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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.

Background Snails of the Lymnaeidae family are the intermediate hosts of Fasciola species, the causative agents of fascioliasis. The purpose of this study was to determine the prevalence of Fasciola species in lymnaeid snails and to investigate the association of geoclimatic factors and Fasciola species distribution in northwestern provinces of Iran using geographical information system (GIS) data. Methods A total of 2000 lymnaeid snails were collected from 33 permanent and seasonal habitats in northwestern Iran during the period from June to November 2021. After identification by standard morphological keys, they were subjected to shedding and crushing methods. Different stages of Fasciola obtained from these snails were subjected to the ITS1 polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method for species identification. The associations of weather temperature, rainfall, humidity, evaporation, air pressure, wind speed, elevation, and land cover with the distribution of Fasciola species were investigated. Geographical and statistical analysis was performed using ArcMap and SPSS software, respectively, to determine factors related to Fasciola species distribution. Results Of the 2000 snails collected, 19 were infected with Fasciola hepatica (0.09%), six with F. gigantica (0.03%), and 13 with other trematodes. Among geoclimatic and environmental factors, mean humidity, maximum humidity, and wind speed were significantly higher in areas where F. hepatica was more common than F. gigantica . The altitude of F. hepatica -prevalent areas was generally lower than F. gigantica areas. No significant relationship was observed between other investigated geoclimatic factors and the distribution of infected snails. Conclusions The present study showed the relationship of humidity and wind speed with the distribution of snails infected with F. hepatica or F. gigantica in the northwestern regions of Iran. In contrast to F. gigantica , F. hepatica was more prevalent in low-altitude areas. Further research is recommended to elucidate the relationship between geoclimatic factors and the presence of intermediate hosts of the two Fasciola species. Graphical Abstract

Fasciola hepatica causes fasciolosis, a parasitic disease that poses significant animal and human health challenges. Control relies on flukicides, most of which are adulticides, with only triclabendazole effective against the pathogenic migratory juvenile. Classical neurotransmitter pathways are widely targeted by anthelmintics yet remain underexplored for flukicide development. Here we explore the importance of serotonin (5-HT) signaling in juvenile fluke. In silico analyses confirmed all F. hepatica life stages express a complete 5-HT signaling pathway encompassing genes encoding proteins for 5-HT synthesis, transport, and reuptake, as well as five putative 5-HT G protein-coupled receptors (GPCRs). Homology and binding motif analyses supported the presence of two 5-HT 1 (Fh5HT 1A , Fh5HT 1B ) and three 5-HT 7 (Fh5HT 7A , -7B , -7C ) GPCRs. Immunocytochemistry and in situ hybridization revealed widespread neuronal expression of 5-HT, its synthetic enzyme tryptophan hydroxylase (FhTPH), and the GPCR Fh5HT 7C . 5-HT addition stimulated juvenile fluke motility; consistent with this observation, serotonin reuptake inhibition, which causes 5-HT persistence at synaptic junctions, also enhanced juvenile movement. Silencing of FhTPH, a key enzyme in 5-HT synthesis, blunted juvenile motility, a phenotype reversed by the addition of 5-HT. Silencing the fluke vesicular monoamine transporter (FhVMAT), which packages 5-HT into synaptic vesicles, reduced juvenile motility, whilst silencing the 5-HT reuptake transporter (FhSERT) which recycles synaptic 5-HT increased juvenile motility and growth, consistent with 5-HT accumulation enhancing effects. Whilst combinatorial silencing of Fh5HT 1 receptors reduced fluke motility, silencing Fh5HT 7 receptors led to a greater reduction in motility. Exogenous addition of 5-HT partially rescued motility deficits of juveniles with silenced Fh5HT 1 receptors, but 5-HT excitation was abolished in Fh5HT 7 -RNAi juveniles, exposing their importance to fluke motility. Notably, sustained 5-HT exposure promoted juvenile growth, but these effects were not blunted by receptor-RNAi. The findings emphasize a central role of serotonin signaling in both juvenile motility and growth, exposing novel aspects of receptor function and encouraging therapeutic exploitation for liver fluke control.

Infection by the liver fluke, Fasciola hepatica , places a substantial burden on the global agri-food industry and poses a significant threat to human health in endemic regions. Widespread resistance to a limited arsenal of chemotherapeutics, including the frontline flukicide triclabendazole (TCBZ), renders F. hepatica control unsustainable and accentuates the need for novel therapeutic target discovery. A key facet of F. hepatica biology is a population of specialised stem cells which drive growth and development - their dysregulation is hypothesised to represent an appealing avenue for control. The exploitation of this system as a therapeutic target is impeded by a lack of understanding of the molecular mechanisms underpinning F. hepatica growth and development. Wnt signalling pathways govern a myriad of stem cell processes during embryogenesis and drive tumorigenesis in adult tissues in animals. Here, we identify five putative Wnt ligands and five Frizzled receptors in liver fluke transcriptomic datasets and find that Wnt/β-catenin signalling is most active in juveniles, the most pathogenic life stage. FISH-mediated transcript localisation revealed partitioning of the five Wnt ligands, with each displaying a distinct expression pattern, consistent with each Wnt regulating the development of different cell/tissue types. The silencing of each individual Wnt or Frizzled gene yielded significant reductions in juvenile worm growth and, in select cases, blunted the proliferation of neoblast-like cells. Notably, silencing Fh CTNNB1, the key effector of the Wnt/β-catenin signal cascade led to aberrant development of the neuromuscular system which ultimately proved lethal - the first report of a lethal RNAi-induced phenotype in F. hepatica . The absence of any discernible phenotypes following the silencing of the inhibitory Wnt/β-catenin destruction complex components is consistent with low destruction complex activity in rapidly developing juvenile worms, corroborates transcriptomic expression profiles and underscores the importance of Wnt signalling as a key molecular driver of growth and development in early-stage juvenile fluke. The putative pharmacological inhibition of Wnt/β-catenin signalling using commercially available inhibitors phenocopied RNAi results and provides impetus for drug repurposing. Taken together, these data functionally and chemically validate the targeting of Wnt signalling as a novel strategy to undermine the pathogenicity of juvenile F. hepatica .

The liver fluke Fasciola hepatica is a major pathogen of livestock worldwide, causing huge economic losses to agriculture, as well as 2.4 million human infections annually. Here we provide a draft genome for F. hepatica, which we find to be among the largest known pathogen genomes at 1.3 Gb. This size cannot be explained by genome duplication or expansion of a single repeat element, and remains a paradox given the burden it may impose on egg production necessary to transmit infection. Despite the potential for inbreeding by facultative self-fertilisation, substantial levels of polymorphism were found, which highlights the evolutionary potential for rapid adaptation to changes in host availability, climate change or to drug or vaccine interventions. Non-synonymous polymorphisms were elevated in genes shared with parasitic taxa, which may be particularly relevant for the ability of the parasite to adapt to a broad range of definitive mammalian and intermediate molluscan hosts. Large-scale transcriptional changes, particularly within expanded protease and tubulin families, were found as the parasite migrated from the gut, across the peritoneum and through the liver to mature in the bile ducts. We identify novel members of anti-oxidant and detoxification pathways and defined their differential expression through infection, which may explain the stage-specific efficacy of different anthelmintic drugs. The genome analysis described here provides new insights into the evolution of this important pathogen, its adaptation to the host environment and external selection pressures. This analysis also provides a platform for research into novel drugs and vaccines.

Fasciolosis is a parasitic disease affecting humans and livestock, caused by digenean trematodes of the genus Fasciola , primarily F. hepatica and F. gigantica . This study investigates the coexistence of these species and their hybrids in yaks from Tibet, China. We analyzed the nuclear rDNA internal transcribed spacer (ITS) regions, including ITS1 and ITS2, through Sanger sequencing and Next-Generation Sequencing (NGS) to assess single-nucleotide polymorphisms (SNPs). Our results reveal that one specimen (NM008B) is identical to pure F. hepatica , while another (NM008A) contains genetic information from both F. hepatica and F. gigantica , indicating potential hybridization or introgression. The morphological analysis reveals that the collected adult F. hepatica specimens exhibit distinct characteristics, while the hybrid specimens display “intermediate” features of F. hepatica and F. gigantica . This study is the first to document the coexistence of F. hepatica and hybrid Fasciola forms in a single yak. The findings underscore the complexities of hybridization dynamics and the necessity for advanced molecular techniques in accurately identifying Fasciola species. Future research should focus on mitochondrial DNA and other nuclear gene analysis to further elucidate the nature of these hybrids and their ecological implications.

The absence of an established in vitro platform is a major obstacle in research on parasitic flatworms, including Fasciola flukes. Fasciola flukes cause zoonotic infections that primarily affect the liver and the bile ducts. Infected juveniles can cause severe liver damage in animals, occasionally leading to sudden death. Although resistance to the only drug for the acute liver stage has been reported worldwide, the search for new drugs has been unsuccessful owing to the critical limitations of previous in vitro cultures. Previous studies have been unable to reproduce liver-stage development in vitro , hindering research on this stage. This study aimed to provide a novel in vitro research platform using a laboratory strain of Fasciola hepatica/gigantica hybrid. Juveniles derived from the livers of mice at 7 and 11 days post-infection (dpi) survived for nearly 100 days in the basic medium consisting of Roswell Park Memorial Institute (RPMI) 1640 supplemented with 50% fetal bovine serum. Bovine red blood cells (RBC) and sex-inducing substances (SIS) that induce sexualization in a free-living flatworm (planarian) were supplemented to examine their effects on the developmental processes in the liver stage, including growth, body shape change, and reproductive development. SIS induced all three processes, although the last was incomplete, suggesting that the sex-inducing ability of SIS is conserved between free-living and parasitic flatworms. However, RBC was somewhat toxic and less effective than SIS for both growth and reproductive development and could not alter body shape. Furthermore, the combined effects of the two supplements were not observed. In this study, the reproducibility of the development was carefully confirmed, and it was shown that a single SIS supplementation is currently the best condition and more closely mimics liver-stage development. This study provides a preliminary but outstanding in vitro research platform for liver-stage juveniles and will facilitate further drug development.

Background Fascioliasis is a zoonosis and neglected tropical disease with worldwide distribution and significant economic impact. The search for anthelmintic compounds against Fasciola hepatica , one of the parasitic liver flukes responsible for this disease, has become important due to widespread resistances against existing drugs. To facilitate drug discovery, a useful strategy is the identification of proteins that are vital for the parasite. Protein kinases (PKs) emerged as potential targets in several parasites, given their critical role in many biological processes. To date, knowledge of the PKs present in F. hepatica is fragmentary. Results We curated and classified the kinome of F. hepatica using a refined bioinformatics pipeline and found 245 PKs, which represented 2.14% of the parasite’s proteome. Classification of all PKs into their families and sub-families revealed the CMGC group as the largest PK group. A comparison of the kinomes of F. hepatica with medically important Schistosoma species and the human host revealed key similarities and differences. Based on orthology to human sequences, KEGG functional annotation predicted that 25% of 110 annotated PKs in F. hepatica are involved in cancer pathways. We prioritized a panel of related, small-molecule PK inhibitors to assess their efficacy against different F. hepatica life stages in vitro. Among these, vandetanib and ruboxistaurin showed lethal effects on immature flukes in vitro at 50 µM concentration, and ruboxistaurin significantly reduced the motility of adult liver flukes. Conclusion These findings suggest that repurposing small-molecule PK inhibitors could be a good strategy for obtaining compounds to combat fascioliasis. The newly established F. hepatica kinome represents a resource for future target discovery.

The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30-100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents.

The spatial organization of gene expression dictates tissue functions in multicellular parasites. Here, we present the spatial transcriptome of a parasitic flatworm, the common liver fluke Fasciola hepatica . We identify gene expression profiles and marker genes for eight distinct tissues and validate the latter by in situ hybridization. To demonstrate the power of our spatial atlas, we focus on genes with substantial medical importance, including vaccine candidates (Ly6 proteins) and drug resistance genes (glutathione S-transferases, ABC transporters). Several of these genes exhibit unique expression patterns, indicating tissue-specific biological functions. Notably, the prioritization of tegumental protein kinases identifies a PKCβ, for which small-molecule targeting causes parasite death. Our comprehensive gene expression map provides unprecedented molecular insights into the organ systems of this complex parasitic organism, serving as a valuable tool for both basic and applied research. Fasciola hepatica is a complex multicellular pathogen and the causative agent of fascioliasis, a zoonotic disease that compromises liver function. Using spatial transcriptomics, the authors uncover the tissue-specific gene expression of F. hepatica and identify genes with expression in the surface layer for drug-target discovery.