptide complement of many helminth groups is still limited, especially for parasitic flatworms (cestodes, trematodes, and monogeneans). In this work, we have characterized the neuropeptide complement of the model cestode Hymenolepis microstoma. Peptidomic characterization of adults of H. microstoma validated many of the neuropeptide precursor (npp) genes previously predicted in silico, and identified novel neuropeptides that are conserved in parasitic flatworms. Most neuropeptides from parasitic flatworms lack significant similarity to those from other animals, confirming the uniqueness of their peptidergic signaling. Analysis of gene expression of ten npp genes by in situ hybridization confirmed that all of them are expressed in the nervous system and identified cryptic features, including the first evidence of dorsoventral asymmetry, as well as a new population of peripheral peptidergic cells that appears to be conserved in the trematode Schistosoma mansoni. Finally, we characterized in greater detail Attachin, an SIFamide homolog. Although its expression is largely restricted to the longitudinal nerve cords and cerebral commissure in H. microstoma, it shows widespread localization in the larval nervous system of Echinococcus multilocularis and Mesocestoides corti. Exogenous addition of a peptide corresponding to the highly conserved C-terminus of Attachin stimulated motility and attachment of M. corti larvae. Altogether, this work provides a robust experimental foothold for the characterization of peptidergic signaling in parasitic flatworms. Cover Image for this issue: https://doi.org/10.1111/jnc.15418.

Echinococcus granulosus, the etiological agent of human cystic echinococcosis (formerly known as hydatid disease), represents a serious worldwide public health problem with limited treatment options. The essential role played by the neuromuscular system in parasite survival and the relevance of serotonin (5-HT) in parasite movement and development, makes the serotonergic system an attractive source of drug targets. In this study, we cloned and sequenced a cDNA coding for the serotonin transporter from E. granulosus (EgSERT). Bioinformatic analyses suggest that EgSERT has twelve transmembrane domains with highly conserved ligand and ionic binding sites but a less conserved allosteric site compared with the human orthologue (HsSERT). Modeling studies also suggest a good degree of conservation of the overall structure compared with HsSERT. Functional and pharmacological studies performed on the cloned EgSERT confirm that this protein is indeed a serotonin transporter. EgSERT is specific for 5-HT and does not transport other neurotransmitters. Typical monoamine transport inhibitors also displayed inhibitory activities toward EgSERT, but with lower affinity than for the human SERT (HsSERT), suggesting a high divergence of the cestode transporter compared with HsSERT. In situ hybridization studies performed in the larval protoscolex stage suggest that EgSERT is located in discrete regions that are compatible with the major ganglia of the serotonergic nervous system. The pharmacological properties, the amino acidic substitutions at important functional regions compared with the HsSERT, and the putative role of EgSERT in the nervous system, suggest that it could be an important target for pharmacological intervention.

White adipose tissue hyperplasia has been shown to be crucial for handling excess energy in healthy ways. Though adipogenesis mechanisms have been underscored in vitro, we lack information on how tissue and systemic factors influence the differentiation of new adipocytes. While this could be studied in zebrafish, adipocyte identification currently relies on neutral lipid labeling, thus precluding access to cells in early stages of differentiation. Here we report the generation and analysis of a zebrafish line with the transgene fabp4a(-2.7):EGFPcaax. In vivo confocal microscopy of the pancreatic and abdominal visceral depots of transgenic larvae, revealed the presence of labeled mature adipocytes as well as immature cells in earlier stages of differentiation. Through co-labeling for blood vessels, we observed a close interaction of differentiating adipocytes with endothelial cells through cell protrusions. Finally, we implemented hyperspectral imaging and spectral phasor analysis in Nile Red-labeled transgenic larvae and revealed the lipid metabolic transition towards neutral lipid accumulation of differentiating adipocytes. Altogether our work presents the characterization of a novel adipocyte-specific label in zebrafish and uncovers previously unknown aspects of in vivo adipogenesis. This article has an associated First Person interview with the first author of the paper., Summary: Analysis of the differentiation of adipocytes in vivo through cell-specific labeling in zebrafish, revealed their early interaction with blood vessels as well as early lipid metabolic changes.

Parasitic flatworms have complex neuromuscular systems that serve important functions in their life cycles. However, our understanding of neurotransmission in parasitic flatworms is limited. Pioneering studies have suggested the presence of several classical neurotransmitter systems, but their molecular components have not been characterized in most cases. Because these components are conserved in bilaterian animals, we searched the genomes of parasitic flatworms for orthologs of genes required for neurotransmitter synthesis, vesicular transport, reuptake, and reception. Our results indicate that tapeworms have lost the genes that are specifically required in other animals for synaptic signaling using the classical neurotransmitters dopamine, tyramine, octopamine, histamine and gamma-aminobutyric acid (GABA). These results imply that these signaling pathways are either absent in these parasites, or that they require completely different molecular components in comparison with other animals. The orthologs of genes related to histaminergic and GABA signaling are also missing in trematodes (although Schistosoma-specific histaminergic receptors have been previously described). In contrast, conserved genes required for glutamatergic, serotonergic and cholinergic signaling could be found in all analyzed flatworms. We analyzed the expression of selected markers of each pathway in the tapeworm Hymenolepis microstoma by whole-mount in situ hybridization. Each marker was specifically expressed in the nervous system, although with different patterns. In addition, we analyzed the expression of proprotein convertase 2 as a marker of peptidergic cells. This gene showed the widest expression in the nervous system, but was also expressed in other tissues, suggesting additional roles of peptidergic signaling in tapeworm development and reproduction.

Cestodes are platyhelminth parasites with a wide range of hosts that cause neglected diseases. Neurotransmitter signaling is of critical importance for these parasites which lack circulatory, respiratory and digestive systems. For example, serotonin (5-HT) and serotonergic G-protein coupled receptors (5-HT GPCRs) play major roles in cestode motility, development and reproduction. In previous work, we deorphanized a group of 5-HT7 type GPCRs from cestodes. However, little is known about another type of 5-HT GPCR, the 5-HT1 clade, which has been studied in several invertebrate phyla but not in platyhelminthes. Three putative 5-HT GPCRs from Echinococcus canadensis, Mesocestoides vogae (syn. M. corti) and Hymenolepis microstoma were cloned, sequenced and bioinformatically analyzed. Evidence grouped these new sequences within the 5-HT1 clade of GPCRs but differences in highly conserved GPCR motifs were observed. Transcriptomic analysis, heterologous expression and immunolocalization studies were performed to characterize the E. canadensis receptor, called Eca-5-HT1a. Functional heterologous expression studies showed that Eca-5-HT1a is highly specific for serotonin. 5-Methoxytryptamine and α-methylserotonin, both known 5-HT GPCR agonists, give stimulatory responses whereas methysergide, a known 5-HT GPCR ligand, give an antagonist response in Eca-5-HT1a. Mutants obtained by the substitution of key predicted residues resulted in severe impairment of receptor activity, confirming that indeed, these residues have important roles in receptor function. Immunolocalization studies on the protoscolex stage from E. canadensis, showed that Eca-5-HT1a is localized in branched fibers which correspond to the nervous system of the parasite. The patterns of immunoreactive fibers for Eca-5-HT1a and for serotonin were intimately intertwined but not identical, suggesting that they are two separate groups of fibers. These data provide the first functional, pharmacological and localization report of a serotonergic receptor that putatively belongs to the 5-HT1 type of GPCRs in cestodes. The serotonergic GPCR characterized here may represent a new target for antiparasitic intervention.

Racemose neurocysticercosis is an aggressive disease caused by the aberrant expansion of the cyst form of Taenia solium within the subarachnoid spaces of the human brain and spinal cord resulting in a mass effect and chronic inflammation. Although expansion is likely caused by the proliferation and growth of the parasite bladder wall, there is little direct evidence of the mechanisms that underlie these processes. Since the development and growth of cysts in related cestodes involves totipotential germinative cells, we hypothesized that the expansive growth of the racemose larvae is organized and maintained by germinative cells. Here, we identified proliferative cells expressing the serine/threonine-protein kinase plk1 by in situ hybridization. Proliferative cells were present within the bladder wall of racemose form and absent from the homologous tissue surrounding the vesicular form. Cyst proliferation in the related model species Taenia crassiceps (ORF strain) occurs normally by budding from the cyst bladder wall and proliferative cells were concentrated within the growth buds. Cells isolated from bladder wall of racemose larvae were established in primary cell culture and insulin stimulated their proliferation in a dose-dependent manner. These findings indicate that the growth of racemose larvae is likely due to abnormal cell proliferation. The different distribution of proliferative cells in the racemose larvae and their sensitivity to insulin may reflect significant changes at the cellular and molecular levels involved in their tumor-like growth. Parasite cell cultures offer a powerful tool to characterize the nature and formation of the racemose form, understand the developmental biology of T. solium, and to identify new effective drugs for treatment., Author summary Racemose neurocysticercosis is the most aggressive and lethal form of the disease characterized by progressive expansion of the larval stage of Taenia solium within the subarachnoid spaces of the brain. Currently, the cellular or molecular events that promote its formation and development are unknown. Our observations, employing tissue samples, revealed the presence of mitotically active cells in the bladder wall of the racemose larvae. Finally, we isolated and established primary cell cultures and observed that these cells are sensitive (proliferate in response to insulin). These results suggest that the proliferative cells stimulated by host biomolecules like hormones would be responsible for the abnormal growth of racemose larvae. Further studies are needed to identify all genes and pathways altered in the racemose form. The primary cell cultures will be used as a therapeutic target and allow us in future works to understand the developmental biology of the parasite.

The neglected zoonotic disease alveolar echinococcosis (AE) is caused by the metacestode stage of the tapeworm parasite Echinococcus multilocularis. MicroRNAs (miRNAs) are small non-coding RNAs with a major role in regulating gene expression in key biological processes. We analyzed the expression profile of E. multilocularis miRNAs throughout metacestode development in vitro, determined the spatial expression of miR-71 in metacestodes cultured in vitro and predicted miRNA targets. Small cDNA libraries from different samples of E. multilocularis were sequenced. We confirmed the expression of 37 miRNAs in E. multilocularis being some of them absent in the host, such as miR-71. We found a few miRNAs highly expressed in all life cycle stages and conditions analyzed, whereas most miRNAs showed very low expression. The most expressed miRNAs were miR-71, miR-9, let-7, miR-10, miR-4989 and miR-1. The high expression of these miRNAs was conserved in other tapeworms, suggesting essential roles in development, survival, or host-parasite interaction. We found highly regulated miRNAs during the different transitions or cultured conditions analyzed, which might suggest a role in the regulation of developmental timing, host-parasite interaction, and/or in maintaining the unique developmental features of each developmental stage or condition. We determined that miR-71 is expressed in germinative cells and in other cell types of the germinal layer in E. multilocularis metacestodes cultured in vitro. MiRNA target prediction of the most highly expressed miRNAs and in silico functional analysis suggested conserved and essential roles for these miRNAs in parasite biology. We found relevant targets potentially involved in development, cell growth and death, lifespan regulation, transcription, signal transduction and cell motility. The evolutionary conservation and expression analyses of E. multilocularis miRNAs throughout metacestode development along with the in silico functional analyses of their predicted targets might help to identify selective therapeutic targets for treatment and control of AE., Author summary Alveolar echinococcosis (AE) is a zoonotic disease caused by the metacestode stage of the helminth parasite Echinococcus multilocularis. Current treatment requires surgery and/or prolonged drug therapy. Thus, novel strategies for the treatment of AE are needed. MicroRNAs (miRNAs), a class of small ~22-nucleotide (nt) non-coding RNAs with a major role in regulating gene expression, have been suggested as potential therapeutic targets for treatment and control of helminth parasite infections. In this work, we analyzed the expression profile of E. multilocularis miRNAs throughout metacestode development in vitro. We predicted functional roles for highly expressed miRNAs and found that they could be involved in essential roles for survival and development in the host. We determined that E. multilocularis miR-71, a highly expressed miRNA that is absent in the human host, is expressed in germinative cells and in other cell types of the germinal layer in E. multilocularis metacestodes cultured in vitro. Germinative cells are a relevant cell type to target for anti-echinococcosis drug development. MiRNAs that are absent in the human host, involved in essential functions, highly expressed and/or expressed in germinative cells in E. multilocularis metacestodes may represent selective therapeutic targets for treatment and control of AE.

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.

In situ hybridization (ISH) is a technique used for the spatial localization of nucleic acids within tissues and cells. It is based on the ability of labeled nucleic acids (probes) to hybridize under the right conditions with the nucleic acids present in fixed biological specimens. In this chapter, we describe protocols for detection of RNA by ISH using digoxigenin (DIG)-labeled probes for Fasciola hepatica adults (in cryosections, given their large size) and for newly excysted juveniles (NEJs, which are ideally suited given their small size for whole-mount ISH). We describe fluorogenic and chromogenic protocols, respectively, but the detection methods can be easily interchanged by using the appropriate enzyme-conjugated antibodies and detection solutions.

Background Spliced Leader trans-splicing is an important mechanism for the maturation of mRNAs in several lineages of eukaryotes, including several groups of parasites of great medical and economic importance. Nevertheless, its study across the tree of life is severely hindered by the problem of identifying the SL sequences that are being trans-spliced. Results In this paper we present SLFinder, a four-step pipeline meant to identify de novo candidate SL sequences making very few assumptions regarding the SL sequence properties. The pipeline takes transcriptomic de novo assemblies and a reference genome as input and allows the user intervention on several points to account for unexpected features of the dataset. The strategy and its implementation were tested on real RNAseq data from species with and without SL Trans-Splicing. Conclusions SLFinder is capable to identify SL candidates with good precision in a reasonable amount of time. It is especially suitable for species with unknown SL sequences, generating candidate sequences for further refining and experimental validation.

The oncosphere larvae of tapeworms cyclically extend and retract their hooks during the penetration of the intestine of their intermediate hosts. The mechanisms regulating these movements are essentially unknown, in part due to the biohazardous nature of oncospheres from human pathogens. In this work, we standardized a method for the analysis of motility of hatched oncospheres (hexacanths) of the model tapeworm Hymenolepis microstoma. We used this assay to explore the relevance of protein kinases C (PKC) and A (PKA) in these processes. Pharmacological inhibition of the PKC pathway resulted in impaired larval motility. On the other hand, the PKA inhibitor H-89 potently blocked larval motility, as well as the motility of other life stages, although other inhibitors of the PKA pathway were not effective. This work represents the first study of the mechanisms that regulate the motility of oncospheres, and provides a path for further exploration.

Neuropeptide mediated signalling is an ancient mechanism found in almost all animals and has been proposed as a promising target for the development of novel drugs against helminths. However, identification of neuropeptides from genomic data is challenging, and knowledge of the neuropeptide complement of parasitic flatworms is still fragmentary. In this work, we have developed an evolution-based strategy for the de novo discovery of neuropeptide precursors, based on the detection of localised sequence conservation between possible prohormone convertase cleavage sites. The method detected known neuropeptide precursors with good precision and specificity in the models Drosophila melanogaster and Caenorhabditis elegans. Furthermore, it identified novel putative neuropeptide precursors in nematodes, including the first description of allatotropin homologues in this phylum. Our search for neuropeptide precursors in the genomes of parasitic flatworms resulted in the description of 34 conserved neuropeptide precursor families, including 13 new ones, and of hundreds of new homologues of known neuropeptide precursor families. Most neuropeptide precursor families show a wide phylogenetic distribution among parasitic flatworms and show little similarity to neuropeptide precursors of other bilaterian animals. However, we could also find orthologs of some conserved bilaterian neuropeptides including pyrokinin, crustacean cardioactive peptide, myomodulin, neuropeptide-Y, neuropeptide KY and SIF-amide. Finally, we determined the expression patterns of seven putative neuropeptide precursor genes in the protoscolex of Echinococcus multilocularis. All genes were expressed in the nervous system with different patterns, indicating a hidden complexity of peptidergic signalling in cestodes.

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.

Background Alveolar echinococcosis (AE) is a lethal zoonosis caused by the metacestode larva of the tapeworm Echinococcus multilocularis. The infection is characterized by tumour-like growth of the metacestode within the host liver, leading to extensive fibrosis and organ-failure. The molecular mechanisms of parasite organ tropism towards the liver and influences of liver cytokines and hormones on parasite development are little studied to date. Methodology/Principal findings We show that the E. multilocularis larval stage expresses three members of the fibroblast growth factor (FGF) receptor family with homology to human FGF receptors. Using the Xenopus expression system we demonstrate that all three Echinococcus FGF receptors are activated in response to human acidic and basic FGF, which are present in the liver. In all three cases, activation could be prevented by addition of the tyrosine kinase (TK) inhibitor BIBF 1120, which is used to treat human cancer. At physiological concentrations, acidic and basic FGF significantly stimulated the formation of metacestode vesicles from parasite stem cells in vitro and supported metacestode growth. Furthermore, the parasite’s mitogen activated protein kinase signalling system was stimulated upon addition of human FGF. The survival of metacestode vesicles and parasite stem cells were drastically affected in vitro in the presence of BIBF 1120. Conclusions/Significance Our data indicate that mammalian FGF, which is present in the liver and upregulated during fibrosis, supports the establishment of the Echinococcus metacestode during AE by acting on an evolutionarily conserved parasite FGF signalling system. These data are valuable for understanding molecular mechanisms of organ tropism and host-parasite interaction in AE. Furthermore, our data indicate that the parasite’s FGF signalling systems are promising targets for the development of novel drugs against AE., Author summary To ensure proper communication between their different cell populations, animals rely on secreted hormones and cytokines that act on receptors of target cells. Most of the respective cytokines, such as FGFs, evolved over 500 million years ago and are present in similar form in all animals, including parasitic worms. The authors of this study show that the metacestode larva of the tapeworm E. multilocularis, which grows like a malignant tumor within the host liver, expresses molecules with homology to FGF receptors from mammals. The authors show that human FGF, which is abundantly present in the liver, stimulates metacestode development and that all parasite FGF receptors are activated by human FGF, despite 500 million years of evolutionary distance between both systems. This indicates that cells of the Echinococcus metacestode can directly communicate with cells of the mammalian host using evolutionarily conserved signaling molecules. This mode of host-pathogen interaction is unique for helminths and does not occur between mammals and single-celled pathogens such as protozoans or bacteria. The authors finally demonstrate that BIBF 1120, a drug used to treat human cancer, targets the Echinococcus FGF receptors and leads to parasite death. This opens new ways for the development of anti-parasitic drugs.

The larval stages of the cestodes Echinococcus multilocularis and Echinococcus granulosus cause the important zoonoses known as larval echinococcoses. These larvae are protected by a unique, massive, mucin-based structure known as the laminated layer. The mucin glycans of the E. granulosus laminated layer are core 1- or core 2-based O-glycans in which the core Galpβ1-3 residue can initiate a chain comprising one to three additional Galpβ1-3 residues, a motif not known in mammalian carbohydrates. This chain can be capped with a Galpα1-4 residue, and can be ramified with GlcNAcpβ1-6 residues. These, as well as the GlcNAcpβ1-6 residue in core 2, can be decorated with the Galpα1-4Galpβ1-4 disaccharide. Here we extend our analysis to the laminated layer of E. multilocularis, showing that the non-decorated cores, together with Galpβ1-3(Galpα1-4Galpβ1-4GlcNAcpβ1-6)GalNAc, comprise over 96% of the glycans in molar terms. This simple laminated layer glycome is exhibited by E. multilocularis grown either in vitro or in vivo. Interestingly, all the differences with the complex laminated layer glycome found in E. granulosus may be explained in terms of strongly reduced activity in E. multilocularis of a putative glycosyltransferase catalysing the elongation with Galpβ1-3. Comparative inter-species analysis of available genomic and transcriptomic data suggested a candidate for this enzyme, amongst more than 20 putative (non-core 1) Gal/GlcNAc β1-3 transferases present in each species as a result of a taeniid-specific gene expansion. The candidate gene was experimentally verified to be transcribed at much higher levels in the larva of E. granulosus than that of E. multilocularis.

Tardigrades are a key group for understanding the evolution of the Ecdysozoa, a large clade of molting animals that also includes arthropods and nematodes. However, little is known about most aspects of their basic biology. Neuropeptide and peptide hormone signaling has been extensively studied in arthropods and nematodes (particularly regarding their roles in molting in arthropods), but very little is known about neuropeptide signaling in other ecdysozoans. In this work, different strategies were used to search for neuropeptide and peptide hormone precursors in the genomes of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus. In general, there is a remarkable similarity in the complement of neuropeptides and their sequences between tardigrades and arthropods. The precursors found in tardigrades included homologs of achatin, allatostatins A, B and C, allatotropin, calcitonin, CCHamide, CCRFa, corazonin, crustacean cardioactive peptide, diuretic hormone 31, diuretic hormone 44, ecdysis triggering hormone, eclosion hormone, gonadotropin-releasing hormone (GnRH), GSEFLamide, insulin-like peptides, ion transport peptide, kinin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, RYamide, short neuropeptide F, sulfakinin, tachykinin, trissin and vasopressin. In most cases, homologs of known cognate receptors for each neuropeptide family could only be identified when the precursors were also present in the genome, further supporting their identification. Some neuropeptide precursor genes have undergone several duplications in tardigrades, including allatostatin A and C, corazonin, GnRH, eclosion hormone, sulfakinin and trissin. Furthermore, four novel families of candidate neuropeptide precursors were identified (two of which could also be found in several arthropods). To the best of my knowledge, this work represents the first genome-wide search for neuropeptide precursors in any ecdysozoan species outside arthropods and nematodes, and is a necessary first step towards understanding neuropeptide function in tardigrades.

Taeniid cestodes (including the human parasites Echinococcus spp. and Taenia solium) have very few mobile genetic elements (MGEs) in their genome, despite lacking a canonical PIWI pathway. The MGEs of these parasites are virtually unexplored, and nothing is known about their expression and silencing. In this work, we report the discovery of a novel family of small nonautonomous long terminal repeat retrotransposons (also known as terminal-repeat retrotransposons in miniature, TRIMs) which we have named ta-TRIM (taeniid TRIM). ta-TRIMs are only the second family of TRIM elements discovered in animals, and are likely the result of convergent reductive evolution in different taxonomic groups. These elements originated at the base of the taeniid tree and have expanded during taeniid diversification, including after the divergence of closely related species such as Echinococcus multilocularis and Echinococcus granulosus. They are massively expressed in larval stages, from a small proportion of full-length copies and from isolated terminal repeats that show transcriptional read-through into downstream regions, generating novel noncoding RNAs and transcriptional fusions to coding genes. In E. multilocularis, ta-TRIMs are specifically expressed in the germinative cells (the somatic stem cells) during asexual reproduction of metacestode larvae. This would provide a developmental mechanism for insertion of ta-TRIMs into cells that will eventually generate the adult germ line. Future studies of active and inactive ta-TRIM elements could give the first clues on MGE silencing mechanisms in cestodes.

Surveillance by RNA interference is central to controlling the mobilization of transposable elements (TEs). In stem cells, Piwi argonaute (Ago) proteins and associated proteins repress mobilization of TEs to maintain genome integrity. This defense mechanism targeting TEs is termed the Piwi-interacting RNA (piRNA) pathway. In this opinion article, we draw attention to the situation that the genomes of cestodes and trematodes have lost the piwi and vasa genes that are hallmark characters of the germline multipotency program. This absence of Piwi-like Agos and Vasa helicases prompts the question: how does the germline of these flatworms withstand mobilization of TEs? Here, we present an interpretation of mechanisms likely to defend the germline integrity of parasitic flatworms.

Searching for hsp70 genes in Echinococcus granulosus, a divergent cytoplasmic hsp70-like sequence (EgpsiHsp70) was isolated, possessing a small truncation in the region coding for the C-terminal glycine-rich linker and EEVD-Ct motif. Southern Blot analyses of E. granulosus, and in silico analyses of E. multilocularis indicate that this truncated sequence is repeated several times in both genomes, in some cases containing clear cut features of pseudogenization. Phylogenetic analyses and comparison of surrounding regions indicate that all these copies originated by successive genomic duplications of one originally truncated copy. These copies are diverging at an increased rate compared to functional cytoplasmic hsp70 genes, and ratios of non-synonymous over synonymous substitutions rates (dN/dS) point to a relaxation of sequence constraint, suggesting that these sequences are pseudogenes. Interestingly, RT-PCR demonstrates that EgpsiHsp70 is transcribed in protoscoleces and adult individuals of E. granulosus. We suggest that this sequence does not code for a functional polypeptide, although some features are unexpected for a sequence evolving under a strictly neutral mode. Transcription could either be vestigial or have a specific, non-coding function.

Little is known about the Hox gene complement in parasitic platyhelminthes (Neodermata). With the aim of identifying Hox genes in this group we performed two independent strategies: we performed a PCR survey with degenerate primers directed to the Hox homeobox in the cestode Mesocestoides corti, and we searched genomic assemblies of Echinococcus multilocularis and Schistosoma mansoni. We identified two Hox genes in M. corti, seven in E. multilocularis, and nine in S. mansoni (including five previously reported). The affinities of these sequences, and other previously reported Hox sequences from flatworms, were determined according to phylogenetic analysis, presence of characteristic parapeptide sequences, and unusual intron positions. Our results suggest that the last common ancestor of triclads and neodermatans had a Hox gene complement of at least seven genes, and that this was probably derived by gene loss from a larger ancestral Hox complement in lophotrochozoans.

Pumilio proteins are proposed to have a conserved primordial function in the maintenance of proliferation in stem cells through post-transcriptional regulation. In this work, a search for pumilio homology domain (PUM-HD) sequences of pumilio genes from several Platyhelminthes species was performed, including representatives form Cestoda, Trematoda and Tricladida. Only one PUM-HD sequence was found in each triclad species; however, two PUM-HD homologues were found in all the parasitic species. These sequences formed two clearly separated clades: PlatyPum1, with sequences from all species, and PlatyPum2, composed exclusively of neodermatan sequences. Therefore, at least one duplication of the pumilio gene must have occurred before the divergence of cestodes and trematodes. Further duplications of PUM-HD were found in Fasciola hepatica, but these consist of retropseudogenes. This is the first comparative analysis of PUM-HD sequences in the Platyhelminthes and, more generally, in any lophotrochozoan phylum.

The life-threatening diseases alveolar and cystic echinococcoses are caused by larvae of the tapeworms Echinococcus multilocularis and E. granulosus, respectively. In both cases, intermediate hosts, such as humans, are infected by oral uptake of oncosphere larvae, followed by asexual multiplication and almost unrestricted growth of the metacestode within host organs. Besides surgery, echinococcosis treatment relies on benzimidazole-based chemother- apy, directed against parasite beta-tubulin. However, since beta-tubulins are highly similar between cestodes and humans, benzimidazoles can only be applied at parasitostatic doses and are associated with adverse side effects. Mostly aiming at identifying alternative drug targets, the nuclear genome sequences of E. multilocularis and E. granulosus have recently been characterized, revealing a large number of druggable targets that are expressed by the metacestode. Furthermore, recent cell biological investigations have demonstrated that E. multilocularis employs pluripotent stem cells, called germinative cells, which are the only parasite cells capable of proliferation and which give rise to all differentiated cells. Hence, the germinative cells are the crucial cell type mediating prolif- eration of E. multilocularis, and most likely also E. granulosus, within host organs and should also be responsible for parasite recurrence upon discontinuation of chemotherapy. Interestingly, recent investigations have also indicated that germinative cells might be less sensitive to chemotherapy because they express a beta-tubulin isoform with limited affinity to benzimidazoles. In this article, we briefly review the recent findings concerning Echinococcus genomics and stem cell research and propose that future research into anti-echinococcosis drugs should also focus on the parasite's stem cell population.

Cestodes show a remarkable proliferative capability that sustains the constant growth and differentiation of proglottids essential for their lifestyle. It is believed that a separate population of undifferentiated stem cells (the so-called germinative cells) are the only cells capable of proliferation during growth and development. The study of this particular cell subpopulation is hampered by the current lack of methods to isolate it. In this work, we developed a reproducible flow cytometry and cell sorting method to quantify and isolate the proliferating cells in the tetrathyridia larvae of the model cestode Mesocestoides corti, based on the DNA content of the cells. The isolated cells display the typical germinative cell morphology, and can be used for RNA isolation with a yield in the ng to μg range. We expect that this approach may facilitate the characterization of the germinative cells in M. corti and other model tapeworms.

Tropomyosins are a family of actin-binding proteins with diverse roles in actin filament function. One of the best characterized roles is the regulation of muscle contraction. Tropomyosin isoforms can be generated from different genes, and from alternative promoters and alternative splicing from the same gene. In this work, we have isolated sequences for tropomyosin isoforms from the cestode Mesocestoides corti, and searched for tropomyosin genes and isoforms in other flatworms. Two genes are conserved in the cestodes M. corti and Echinococcus multilocularis, and in the trematode Schistosoma mansoni. Both genes have the same structure, and each gene gives rise to at least two different isoforms, a high molecular weight (HMW) and a low molecular weight (LMW) one. Because most exons are duplicated and spliced in a mutually exclusive fashion, isoforms from one gene only share one exon and are highly divergent. The gene duplication preceded the divergence of neodermatans and the planarian Schmidtea mediterranea. Further duplications occurred in Schmidtea, coupled to the selective loss of duplicated exons, resulting in genes that only code for HMW or LMW isoforms. A polyclonal antibody raised against a HMW tropomyosin from Echinococcus granulosus was demonstrated to specifically recognize HMW tropomyosin isoforms of M. corti, and used to study their expression during segmentation. HMW tropomyosins are expressed in muscle layers, with very low or absent levels in other tissues. No expression of HMW tropomyosins is present in early or late genital primordia, and expression only begins once muscle fibers develop in the genital ducts. Therefore, HMW tropomyosins are markers for the development of muscles during the final differentiation of genital primordia.

Alveolar and cystic echinococcosis, caused by the metacestode larval stages of the tapeworms Echinococcus multilocularis and Echinococcus granulosus, respectively, are life-threatening diseases and very difficult to treat. The introduction of benzimidazole-based chemotherapy, which targets parasite β-tubulin, has significantly improved the life-span and prognosis of echinococcosis patients. However, benzimidazoles show only parasitostatic activity, are associated with serious adverse side effects and have to be administered for very long time periods, underlining the need for new drugs. Very recently, the nuclear genomes of E. multilocularis and E. granulosus have been characterised, revealing a plethora of data for gaining a deeper understanding of host-parasite interaction, parasite development and parasite evolution. Combined with extensive transcriptome analyses of Echinococcus life cycle stages these investigations also yielded novel clues for targeted drug design. Recent years also witnessed significant advancements in the molecular and cellular characterisation of the Echinococcus ‘germinative cell’ population, which forms a unique stem cell system that differs from stem cells of other organisms in the expression of several genes associated with the maintenance of pluripotency. As the only parasite cell type capable of undergoing mitosis, the germinative cells are central to all developmental transitions of Echinococcus within the host and to parasite expansion via asexual proliferation. In the present article, we will briefly introduce and discuss recent advances in Echinococcus genomics and stem cell research in the context of drug design and development. Interestingly, it turns out that benzimidazoles seem to have very limited effects on Echinococcus germinative cells, which could explain the high recurrence rates observed after chemotherapeutic treatment of echinococcosis patients. This clearly indicates that future efforts into the development of parasitocidal drugs should also target the parasite's stem cell system.