Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis’ gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in ruminants and humans.

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Liver and intestinal flukes of the family Fasciolidae cause zoonotic food–borne infections that impact both agriculture and human health throughout the world. Their evolutionary history and the genetic basis underlying their phenotypic and ecological diversity are not well understood. To close that knowledge gap, we compared the whole genomes of Fasciola hepatica, Fasciola gigantica, and Fasciolopsis buski and determined that the split between Fasciolopsis and Fasciola took place ∼90 Ma in the late Cretaceous period, and that between 65 and 50 Ma an intermediate host switch and a shift from intestinal to hepatic habitats occurred in the Fasciola lineage. The rapid climatic and ecological changes occurring during this period may have contributed to the adaptive radiation of these flukes. Expansion of cathepsins, fatty-acid-binding proteins, protein disulfide-isomerases, and molecular chaperones in the genus Fasciola highlights the significance of excretory–secretory proteins in these liver-dwelling flukes. Fasciola hepatica and Fasciola gigantica diverged ∼5 Ma near the Miocene–Pliocene boundary that coincides with reduced faunal exchange between Africa and Eurasia. Severe decrease in the effective population size ∼10 ka in Fasciola is consistent with a founder effect associated with its recent global spread through ruminant domestication. G-protein-coupled receptors may have key roles in adaptation of physiology and behavior to new ecological niches. This study has provided novel insights about the genome evolution of these important pathogens, has generated genomic resources to enable development of improved interventions and diagnosis, and has laid a solid foundation for genomic epidemiology to trace drug resistance and to aid surveillance.

BackgroundSmall non-coding RNAs, including miRNAs, and gene silencing mediated by RNA interference have been described in free-living and parasitic lineages of flatworms, but only few key factors of the small RNA pathways have been exhaustively investigated in a limited number of species. The availability of flatworm draft genomes and predicted proteomes allowed us to perform an extended survey of the genes involved in small non-coding RNA pathways in this phylum.ResultsOverall, findings show that the small non-coding RNA pathways are conserved in all the analyzed flatworm linages; however notable peculiarities were identified. While Piwi genes are amplified in free-living worms they are completely absent in all parasitic species. Remarkably all flatworms share a specific Argonaute family (FL-Ago) that has been independently amplified in different lineages. Other key factors such as Dicer are also duplicated, with Dicer-2 showing structural differences between trematodes, cestodes and free-living flatworms. Similarly, a very divergent GW182 Argonaute interacting protein was identified in all flatworm linages. Contrasting to this, genes involved in the amplification of the RNAi interfering signal were detected only in the ancestral free living species Macrostomum lignano. We here described all the putative small RNA pathways present in both free living and parasitic flatworm lineages.ConclusionThese findings highlight innovations specifically evolved in platyhelminths presumably associated with novel mechanisms of gene expression regulation mediated by small RNA pathways that differ to what has been classically described in model organisms. Understanding these phylum-specific innovations and the differences between free living and parasitic species might provide clues to adaptations to parasitism, and would be relevant for gene-silencing technology development for parasitic flatworms that infect hundreds of million people worldwide.Electronic supplementary materialThe online version of this article (10.1186/s12862-017-1061-5) contains supplementary material, which is available to authorized users.

Platyhelminthes comprise one of the major phyla of invertebrate animals, inhabiting a wide range of ecosystems, and one of the most successful in adapting to parasitic life. Small non-coding RNAs have been implicated in regulating complex developmental transitions in model parasitic species. Notably, parasitic flatworms have lost Piwi RNA pathways but gained a novel Argonaute gene. Herein, we analyzed, contrasted and compared the conservation of small RNA pathways among several free-living species (a paraphyletic group traditionally known as ‘turbellarians’) and parasitic species (organized in the monophyletic clade Neodermata) to disentangle possible adaptations during the transition to parasitism. Our findings showed that complete miRNA and RNAi pathways are present in all analyzed free-living flatworms. Remarkably, whilst all ‘turbellarians’ have Piwi proteins, these were lost in parasitic Neodermantans. Moreover, two clusters of Piwi class Argonaute genes are present in all ‘turbellarians’. Interestingly, we identified a divergent Piwi class Argonaute in free living flatworms exclusively, which we named ‘Fliwi’. In addition, other key proteins of the Piwi pathways were conserved in ‘turbellarians’, while none of them were detected in Neodermatans. Besides Piwi and the canonical Argonaute proteins, a flatworm-specific class of Argonautes (FL-Ago) was identified in the analyzed species confirming its ancestrallity to all Platyhelminthes. Remarkably, this clade was expanded in parasitic Neodermatans, but not in free-living species. These phyla-specific Argonautes showed lower sequence conservation compared to other Argonaute proteins, suggesting that they might have been subjected to high evolutionary rates. However, key residues involved in the interaction with the small RNA and mRNA cleavage in the canonical Argonautes were more conserved in the FL-Agos than in the Piwi Argonautes. Whether this is related to specialized functions and adaptations to parasitism in Neodermatans remains unclear. In conclusion, differences detected in gene conservation, sequence and structure of the Argonaute family suggest tentative biological and evolutionary diversifications that are unique to Platyhelminthes. The remarkable divergencies in the small RNA pathways between free-living and parasitic flatworms indicate that they may have been involved in the adaptation to parasitism of Neodermatans.

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.

BackgroundFasciola hepatica is the main agent of fasciolosis, a zoonotic disease affecting livestock worldwide, and an emerging food-borne disease in humans. Even when effective treatments are available, drugs are costly and can result in tolerance, liver damage and normally they do not prevent reinfection. Drug-resistant strains in livestock have been reported in various countries and, more worryingly, drug resistance in human cases has emerged in South America. The present study aims to characterize the transcriptome of two South American resistant isolates, the Cajamarca isolate from Peru, resistant to both triclabendazole and albendazole (TCBZR/ABZR) and the Rubino isolate from Uruguay, resistant to ABZ (TCBZS/ABZR), and compare them to a sensitive strain (Cenapa, Mexico, TCBZS/ABZS) to reveal putative molecular mechanisms leading to drug resistance.ResultsWe observed a major reduction in transcription in the Cajamarca TCBZR/ABZR isolate in comparison to the other isolates. While most of the differentially expressed genes are still unannotated, several trends could be detected. Specific reduction in the expression levels of cytoskeleton proteins was consistent with a role of tubulins as putative targets of triclabendazole (TCBZ). A marked reduction of adenylate cyclase might be underlying pleiotropic effects on diverse metabolic pathways of the parasite. Upregulation of GST mu isoforms suggests this detoxifying mechanism as one of the strategies associated with resistance.ConclusionsOur results stress the value of transcriptomic approaches as a means of providing novel insights to advance the understanding of drug mode of action and drug resistance. The results provide evidence for pleiotropic variations in drug-resistant isolates consistent with early observations of TCBZ and ABZ effects and recent proteomic findings.Electronic supplementary materialThe online version of this article (10.1186/s13071-017-2553-2) contains supplementary material, which is available to authorized users.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression being involved in many different biological processes and play a key role in developmental timing. Additionally, recent studies have shown that miRNAs released from parasites are capable of regulating the expression of host genes. In the present work, we studied the expression patterns of ncRNAs of various intra-mammalian life-cycle stages of the liver fluke, Fasciola hepatica, as well as those packaged into extracellular vesicles and shed by the adult fluke. The miRNA expression profile of the intra-mammalian stages shows important variations, despite a set of predominant miRNAs that are highly expressed across all stages. No substantial variations in miRNA expression between dormant and activated metacercariae were detected, suggesting that they might not be central players in regulating fluke gene expression during this crucial step in the invasion of the definitive host. We generated a curated pipeline for the prediction of putative target genes that reports only sites conserved between three different prediction approaches. This pipeline was tested against an iso-seq curated database of the 3’ UTR regions of F. hepatica genes to detect miRNA regulation networks within liver fluke. Several functions related to the host immune response or modulation were enriched among the targets of the most highly expressed parasite miRNAs, stressing that they might be key players during the establishment and maintenance of infection. Additionally, we detected fragments derived from the processing of tRNAs, in all developmental stages analyzed, and documented the presence of novel long tRNA fragments enriched in vesicles. We confirmed the presence of at least 5 putative vault RNAs (vtRNAs), that are expressed across different stages and enriched in vesicles. The presence of tRNA fragments and vtRNAs in vesicles raise the possibility that they could be involved in the host-parasite interaction.