In vitro system for the growth and asexual multiplication of Taenia crassiceps cysticerci.

Taenia solium is the aetiological agent of cysticercosis, a zoonosis that causes severe health and economic losses across Latin America, Africa and Asia. The most serious manifestation of the disease is neurocysticercosis, which occurs when the larval stage (cysticercus) establishes in the central nervous system. Using Taenia crassiceps as an experimental model organism for the study of cysticercosis, we aimed to identify the in vitro conditions necessary to allow parasite development at the short- and long terms. First, cysticerci were incubated for 15 days in different media and parasite densities. The number of buddings and cysticerci diameter were measured to evaluate asexual multiplication and parasite growth, respectively. Vitality was determined by trypan blue staining and morphology analysis. As a result, high cysticerci density and medium containing FBS and the excretion/secretion (E/S) products of feeder cells induced parasite survival, growth and multiplication. Then, the long-term (5 weeks) incubation of the parasites in co-culture with feeder cells was evaluated. Consequently, the mammalian cell lines induced a significant increase in total parasite volume while axenic cultures did not show any statistically significant change over time. In this study, the proper conditions to maintain T. crassiceps in vitro are described for the first time in a simpler and more controlled setting other than experimental infections. In addition, it was shown that cysticerci growth, survival and asexual multiplication depend on a complex network of secreted factors from both parasite and host.

Cestodes in the genomic era.

The first cestode genomes were obtained by an international consortium led by the Wellcome Sanger Institute that included representative institutions from countries where the sequenced parasites have been studied for decades, in part because they are etiological agents of endemic diseases (Argentina, Uruguay, Mexico, Canada, UK, Germany, Switzerland, Ireland, USA, Japan, and China). After this, several complete genomes were obtained reaching 16 species to date. Cestode genomes have smaller relative size compared to other animals including free-living flatworms. Moreover, the features genome size and repeat content seem to differ in the two analyzed orders. Cyclophyllidean species have smaller genomes and with fewer repetitive content than Diphyllobothriidean species. On average, cestode genomes have 13,753 genes with 6 exons per gene and 41% GC content. More than 5,000 shared cestode proteins were accurately annotated by the integration of gene predictions and transcriptome evidence being more than 40% of these proteins of unknown function. Several gene losses and reduction of gene families were found and could be related to the extreme parasitic lifestyle of these species. The application of cutting-edge sequencing technology allowed the characterization of the terminal sequences of chromosomes that possess unique characteristics. Here, we review the current status of knowledge of complete cestode genomes and place it within a comparative genomics perspective. Multidisciplinary work together with the implementation of new technologies will provide valuable information that can certainly improve our chances to finally eradicate or at least control diseases caused by cestodes.

The genomes of four tapeworm species reveal adaptations to parasitism.

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.

The Echinococcus canadensis (G7) genome: a key knowledge of parasitic platyhelminth human diseases.

BACKGROUND: The parasite Echinococcus canadensis (G7) (phylum Platyhelminthes, class Cestoda) is one of the causative agents of echinococcosis. Echinococcosis is a worldwide chronic zoonosis affecting humans as well as domestic and wild mammals, which has been reported as a prioritized neglected disease by the World Health Organisation. No genomic data, comparative genomic analyses or efficient therapeutic and diagnostic tools are available for this severe disease. The information presented in this study will help to understand the peculiar biological characters and to design species-specific control tools. RESULTS: We sequenced, assembled and annotated the 115-Mb genome of E. canadensis (G7). Comparative genomic analyses using whole genome data of three Echinococcus species not only confirmed the status of E. canadensis (G7) as a separate species but also demonstrated a high nucleotide sequences divergence in relation to E. granulosus (G1). The E. canadensis (G7) genome contains 11,449 genes with a core set of 881 orthologs shared among five cestode species. Comparative genomics revealed that there are more single nucleotide polymorphisms (SNPs) between E. canadensis (G7) and E. granulosus (G1) than between E. canadensis (G7) and E. multilocularis. This result was unexpected since E. canadensis (G7) and E. granulosus (G1) were considered to belong to the species complex E. granulosus sensu lato. We described SNPs in known drug targets and metabolism genes in the E. canadensis (G7) genome. Regarding gene regulation, we analysed three particular features: CpG island distribution along the three Echinococcus genomes, DNA methylation system and small RNA pathway. The results suggest the occurrence of yet unknown gene regulation mechanisms in Echinococcus. CONCLUSIONS: This is the first work that addresses Echinococcus comparative genomics. The resources presented here will promote the study of mechanisms of parasite development as well as new tools for drug discovery. The availability of a high-quality genome assembly is critical for fully exploring the biology of a pathogenic organism. The E. canadensis (G7) genome presented in this study provides a unique opportunity to address the genetic diversity among the genus Echinococcus and its particular developmental features. At present, there is no unequivocal taxonomic classification of Echinococcus species; however, the genome-wide SNPs analysis performed here revealed the phylogenetic distance among these three Echinococcus species. Additional cestode genomes need to be sequenced to be able to resolve their phylogeny.

Trypanosoma cruzi Infection Imparts a Regulatory Program in Dendritic Cells and T Cells via Galectin-1-Dependent Mechanisms.

Galectin-1 (Gal-1), an endogenous glycan-binding protein, is widely distributed at sites of inflammation and microbial invasion. Despite considerable progress regarding the immunoregulatory activity of this lectin, the role of endogenous Gal-1 during acute parasite infections is uncertain. In this study, we show that Gal-1 functions as a negative regulator to limit host-protective immunity following intradermal infection with Trypanosoma cruzi. Concomitant with the upregulation of immune inhibitory mediators, including IL-10, TGF-ß1, IDO, and programmed death ligand 2, T. cruzi infection induced an early increase of Gal-1 expression in vivo. Compared to their wild-type (WT) counterpart, Gal-1-deficient (Lgals1(-/-)) mice exhibited reduced mortality and lower parasite load in muscle tissue. Resistance of Lgals1(-/-) mice to T. cruzi infection was associated with a failure in the activation of Gal-1-driven tolerogenic circuits, otherwise orchestrated by WT dendritic cells, leading to secondary dysfunction in the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells. This effect was accompanied by an increased number of CD8(+) T cells and higher frequency of IFN-γ-producing CD4(+) T cells in muscle tissues and draining lymph nodes as well as reduced parasite burden in heart and hindlimb skeletal muscle. Moreover, dendritic cells lacking Gal-1 interrupted the Gal-1-mediated tolerogenic circuit and reinforced T cell-dependent anti-parasite immunity when adoptively transferred into WT mice. Thus, endogenous Gal-1 may influence T. cruzi infection by fueling tolerogenic circuits that hinder anti-parasite immunity.

Cystic echinococcosis in South America: systematic review of species and genotypes of Echinococcus granulosus sensu lato in humans and natural domestic hosts.

OBJECTIVE: To systematically review publications on Echinococcus granulosus sensu lato species/genotypes reported in domestic intermediate and definitive hosts in South America and in human cases worldwide, taking into account those articles where DNA sequencing was performed; and to analyse the density of each type of livestock that can act as intermediate host, and features of medical importance such as cyst organ location. METHODS: Literature search in numerous databases. We included only articles where samples were genotyped by sequencing since to date it is the most accurate method to unambiguously identify all E. granulosus s. l. genotypes. Also, we report new E. granulosus s. l. samples from Argentina and Uruguay analysed by sequencing of cox1 gene. RESULTS: In South America, five countries have cystic echinococcosis cases for which sequencing data are available: Argentina, Brazil, Chile, Peru and Uruguay, adding up 1534 cases. E. granulosus s. s. (G1) accounts for most of the global burden of human and livestock cases. Also, E. canadensis (G6) plays a significant role in human cystic echinococcosis. Likewise, worldwide analysis of human cases showed that 72.9% are caused by E. granulosus s. s. (G1) and 12.2% and 9.6% by E. canadensis G6 and G7, respectively. CONCLUSIONS: E. granulosus s. s. (G1) accounts for most of the global burden followed by E. canadensis (G6 and G7) in South America and worldwide. This information should be taken into account to suit local cystic echinococcosis control and prevention programmes according to each molecular epidemiological situation.

Mouse model of secondary cystic echinococcosis.

Cystic echinococcosis (CE) is a parasitic zoonosis caused by the larval stage of the cestode Echinococcus granulosus sensu lato (s. l.), a genetic complex composed of five species: E. granulosus sensu stricto (s. s.), E. equinus, E. ortleppi, E. canadensis, and E. felidis. The parasite requires two mammalian hosts to complete its life cycle: a definitive host (mainly dogs) harboring the adult parasite in its intestines, and an intermediate host (mostly farm and wild ungulates) where hydatid cysts develop mainly in the liver and lungs. Humans are accidental intermediate hosts, being susceptible to either primary or secondary forms of CE; the first one due to the ingestion of oncospheres, and the second one because of the spillage of protoscoleces (PSC) contained within a primary cyst. Secondary CE is a serious medical problem, and can be modeled in immunocompetent mice (a non-natural intermediate host) through the intraperitoneal inoculation of viable PSC from E. granulosus s. l. This model is useful to study not only the immunobiology of CE, but also to test new chemotherapeutics or therapeutical protocols, to explore novel vaccine candidates, and to evaluate alternative diagnostic and/or follow-up tools. The mouse model of secondary CE involves two sequential stages: an early stage of parasite pre-encystment (PSC develop into hydatid cysts in the peritoneal cavity of mice), and a late or chronic stage of parasite post-encystment (already differentiated cysts slowly grow during the whole host lifespan). This model is a time-consuming infection, whose outcome depends on several factors like the parasite infective dose, the mouse strain, and the parasite species/genotype. Thus, such variables should always be adjusted according to the research objectives. Herein, the general materials and procedures needed to establish secondary CE in mice are described, as well as several useful tips and recommendations.

Using the model cestode Taenia crassiceps for the study of cysticercosis.

Taenia solium is the aetiological agent of taeniasis/cysticercosis, one of the most severe neglected tropical diseases (NTD) according to the World Health Organization (WHO). The life cycle of T. solium alternates between pigs (intermediate host) and humans (definitive host). In addition, humans can act as accidental intermediate hosts if they ingest infective eggs. In this case, the most severe condition of the disease occurs when parasites invade the central nervous system, causing neurocysticercosis (NCC). The complexity of the life cycle of T. solium imposes a barrier to study this pathogen thoroughly. Thus, related species, such as T. crassiceps are commonly used. Due to its capacity to multiply asexually, T. crassiceps can be maintained by serial passage in laboratory mice in standard biosecurity level facilities. In addition, an in vitro system to generate cysticerci in the presence of feeder cells has been recently developed. Despite model species display biological differences with their zoonotic counterparts, they have historically helped to understand the biology of the related pathogenic species and hence, generate improvements in NTD detection and control. In this chapter, we describe the procedures to carry out both in vivo and in vitro systems for T. crassiceps in the laboratory.

[First report of Echinococcus vogeli in a paca in Misiones province, Argentina]. / Primera identificación de Echinococcus vogeli en una paca en la provincia de Misiones, Argentina.

We report the first finding of Echinococcus vogeli in a paca, Cuniculus paca, in the tropical forest of Misiones, in the north of Argentina. The presence of the bush dog, Speothos venaticus, E. vogelís only natural definitive host, was also reported. The polycystic hydatids, 2 to 3 cm in diameter, were only found in the liver of an adult paca. The size range of the hooks and the relative proportion blade/handle did not show significant differences with respect to the ones reported for E. vogeli. The size of E. granulosus hooks, measured for comparison purposes, was significantly smaller (p E. vogeli in Argentina. The probability of finding neotropical echinococcosis in humans reinforces the need to expand the search for E. vogeli in Argentina. Echinococcosis due to E. vogeli is very aggressive and may cause death in about a third of the human population affected.

microRNA silencing in a whole worm cestode model provides insight into miR-71 function.

Parasites belonging to the class Cestoda include zoonotic species such as Echinococcus spp. and Taenia spp. that cause morbidity and mortality in endemic areas, mainly affecting pastoral and rural communities in low income countries but also upper middle income countries. Cestodes show remarkable developmental plasticity, implying tight regulation of gene expression throughout their complex life cycles. Despite the recent availability of genomic data for cestodes, little progress was made on postgenomic functional studies. MicroRNAs (miRNAs) are key components of gene regulatory systems that guide diverse developmental processes in multicellular organisms. miR-71 is a highly expressed miRNA in cestodes, which is absent in vertebrates and targets essential parasite genes, representing a potential key player in understanding the role of miRNAs in cestodes biology. Here we used transfection with antisense oligonucleotides to perform whole worm miRNA knockdown in tetrathyridia of Mesocestoides vogae (syn. Mesocestoides corti), a laboratory model of cestodes. We believe this is the first report of miRNA knockdown at the organism level in these parasites. Our results showed that M. vogae miR-71 is involved in the control of strobilation in vitro and in the establishment of murine infection. In addition, we identified miR-71 targets in M. vogae, several of them being de-repressed upon miR-71 knockdown. This study provides new knowledge on gene expression regulation in cestodes and suggests that miRNAs could be evaluated as new selective therapeutic targets for treating Neglected Tropical Diseases prioritised by the World Health Organization.

Circulating Small RNA Profiling of Patients with Alveolar and Cystic Echinococcosis.

Alveolar (AE) and cystic (CE) echinococcosis are two parasitic diseases caused by the tapeworms Echinococcus multilocularis and E. granulosus sensu lato (s. l.), respectively. Currently, AE and CE are mainly diagnosed by means of imaging techniques, serology, and clinical and epidemiological data. However, no viability markers that indicate parasite state during infection are available. Extracellular small RNAs (sRNAs) are short non-coding RNAs that can be secreted by cells through association with extracellular vesicles, proteins, or lipoproteins. Circulating sRNAs can show altered expression in pathological states; hence, they are intensively studied as biomarkers for several diseases. Here, we profiled the sRNA transcriptomes of AE and CE patients to identify novel biomarkers to aid in medical decisions when current diagnostic procedures are inconclusive. For this, endogenous and parasitic sRNAs were analyzed by sRNA sequencing in serum from disease negative, positive, and treated patients and patients harboring a non-parasitic lesion. Consequently, 20 differentially expressed sRNAs associated with AE, CE, and/or non-parasitic lesion were identified. Our results represent an in-depth characterization of the effect E. multilocularis and E. granulosus s. l. exert on the extracellular sRNA landscape in human infections and provide a set of novel candidate biomarkers for both AE and CE detection.

A Comparative Analysis of the Protein Cargo of Extracellular Vesicles from Helminth Parasites.

Helminth parasites cause debilitating-sometimes fatal-diseases in humans and animals. Despite their impact on global health, mechanisms underlying host-parasite interactions are still poorly understood. One such mechanism involves the exchange of extracellular vesicles (EVs), which are membrane-enclosed subcellular nanoparticles. To date, EV secretion has been studied in helminth parasites, including EV protein content. However, information is highly heterogeneous, since it was generated in multiple species, using varied protocols for EV isolation and data analysis. Here, we compared the protein cargo of helminth EVs to identify common markers for each taxon. For this, we integrated published proteomic data and performed a comparative analysis through an orthology approach. Overall, only three proteins were common in the EVs of the seven analyzed species. Additionally, varied repertoires of proteins with moonlighting activity, vaccine antigens, canonical and non-canonical proteins related to EV biogenesis, taxon-specific proteins of unknown function and RNA-binding proteins were observed in platyhelminth and nematode EVs. Despite the lack of consensus on EV isolation protocols and protein annotation, several proteins were shown to be consistently detected in EV preparations from organisms at different taxa levels, providing a starting point for a selective biochemical characterization.

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Extracellular vesicles from Trypanosoma cruzi-dendritic cell interaction show modulatory properties and confer resistance to lethal infection as a cell-free based therapy strategy.

Extracellular vesicles (EVs) include a heterogeneous group of particles. Microvesicles, apoptotic bodies and exosomes are the most characterized vesicles. They can be distinguished by their size, morphology, origin and molecular composition. To date, increasing studies demonstrate that EVs mediate intercellular communication. EVs reach considerable interest in the scientific community due to their role in diverse processes including antigen-presentation, stimulation of anti-tumoral immune responses, tolerogenic or inflammatory effects. In pathogens, EV shedding is well described in fungi, bacteria, protozoan and helminths parasites. For Trypanosoma cruzi EV liberation and protein composition was previously described. Dendritic cells (DCs), among other cells, are key players promoting the immune response against pathogens and also maintaining self-tolerance. In previous reports we have demonstrate that T. cruzi downregulates DCs immunogenicity in vitro and in vivo. Here we analyze EVs from the in vitro interaction between blood circulating trypomastigotes (Tp) and bone-marrow-derived DCs. We found that Tp incremented the number and the size of EVs in cultures with DCs. EVs displayed some exosome markers and intracellular RNA. Protein analysis demonstrated that the parasite changes the DC protein-EV profile. We observed that EVs from the interaction of Tp-DCs were easily captured by unstimulated-DCs in comparison with EVs from DCs cultured without the parasite, and also modified the activation status of LPS-stimulated DCs. Noteworthy, we found protection in animals treated with EVs-DCs+Tp and challenged with T. cruzi lethal infection. Our goal is to go deep into the molecular characterization of EVs from the DCs-Tp interaction, in order to identify mediators for therapeutic purposes.

The challenging world of extracellular RNAs of helminth parasites.

In the last years, cell free or extracellular RNAs (ex-RNAs) have emerged as novel intercellular messengers between animal cells, including pathogens. In infectious diseases, ex-RNAs represent novel players in the host-pathogen and pathogen-pathogen interplays and have been described in parasitic helminths from the three major taxonomic groups: nematodes, trematodes and cestodes. Altogether, it is estimated that approximately 30 percent of the world's population is infected with helminths, which cause debilitating diseases and syndromes. Ex-RNAs are protected from degradation by encapsulation in extracellular vesicles (EV), or association to proteins or lipoproteins, and have been detected in the excretion/secretion products of helminth parasites, with EV as the preferred extracellular compartment under study. EV is the generic term used to describe a heterogenous group of subcellular membrane-bound particles, with varying sizes, biogenesis, density and composition. However, recent data suggests that this is not the only means used by helminth parasites to secrete RNAs since ex-RNAs can also be found in EV-depleted samples. Furthermore, the use of pathogen ex-RNAs as biomarkers promise the advent of new diagnostic tools though this field is still in early stages of exploration. In this review, we summarize current knowledge of vesicular and non-vesicular ex-RNAs secretion in helminth parasites, their potential as biomarkers and the evidence of their role in parasite and host reciprocal communication, together with unanswered questions in the field.

Expression profiling of Echinococcus multilocularis miRNAs throughout metacestode development in vitro.

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.

Ultrastructural characterization of the tegument in protoscoleces of Echinococcus ortleppi.

Cystic echinococcosis is a globally distributed zoonosis caused by cestodes of the Echinococcus granulosus sensu lato (s.l.) complex, with Echinococcus ortleppi mainly involved in cattle infection. Protoscoleces show high developmental plasticity, being able to differentiate into either adult worms or metacestodes within definitive or intermediate hosts, respectively. Their outermost cellular layer is called the tegument, which is important in determining the infection outcome through its immunomodulating activities. Herein, we report an in-depth characterization of the tegument of E. ortleppi protoscoleces performed through a combination of scanning and transmission electron microscopy techniques. Using electron tomography, a three-dimensional reconstruction of the tegumental cellular territories was obtained, revealing a novel structure termed the 'tegumental vesicular body' (TVB). Vesicle-like structures, possibly involved in endocytic/exocytic routes, were found within the TVB as well as in the parasite glycocalyx, distal cytoplasm and close inner structures. Furthermore, parasite antigens (GST-1 and AgB) were unevenly localised within tegumental structures, with both being detected in vesicles found within the TBV. Finally, the presence of host (bovine) IgG was also assessed, suggesting a possible endocytic route in protoscoleces. Our data forms the basis for a better understanding of E. ortleppi and E. granulosus s.l. structural biology.

The protein and microRNA cargo of extracellular vesicles from parasitic helminths - current status and research priorities.

Helminth parasites have a remarkable ability to persist within their mammalian hosts, which is largely due to their secretion of molecules with immunomodulatory properties. Although the soluble components of helminth secretions have been extensively studied, the discovery that helminths release extracellular vesicles (EVs) has added further complexity to the host-parasite interaction. Whilst several studies have begun to characterise the molecules carried by helminth EVs, work aimed at investigating their biological functions has been hindered by a lack of helminth-specific EV markers. To begin to address this, we summarised helminth EV literature to date. With a focus on the protein and microRNA (miRNA) cargo, we aimed to detect similarities and differences across those major groups of helminths for which data are available; namely nematodes, trematodes and cestodes. Pfam analysis revealed that although there is no universal EV marker for all helminth species, the EF-hand protein family was present in all EV datasets from cestodes and trematodes, and could serve as a platyhelminth EV biomarker. In contrast, M13 metallopeptidases and actin may have potential as markers for nematode EVs. As with proteins, many miRNA families appeared to be species-, stage-, or dataset-specific. Two miRNA families were common to nematode EVs (mir-10 and let-7); the miRNA cargo of EVs secreted by clade I species appeared somewhat different from species from other clades. Five miRNA families (mir-71, mir-10, mir-190, let-7 and mir-2) were shared by all trematode species examined. Our analysis has identified novel markers that may be used in studies aimed at characterising helminth EVs and interrogating their function at the host-parasite interface. In addition, we discuss the heterogeneity of methods used for helminth EV isolation and emphasise the need for a standardised approach in reporting on helminth EV data.

Extracellular non-coding RNA signatures of the metacestode stage of Echinococcus multilocularis.

Extracellular RNAs (ex-RNAs) are secreted by cells through different means that may involve association with proteins, lipoproteins or extracellular vesicles (EV). In the context of parasitism, ex-RNAs represent new and exciting communication intermediaries with promising potential as novel biomarkers. In the last years, it was shown that helminth parasites secrete ex-RNAs, however, most work mainly focused on RNA secretion mediated by EV. Ex-RNA study is of special interest in those helminth infections that still lack biomarkers for early and/or follow-up diagnosis, such as echinococcosis, a neglected zoonotic disease caused by cestodes of the genus Echinococcus. In this work, we have characterised the ex-RNA profile secreted by in vitro grown metacestodes of Echinococcus multilocularis, the casuative agent of alveolar echinococcosis. We have used high throughput RNA-sequencing together with RT-qPCR to characterise the ex-RNA profile secreted towards the extra- and intra-parasite milieus in EV-enriched and EV-depleted fractions. We show that a polarized secretion of small RNAs takes place, with microRNAs mainly secreted to the extra-parasite milieu and rRNA- and tRNA-derived sequences mostly secreted to the intra-parasite milieu. In addition, we show by nanoparticle tracking analyses that viable metacestodes secrete EV mainly into the metacestode inner vesicular fluid (MVF); however, the number of nanoparticles in culture medium and MVF increases > 10-fold when metacestodes show signs of tegument impairment. Interestingly, we confirm the presence of host miRNAs in the intra-parasite milieu, implying their internalization and transport through the tegument towards the MVF. Finally, our assessment of the detection of Echinococcus miRNAs in patient samples by RT-qPCR yielded negative results suggesting the tested miRNAs may not be good biomarkers for this disease. A comprehensive study of the secretion mechanisms throughout the life cycle of these parasites will help to understand parasite interaction with the host and also, improve current diagnostic tools.

Identification and expression profiling of microRNAs in Hymenolepis.

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.