Validation of a serum ELISA test for cyathostomin infection in equines.

Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibrators to provide standard curves for quantification of antigen-specific IgG(T) used to generate a CT3-specific 'serum score' for each horse. Validation dataset results were then used to assess the optimised test's performance and select serum score cut-off values for diagnosis of burdens above 1000, 5000 and 10,000 cyathostomins. The test demonstrated excellent performance (Receiver Operating Characteristic Area Under the Curve values >0.9) in diagnosing infection, with >90% sensitivity and >70% specificity at the selected serum score cut-off values. CT3-specific serum IgG(T) profiles in equines in different settings were assessed to provide information for commercial test use. These studies demonstrated maternal transfer of CT3-specific IgG(T) in colostrum to newborns, levels of which declined before increasing as foals consumed contaminated pasture. Studies in geographically distinct populations demonstrated that the proportion of horses that reported as test positive at a 14.37 CT3 serum score (1000-cyathostomin threshold) was associated with parasite transmission risk. Based on the results, inclusion criteria for commercial use were developed. Logistic regression models were developed to predict probabilities that burdens of individuals are above defined thresholds based on the reported serum score. The models performed at a similar level to the serum score cut-off approach. In conclusion, the CT3 test provides an option for veterinarians to obtain evidence of low cyathostomin burdens that do not require anthelmintic treatment and to support diagnosis of infection.

Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

Schistosoma mansoni venom allergen-like protein 6 (SmVAL6) maintains tegumental barrier function.

The Schistosoma mansoni venom allergen-like protein (SmVAL) superfamily is a collection of at least 29 molecules that have been classified into two distinctive groups (Group 1 and Group 2 SmVALs). The fundamental basis for SmVAL segregation relates to signal peptide and conserved cysteine retention (present in all Group 1 SmVALs, but absent in all Group 2 SmVALs). These structural differences have led to the hypothesis that most Group 1 SmVALs, found as components of schistosome excretory/secretory (E/S) products, predominantly interact with their environment (intermediate or definitive hosts) whereas the Group 2 SmVALs are retained within the schistosome to fulfil parasite-related functions. While experimental evidence to support Group 1 SmVAL/host interactions is growing, similar support for identification of parasite-related Group 2 SmVAL functions is currently lacking. By applying a combination of approaches to the study of SmVAL6, we provide the first known evidence for an essential function of a Group 2 SmVAL in schistosome biology. After whole mount in situ hybridisation (WISH) localised Smval6 to the anterior region of the oesophageal gland (AOG) and cells scattered through the mesenchyme in adult schistosomes, short interfering RNA (siRNA)-mediated silencing of Smval6 was employed to assess loss of function phenotypes. Here, siSmval6-mediated knockdown of transcript and protein levels led to an increase in tegumental permeability as assessed by the quantification of TAMRA-labelled dextran throughout sub-tegumental cells/tissues. Yeast two hybrid screening using SmVAL6 as a bait revealed Sm14 (a fatty acid binding protein) and a dynein light chain (DLC) as directly interacting partners. Interrogation of single-cell RNA-seq (scRNA-seq) data supported these protein interactions by demonstrating the spatial co-expression of Smval6/dlc/Sm14 in a small proportion of adult cell types (e.g. neurons, tegumental cells and neoblasts). In silico modelling of SmVAL6 with Sm14 and DLC provided evidence that opposing faces of SmVAL6 were likely responsible for these protein/protein interactions. Our results suggest that SmVAL6 participates in oesophageal biology, formation of higher order protein complexes and maintenance of tegumental barrier function. Further studies of other Group 2 SmVALs may reveal additional functions of this enigmatic superfamily.

Characterisation of serum IgG(T) responses to potential diagnostic antigens for equine cyathostominosis.

Cyathostomins are ubiquitous parasitic nematodes of horses. These worms spend substantial periods as intestinal wall stage encysted larvae, which can comprise up to 90% of the total burden. Several million larvae have been reported in individuals. Emergence of these larvae from the gut wall can lead to life-threatening colitis. Faecal egg count tests, increasingly used by horse owners to inform anthelmintic treatments, do not correlate with the intra-host burden of cyathostomins; this represents a key gap in the diagnostic toolbox. Previously, a cyathostomin Gut Associated Larval Antigen was identified as a promising marker for the intra-host stages of infection. Here, cyathostomin Gut Associated Larval Antigen and an additional protein, Cyathostomin Immuno-diagnostic antigen, were investigated to examine their value in providing information on cyathostomin burden. ELISA analyses examined serum IgG(T) responses to recombinant proteins derived from individual cyathostomin species. Receiver Operator Characteristic curve analysis was performed on the ELISA data; proteins with the highest Area Under the Curve values were selected to test protein combinations to investigate which were the most informative in identifying the infection status of individuals. Three cocktail combinations were tested, comprising: (a) Cy-GALA proteins from two species and a Cy-CID protein from a third species (CT3), (b) Cy-GALA proteins from five species (CT5), and (c) all CT5 components, plus a Cy-CID protein from an additional species (CT6). The best predictive values for infection were obtained using CT3 and CT6, with similar values achieved for both. Proteins in CT3 are derived from the most commonly reported species, Cyathostomum catinatum, Cylicocyclus nassatus and Cylicostephanus longibursatus. This combination was selected for future development since it represents a more commercially viable format for a diagnostic test.

The repositioning of epigenetic probes/inhibitors identifies new anti-schistosomal lead compounds and chemotherapeutic targets.

BACKGROUND: Praziquantel represents the frontline chemotherapy used to treat schistosomiasis, a neglected tropical disease (NTD) caused by infection with macro-parasitic blood fluke schistosomes. While this drug is safe, its inability to kill all schistosome lifecycle stages within the human host often requires repeat treatments. This limitation, amongst others, has led to the search for novel anti-schistosome replacement or combinatorial chemotherapies. Here, we describe a repositioning strategy to assess the anthelmintic activity of epigenetic probes/inhibitors obtained from the Structural Genomics Consortium. METHODOLOGY/PRINCIPLE FINDINGS: Thirty-seven epigenetic probes/inhibitors targeting histone readers, writers and erasers were initially screened against Schistosoma mansoni schistosomula using the high-throughput Roboworm platform. At 10 µM, 14 of these 37 compounds (38%) negatively affected schistosomula motility and phenotype after 72 hours of continuous co-incubation. Subsequent dose-response titrations against schistosomula and adult worms revealed epigenetic probes targeting one reader (NVS-CECR2-1), one writer (LLY-507 and BAY-598) and one eraser (GSK-J4) to be particularly active. As LLY-507/BAY-598 (SMYD2 histone methyltransferase inhibitors) and GSK-J4 (a JMJD3 histone demethylase inhibitor) regulate an epigenetic process (protein methylation) known to be critical for schistosome development, further characterisation of these compounds/putative targets was performed. RNA interference (RNAi) of one putative LLY-507/BAY-598 S. mansoni target (Smp_000700) in adult worms replicated the compound-mediated motility and egg production defects. Furthermore, H3K36me2, a known product catalysed by SMYD2 activity, was also reduced by LLY-507 (25%), BAY-598 (23%) and siSmp_000700 (15%) treatment of adult worms. Oviposition and packaging of vitelline cells into in vitro laid eggs was also significantly affected by GSK-J4 (putative cell permeable prodrug inhibitor of Smp_034000), but not by the related structural analogue GSK-J1 (cell impermeable inhibitor). CONCLUSION/SIGNIFICANCE: Collectively, these results provide further support for the development of next-generation drugs targeting schistosome epigenetic pathway components. In particular, the progression of histone methylation/demethylation modulators presents a tractable strategy for anti-schistosomal control.

Methyl-CpG-binding (SmMBD2/3) and chromobox (SmCBX) proteins are required for neoblast proliferation and oviposition in the parasitic blood fluke Schistosoma mansoni.

While schistosomiasis remains a significant health problem in low to middle income countries, it also represents a recently recognised threat to more economically-developed regions. Until a vaccine is developed, this neglected infectious disease is primarily controlled by praziquantel, a drug with a currently unknown mechanism of action. By further elucidating how Schistosoma molecular components cooperate to regulate parasite developmental processes, next generation targets will be identified. Here, we continue our studies on schistosome epigenetic participants and characterise the function of a DNA methylation reader, the Schistosoma mansoni methyl-CpG-binding domain protein (SmMBD2/3). Firstly, we demonstrate that SmMBD2/3 contains amino acid features essential for 5-methyl cytosine (5mC) binding and illustrate that adult schistosome nuclear extracts (females > males) contain this activity. We subsequently show that SmMBD2/3 translocates into nuclear compartments of transfected murine NIH-3T3 fibroblasts and recombinant SmMBD2/3 exhibits 5mC binding activity. Secondly, using a yeast-two hybrid (Y2H) screen, we show that SmMBD2/3 interacts with the chromo shadow domain (CSD) of an epigenetic adaptor, S. mansoni chromobox protein (SmCBX). Moreover, fluorescent in situ hybridisation (FISH) mediated co-localisation of Smmbd2/3 and Smcbx to mesenchymal cells as well as somatic- and reproductive- stem cells confirms the Y2H results and demonstrates that these interacting partners are ubiquitously expressed and found within both differentiated as well as proliferating cells. Finally, using RNA interference, we reveal that depletion of Smmbd2/3 or Smcbx in adult females leads to significant reductions (46-58%) in the number of proliferating somatic stem cells (PSCs or neoblasts) as well as in the quantity of in vitro laid eggs. Collectively, these results further expand upon the schistosome components involved in epigenetic processes and suggest that pharmacological inhibition of SmMBD2/3 and/or SmCBX biology could prove useful in the development of future schistosomiasis control strategies.

Histone methylation changes are required for life cycle progression in the human parasite Schistosoma mansoni.

Epigenetic mechanisms and chromatin structure play an important role in development. Their impact is therefore expected to be strong in parasites with complex life cycles and multiple, strikingly different, developmental stages, i.e. developmental plasticity. Some studies have already described how the chromatin structure, through histone modifications, varies from a developmental stage to another in a few unicellular parasites. While H3K4me3 profiles remain relatively constant, H3K27 trimethylation and bivalent methylation show strong variation. Inhibitors (A366 and GSK343) of H3K27 histone methyltransferase activity in S. mansoni efficiently blocked miracidium to sporocyst transition indicating that H3K27 trimethylation is required for life cycle progression. As S. mansoni is a multicellular parasite that significantly affects both the health and economy of endemic areas, a better understanding of fluke developmental processes within the definitive host will likely highlight novel disease control strategies. Towards this goal, we also studied H4K20me1 in female cercariae and adults. In particular, we found that bivalent trimethylation of H3K4 and H3K27 at the transcription start site of genes is a landmark of the cercarial stage. In cercariae, H3K27me3 presence and strong enrichment in H4K20me1 over long regions (10-100 kb) is associated with development related genes. Here, we provide a broad overview of the chromatin structure of a metazoan parasite throughout its most important lifecycle stages. The five developmental stages studied here present distinct chromatin structures, indicating that histone methylation plays an important role during development. Hence, components of the histone methylation (and demethylation) machinery may provide suitable Schistosomiasis control targets.

The anti-fecundity effect of 5-azacytidine (5-AzaC) on Schistosoma mansoni is linked to dis-regulated transcription, translation and stem cell activities.

Uncontrolled host immunological reactions directed against tissue-trapped eggs precipitate a potentially lethal, pathological cascade responsible for schistosomiasis. Blocking schistosome egg production, therefore, presents a strategy for simultaneously reducing immunopathology as well as limiting disease transmission in endemic or emerging areas. We recently demonstrated that the ribonucleoside analogue 5-azacytidine (5-AzaC) inhibited Schistosoma mansoni oviposition, egg maturation and ovarian development. While these anti-fecundity effects were associated with a loss of DNA methylation, other molecular processes affected by 5-AzaC were not examined at the time. By comparing the transcriptomes of 5-AzaC-treated females to controls, we provide evidence that this ribonucleoside analogue also modulates other crucial aspects of schistosome egg-laying biology. For example, S. mansoni gene products associated with amino acid-, carbohydrate-, fatty acid-, nucleotide- and tricarboxylic acid (TCA)- homeostasis are all dysregulated in 5-AzaC treated females. To validate the metabolic pathway most significantly affected by 5-AzaC, amino acid metabolism, nascent protein synthesis was subsequently quantified in adult schistosomes. Here, 5-AzaC inhibited this process by 68% ±16.7% (SEM) in male- and 81% ±4.8% (SEM) in female-schistosomes. Furthermore, the transcriptome data indicated that adult female stem cells were also affected by 5-AzaC. For instance, 40% of transcripts associated with proliferating schistosome cells were significantly down-regulated by 5-AzaC. This finding correlated with a considerable reduction (95%) in the number of 5-ethynyl-2'-deoxyuridine (EdU) positive cells found in 5-AzaC-treated females. In addition to protein coding genes, the effect that 5-AzaC had on repetitive element expression was also assessed. Here, 46 repeats were found differentially transcribed between 5-AzaC-treated and control females with long terminal repeat (LTR) and DNA transposon classes being amongst the most significant. This study demonstrates that the anti-fecundity activity of 5-AzaC affects more than just DNA methylation in schistosome parasites. Further characterisation of these processes may reveal novel targets for schistosomiasis control.

Corrigendum: Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

This corrects the article DOI: 10.1038/ncomms15451.

Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.

The Biomphalaria glabrata DNA methylation machinery displays spatial tissue expression, is differentially active in distinct snail populations and is modulated by interactions with Schistosoma mansoni.

BACKGROUND: The debilitating human disease schistosomiasis is caused by infection with schistosome parasites that maintain a complex lifecycle alternating between definitive (human) and intermediate (snail) hosts. While much is known about how the definitive host responds to schistosome infection, there is comparably less information available describing the snail's response to infection. METHODOLOGY/PRINCIPLE FINDINGS: Here, using information recently revealed by sequencing of the Biomphalaria glabrata intermediate host genome, we provide evidence that the predicted core snail DNA methylation machinery components are associated with both intra-species reproduction processes and inter-species interactions. Firstly, methyl-CpG binding domain protein (Bgmbd2/3) and DNA methyltransferase 1 (Bgdnmt1) genes are transcriptionally enriched in gonadal compared to somatic tissues with 5-azacytidine (5-AzaC) treatment significantly inhibiting oviposition. Secondly, elevated levels of 5-methyl cytosine (5mC), DNA methyltransferase activity and 5mC binding in pigmented hybrid- compared to inbred (NMRI)- B. glabrata populations indicate a role for the snail's DNA methylation machinery in maintaining hybrid vigour or heterosis. Thirdly, locus-specific detection of 5mC by bisulfite (BS)-PCR revealed 5mC within an exonic region of a housekeeping protein-coding gene (Bg14-3-3), supporting previous in silico predictions and whole genome BS-Seq analysis of this species' genome. Finally, we provide preliminary evidence for parasite-mediated host epigenetic reprogramming in the schistosome/snail system, as demonstrated by the increase in Bgdnmt1 and Bgmbd2/3 transcript abundance following Bge (B. glabrata embryonic cell line) exposure to parasite larval transformation products (LTP). CONCLUSIONS/SIGNIFICANCE: The presence of a functional DNA methylation machinery in B. glabrata as well as the modulation of these gene products in response to schistosome products, suggests a vital role for DNA methylation during snail development/oviposition and parasite interactions. Further deciphering the role of this epigenetic process during Biomphalaria/Schistosoma co-evolutionary biology may reveal key factors associated with disease transmission and, moreover, enable the discovery of novel lifecycle intervention strategies.

(Epi)genetic Inheritance in Schistosoma mansoni: A Systems Approach.

The G×E concept, in which genotype × environment interactions bring about the phenotype, is widely used to describe biological phenomena. We propose to extend the initial notion of the concept, replacing G by 'inheritance system'. This system, comprised of both genome and epigenome components, collectively interacts with the environment to shape the development of a phenotype. In the case of the human blood fluke Schistosoma mansoni, responsible for intestinal bilharzia, the phenotypic trait that is most relevant to global health is infection success. Taking a systems biology view we show how genetic and epigenetic interactions result in ephemeral, but also heritable, phenotypic variations that are important for infection success.

DISMISS: detection of stranded methylation in MeDIP-Seq data.

BACKGROUND: DNA methylation is an important regulator of gene expression and chromatin structure. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is commonly used to identify regions of DNA methylation in eukaryotic genomes. Within MeDIP-Seq libraries, methylated cytosines can be found in both double-stranded (symmetric) and single-stranded (asymmetric) genomic contexts. While symmetric CG methylation has been relatively well-studied, asymmetric methylation in any dinucleotide context has received less attention. Importantly, no currently available software for processing MeDIP-Seq reads is able to resolve these strand-specific DNA methylation signals. Here we introduce DISMISS, a new software package that detects strand-associated DNA methylation from existing MeDIP-Seq analyses. RESULTS: Using MeDIP-Seq datasets derived from Apis mellifera (honeybee), an invertebrate species that contains more asymmetric- than symmetric- DNA methylation, we demonstrate that DISMISS can identify strand-specific DNA methylation signals with similar accuracy as bisulfite sequencing (BS-Seq; single nucleotide resolution methodology). Specifically, DISMISS is able to confidently predict where DNA methylation predominates (plus or minus DNA strands - asymmetric DNA methylation; plus and minus DNA stands - symmetric DNA methylation) in MeDIP-Seq datasets derived from A. mellifera samples. When compared to DNA methylation data derived from BS-Seq analysis of A. mellifera worker larva, DISMISS-mediated identification of strand-specific methylated cytosines is 80 % accurate. Furthermore, DISMISS can correctly (p <0.0001) detect the origin (sense vs antisense DNA strands) of DNA methylation at splice site junctions in A. mellifera MeDIP-Seq datasets with a precision close to BS-Seq analysis. Finally, DISMISS-mediated identification of DNA methylation signals associated with upstream, exonic, intronic and downstream genomic loci from A. mellifera MeDIP-Seq datasets outperforms MACS2 (Model-based Analysis of ChIP-Seq2; a commonly used MeDIP-Seq analysis software) and closely approaches the results achieved by BS-Seq. CONCLUSIONS: While asymmetric DNA methylation is increasingly being found in growing numbers of eukaryotic species and is the predominant pattern observed in some invertebrate genomes, it has been difficult to detect in MeDIP-Seq datasets using existing software. DISMISS now enables more sensitive examinations of MeDIP-Seq datasets and will be especially useful for the study of genomes containing either low levels of DNA methylation or for genomes containing relatively high amounts of asymmetric methylation.

Cytosine methylation is a conserved epigenetic feature found throughout the phylum Platyhelminthes.

BACKGROUND: The phylum Platyhelminthes (flatworms) contains an important group of bilaterian organisms responsible for many debilitating and chronic infectious diseases of human and animal populations inhabiting the planet today. In addition to their biomedical and veterinary relevance, some platyhelminths are also frequently used models for understanding tissue regeneration and stem cell biology. Therefore, the molecular (genetic and epigenetic) characteristics that underlie trophic specialism, pathogenicity or developmental maturation are likely to be pivotal in our continued studies of this important metazoan group. Indeed, in contrast to earlier studies that failed to detect evidence of cytosine or adenine methylation in parasitic flatworm taxa, our laboratory has recently defined a critical role for cytosine methylation in Schistosoma mansoni oviposition, egg maturation and ovarian development. Thus, in order to identify whether this epigenetic modification features in other platyhelminth species or is a novelty of S. mansoni, we conducted a study simultaneously surveying for DNA methylation machinery components and DNA methylation marks throughout the phylum using both parasitic and non-parasitic representatives. RESULTS: Firstly, using both S. mansoni DNA methyltransferase 2 (SmDNMT2) and methyl-CpG binding domain protein (SmMBD) as query sequences, we illustrate that essential DNA methylation machinery components are well conserved throughout the phylum. Secondly, using both molecular (methylation specific amplification polymorphism, MSAP) and immunological (enzyme-linked immunoabsorbent assay, ELISA) methodologies, we demonstrate that representative species (Echinococcus multilocularis, Protopolystoma xenopodis, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica and Polycelis nigra) within all four platyhelminth classes (Cestoda, Monogenea, Trematoda and 'Turbellaria') contain methylated cytosines within their genome compartments. CONCLUSIONS: Collectively, these findings provide the first direct evidence for a functionally conserved and enzymatically active DNA methylation system throughout the Platyhelminthes. Defining how this epigenetic feature shapes phenotypic diversity and development within the phylum represents an exciting new area of metazoan biology.

Epigenetics: a key regulator of platyhelminth developmental biology?

The Platyhelminthes (flukes/flatworms) are a large group of derived metazoans beautifully adapted for existence in diversely challenging ecosystems. As tractable examples of development and self-regeneration or as causative agents of aquacultural, veterinary and biomedically-relevant parasitic diseases, the platyhelminths are subject to intensive inter-disciplinary research. Given the complex lifestyles exhibited by individuals within this phylum, we postulate that epigenetic processes feature in many aspects of platyhelminth lifecycle diversity, development and environmentally-driven adaptations.

Cytosine methylation regulates oviposition in the pathogenic blood fluke Schistosoma mansoni.

Similar to other metazoan pathogens, Schistosoma mansoni undergoes transcriptional and developmental regulation during its complex lifecycle and host interactions. DNA methylation as a mechanism to control these processes has, to date, been discounted in this parasite. Here we show the first evidence for cytosine methylation in the S. mansoni genome. Transcriptional coregulation of novel DNA methyltransferase (SmDnmt2) and methyl-CpG-binding domain proteins mirrors the detection of cytosine methylation abundance and implicates the presence of a functional DNA methylation machinery. Genome losses in cytosine methylation upon SmDnmt2 silencing and the identification of a hypermethylated, repetitive intron within a predicted forkhead gene confirm this assertion. Importantly, disruption of egg production and egg maturation by 5-azacytidine establishes an essential role for 5-methylcytosine in this parasite. These findings provide the first functional confirmation for this epigenetic modification in any worm species and link the cytosine methylation machinery to platyhelminth oviposition processes.