1.1. Ethics statement

All animal experiments were conducted under Home Office Project Licence No. 80/2596. All protocols were presented and approved by the Animal Welfare and Ethical Review Body (AWERB) of the Wellcome Trust Sanger Institute. The AWERB is constituted as required by the UK Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012.

1.2. Parasite material

Infected Biomphalaria glabrata snails were purchased from BioGlab Ltd. Nottingham, UK and cercariae were harvested by exposure of infected snails to light for two hours in aquarium-conditioned water. S. mansoni intra-mammalian stages were collected from Balb/c mice previously infected with 300-pooled cercariae. For single sex infections (to retrieve unpaired male and female worms), mono-miracidia snail infections were performed and each snail tested for the production of male or female cercariae, by sex-specific PCR [37]. Balb/c mice were infected separately with either male or female cercariae. Juvenile and adult worms were recovered via perfusion of mouse circulatory system [2] at the indicated times post-infection (see Results). Male and female worms were separated, washed in DMEM, concentrated and stored in Trizol reagent (Invitrogen, UK) at -80°C.

1.3. RNA extraction, RNAseq library preparation and sequencing

RNA was isolated from samples stored in Trizol reagent (Invitrogen, UK) following manufacturer instructions. RNAseq libraries were prepared from 1ug of total RNA using TruSeq RNA Library Preparation Kit (Illumina, UK). All samples were processed with three biological replicates–worms from one mouse representing one biological replicate. Libraries were multiplexed and sequenced on Illumina HiSeq 2500 with 100 bp paired-end reads. Sequencing data was submitted to ArrayExpress under accession number E-ERAD-478.

1.4. Processing of poly-A enriched RNAseq sequencing reads and differential expression analysis of paired / unpaired male and female worms

RNAseq data from juvenile and adult, male and female schistosome worms were mapped to the reference S. mansoni assembly version 5.2 of the genome assembly [38] using Tophat2 (default parameters except: -g 1—library-type fr-firststrand -a 6 -i 10 -I 40000—microexon-search—min-segment-intron 10—max-segment-intron 40000). The resulting binary alignment mapping (BAM) files were sorted using Samtools [39] and reads per transcript calculated using HTseq-count [40] (parameters: -f bam -a 30 -t CDS -s reverse -m union). The GTF file used to calculate reads per transcript can be found in Supplementary S1 Dataset; only features starting with “Smp” (corresponding to protein coding genes) were taken into consideration. After mapping and counting, differential expression analysis was performed using DESeq2 [41] in the R environment [42]. Pairwise differential expression values for male and females worms (from mixed infections) between juveniles (21 days post infection d.p.i.) and adult worms (38 d.p.i.) were calculated and used as input for Sylamer (see below). Time-course expression data for male and female, paired and unpaired worms was generated using likelihood ratio test incorporated in DESeq2 package.

1.5. UTR prediction and experimental validation

RNAseq data generated from polyadenylated-enriched samples was used to improve the annotation of 3’-UTRs in the S. mansoni genome. BAM files generated with Tophat2 were merged using Samtools [39] and used as input for Cufflinks [43,44] (using default parameters except: -p 16—library-type fr-firststrand). To generate UTR sequences, RNAseq data and existing annotation were combined using a script developed in-house (https://github.com/sanger-pathogens/Assembly_tools/tree/master/assembly_tools/annotate_utrs_using_cufflinks). Briefly, for each existing gene, the script takes an intersecting annotation from the cufflinks GTK output file and extends the existing gene model using the provided annotation, labelling the new part of the annotation as a UTR. If the potential UTR happens to overlap a second gene, then it is not used. As a result, 3,321 3’-UTRs from a total of 7,373 3’-UTRs were updated using this method. Likewise, 4,081 5’-UTRs from a total of 7,271 5’-UTRs were updated. The total number of protein-coding genes is 10,841. Note that this version of the annotation is a “snapshot” and may not coincide with the current version in http://www.genedb.org/Homepage/Smansoni or http://parasite.wormbase.org/Schistosoma_mansoni_prjea36577/Info/Index/.

A selection of twelve 3’-UTRs with lengths ≥ 600 bp, and a range of RPKM expression values between 9–3,600 in adult worms, were chosen for validation by PCR (Supplementary S1 Fig). Primers were designed using primer3 batch service [45] using default parameters except amplicon length, which was set to 500 bp. PCR reactions were carried out using Qiagen Fast Cycling PCR Mix (Qiagen, UK) using standard conditions with annealing temperature 55C and mix sex adult cDNA as template. The list of primers is shown in Supplementary S1 Table.

1.6. Sylamer analysis

Sylamer [36] is a tool to identify miRNA regulation effects from a list of differentially expressed genes, independently from miRNA measurements. It can either be used to suggest candidate miRNAs for follow-up analysis in the absence of miRNA measurements, or to confirm that putative miRNA target genes are shifted concordantly with miRNA expression changes when miRNA measurements are present. The latter is the case here. It has previously been used, among many more applications, to identify the role of miRNAs in germcell tumors [46], the effect of a miRNA seed mutation on gene expression [47], and identification of miRNA targets in murine Dgcr8-deficient embryonic stem cell lines [48]. Sylamer’s modus operandi is briefly described here, for full details refer to [36]. Several nucleotide words in the eight-nucleotide stretch (8-mer) at the 5' end of a miRNA are core determinants of miRNA binding [34,49]. These are the 8-mer itself, the two 7-mers, the core 6-mer, and the leading 6-mer. The nucleotide sequences that are complementary to these miRNA seed words are called Seed Complementary Regions (SCR). Sylamer considers a list of differentially expressed genes and tests the hypothesis that SCRs in 3’-UTRs are shifted towards one end of the gene list against the null hypothesis that these sites are homogeneously distributed throughout the gene list. It does so whilst taking into account UTR length and correcting for composition biases, using a hypergeometric model of nucleotide word occurrences in UTRs. Analogously to Gene Set Enrichment Analysis [50], Sylamer employs a moving rank cut-off and can hence detect shifts of SCRs that are dispersed to a greater or lesser extent.

Sylamer plots construction.

All information produced by a Sylamer run can be summarised in a single plot, as a collection of lines where each line represents a single SCR. In a single Sylamer run, the word length is kept constant, so different plots are obtained for SCRs of length 6, 7 and 8, respectively. A single line in a Sylamer plot describes a single SCR as follows: at different cut-offs in the list of UTRs, a hypergeometric test is performed considering the summed total length of the UTRs up to that cut-off (representing the number of all SCRs), the summed total length of all UTRs, the number of occurrences of the particular SCR up to the cut-off, and the total number of occurrences of the particular SCR in all UTRs. If the SCR is enriched in the subset of UTRs, the -log10(p-value) is drawn (hence at the positive y-axis). If is instead depleted, the log10(p-value) is drawn (at the negative y-axis). Multiple cut-offs are tested and a large number of SCRs are tested. On the one hand, this requires multiple testing correction, on the other hand this allows a single SCR result to be evaluated against the background of plotted lines for all SCRs. A Bonferroni-adjusted significance threshold of 0.05 (as drawn in Fig 1A and 1B) typically delineates and encloses the background distribution with significant SCRs jutting out.

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Fig 1. MiRNA target prediction based on both miRNA-unaware and miRNA-guided approaches.

(A) Sylamer enrichment landscape plots for 7mers in male (top) and female (bottom) expression data. The x-axis represents a list of transcripts, ranked from more expressed in juveniles to more expressed in adults. The y-axis represents the significance values acquired for each 7mer at each position in the ranked list of transcripts. Coloured boxes represent the fraction of transcripts significantly (adjusted p-value < 0.01) differentially expressed between juvenile and adult worm as found using DESeq2. These transcripts were subsequently filtered based on the presence of the 7mers TGCATTT or GCATTTA as found by Sylamer. The resulting sets are referred to as Male and Female Sylamer genes. (B) Venn Diagram showing the intersection of Male and Female Sylamer genes with schistosome-conserved miRNA targets as found using TargetScan with conservation + miRanda. The overlap represents transcripts with highly conserved sma-miR-277 target sites across the three Schistosome spp (S. mansoni, S. haematobium and S. japonicum) that are also significantly down regulated during worm development.

https://doi.org/10.1371/journal.pntd.0005559.g001

Additional significant SCRs above the Bonferroni-adjust threshold may occur in a Sylamer plot. This can be due to several causes. Firstly, multiple regulatory elements besides SCR are present in UTRs, such as poly-A and ARE (AU-rich element) signals. MiRNA SCRs may share such motifs. Secondly, repeated sequence fragments in UTRs of related genes may cause sharp spikes in Sylamer plots, and stretches of low complexity sequence can cause significant results for SCRs matching such a sequence. This is overcome (as done here) by pre-processing the UTRs to remove low complexity stretches with DUST [51] and repeated sequence fragments with the RSAT [52] interface to the Vmatch program (http://www.vmatch.de). Finally, a significant SCR signal can cause words that are very close to it to piggyback the signal and achieve elevated significance. This is then evident from the words involved.

1.7. miRNA target prediction (miRanda and TargetScan) and gene ontology enrichment

Targets were predicted in the 3’-UTRs of 4,851 genes for previously reported S. mansoni and S. japonicum miRNAs and the novel miRNA from the present study. First, miRanda (v 3.3a, ref. [54]) was run with default parameters against the predicted set of S. mansoni 3’-UTRs. Second, TargetScan (v 6.0, ref. [55]) was used to search the same 3’-UTRs dataset; the script targetscan_60.pl was run with default parameters, using the seed sequences of each Schistosoma spp. miRNA (from miRBase, release 21, ref. [56]) and alignments of orthologous 3’-UTRs (see below) between S. mansoni, S. japonicum and S. haematobium. Using custom scripts, only S. mansoni TargetScan target site predictions conserved among the three Schistosoma species were retained, i.e. requiring the target prediction to be present on the same exact aligned location on the all orthologous 3’-UTRs. Further to this, only a high-confidence set of predictions found by both TargetScan and miRanda were retained. Except for S. mansoni, current 3’-UTR predictions for the other two Schistosoma species used were not reliable; therefore new 3’-UTR predictions for S. haematobium and S. japonicum were created based on orthology with S. mansoni. For each S. mansoni gene the mRNA isoform was selected with the longest spliced sequence, and for genes with annotated 3’-UTRs, orthologous genes in S. japonicum and S. haematobium were identified using a custom-made EnsemblCompara database [57]. For each S. japonicum or S. haematobium orthologue, a predicted 3’-UTR was created with the same length of the 3’-UTR of the respective S. mansoni orthologous gene (up to maximum of 5 kb or to the end of underlying sequence scaffold). These predicted sets of 3’-UTRs were termed “orthologous 3’-UTRs”, even though orthology is based on the underlying gene product to which they correspond. Alignments of orthologous 3’-UTR sets for the TargetScan predictions were produced with MUSCLE v3.8.31 [58], with default parameters. Assemblies and gene sets of the three species used for accessing conservation of miRNA target predictions can be downloaded at WormBase ParaSite release 4 (http://parasite.wormbase.org/ftp.html): S. haematobium ‘S.haematobium.v3.0’ from the University of Melbourne, and S. japonicum ‘ASM15177v1’ from the Chinese National Human Genome Center. The S. mansoni assembly used here is the most updated version reported previously [38].

Gene Ontology enrichment analysis was performed using the TopGO package [59].

1.8. Processing of small RNA-seq libraries and genomic localisation of sma-miR-277/4989 cluster

We used previously generated small RNA-seq libraries from schistosomula stages of S mansoni to assist our description of the sma-miR-277 locus (European Nucleotide Archive study PRJEB3190). Kraken [60] was used to remove adapter contamination from libraries and collapse identical reads into single sequences while maintaining annotated depth information (Supplementary S2 Table). Reads were then mapped against each other using BLASTN [61,62] to determine all pairwise similarities between reads allowing up to two mismatches (E-value < = 0.1). A pairwise similarity matrix was used to cluster reads using MCL [60,63]. Multiple sequence alignment of each cluster was performed using Clustal Omega [64]. From each cluster, a ‘sentinel’ read was chosen with the highest depth and mapped to the reference genome using Bowtie [65], allowing up to two mismatches.

After identifying the genomic location for the candidate and in order to obtain the putative miRNA precursor sequence, the sequences were extended in both directions. The first extension took 50nt upstream and 100nt downstream while the second extension took 100nt upstream and 50nt downstream of the cluster. Secondary structures of these putative miRNA precursors were then assessed using RNAfold from the Vienna package [66], and structures discarded with minimum free energy (MFE) > -20 kcal/mol. For each cluster, the extended sequence with the lowest associated MFE structure was retained.

1.9. RT-qPCR / miRNA expression analysis

Real-time quantitative PCRs were performed across two life cycle stages of S. mansoni representing juvenile (28 days post-infection, d.p.i.) and adult worms (49 d.p.i.) using TaqMan Small RNA Assays (Supplementary S1 Text) purchased from Applied Biosystems (Life Technologies, UK). Reverse transcription and quantitative PCR experiments were performed according to manufacturer’s instructions in a StepOnePlus RT-qPCR machine (Applied Biosystems, Life Technologies, UK). S. mansoni U6 was used as the endogenous control [20] (gene entry sma.U6.1.1 in http://www.genedb.org/Homepage/Smansoni) and fold changes to miRNA expression were estimated using the delta-delta-Ct method [67].

1.10. Whole mount and fluorescence in situ hybridisation of miRNAs

Forty-two to 49 d.p.i. male and female worms from mixed infections were retrieved from infected mice, as described above, and processed using previously published protocols for in situ hybridization [68]. Parasites were hybridized with 21–22 nt antisense LNA-DNA probes conjugated to Digoxigenin (Exiqon, Denmark) at a final concentration between 6.5–50 nM. Arabidopsis thaliana ath-miR-159a or scrambled sequences were used as negative control. For FISH to detect prohormone convertase 2 (Smp_077980) or Tegumental cells, we synthesized FITC-conjugated probes generated from in vitro transcription of cloned cDNA. To robustly detect tegumental cells we employed a mixture of four FITC-conjugated antisense probes targeting the following tegument-specific mRNAs: calpain (Smp_214190), gtp -4 (Smp_105410), annexin (Smp_077720), and npp-5 (Smp_153390) (ref: [69–73]).

2.1. Target-prediction suggests sma-miR-277/4989 are prominent post-regulatory miRNAs in developing juvenile worms

In our analysis, Sylamer [36] was used to search for enriched short sequences, corresponding to potential miRNA target sites, from a list of genes differentially expressed between juvenile and adult male and female worms. Sylamer finds enriched “words” (in our case, Seed Complementary Regions or SCR) in the 3’-UTRs of transcripts with similar expression profiles (i.e. up-regulated or down-regulated). The advantage of this method is that potential targets can be identified without prior knowledge of the specific miRNAs that affect the transcriptome. Sylamer indicated that the 7mers TGCATTT/GCATTTA, corresponding to the SCR of miR-277 family, were significantly enriched in male (p-value = 1.62E-13, Supplementary S3A Table) and female (p-value = 1.22E-06, Supplementary S3B Table) genes that were more highly expressed in juvenile worms compared to adults (Fig 1A).

Genes with 3’-UTRs containing the 7mers TGCATTT/GCATTTA were selected from the list of transcripts significantly upregulated (adjusted p-value ≤ 0.01) in juveniles compared to adult worms, using genes found in males (n = 1,099) and females (n = 1,857) separately. Among these transcripts, 225 male and 429 female transcripts contained the 7mers TGCATTT/GCATTTA in their 3’-UTRs (as predicted by Sylamer) and therefore were regarded as potential targets for miRNAs of the sma-miR-277 family. We refer to these groups as male and female “Sylamer genes” (Supplementary S2 Fig).

Secondly, we employed miRanda [54] and TargetScan with conservation evidence [55] to perform miRNA-guided predictions of potential targets in the transcriptome. This latter approach is based on curated three-way alignments of 3’-UTR sequences from orthologous genes from S. mansoni, S. haematobium and S. japonicum (see Materials and Methods). Unlike Sylamer, the combined results of miRanda (Supplementary S4E Table) and TargetScan with conservation (Supplementary S4F Table) only take into account known miRNA seeds in the 3’-UTRs and not the expression of their transcripts.

We further refined this list by selecting those Sylamer genes that also have conserved target sites across the three schistosome species (Fig 1B) as identified by combining miRanda [54] and TargetScan with conservation [55]. Notably, miRanda and TargetScan with conservation alone found 98 potential targets for the sma-miR-277 family in the Schistosoma transcriptome (Supplementary S4 Table), considerably higher than for any other miRNA in this study. Of these, 46 genes were also “Sylamer genes” (26 male and 34 female gene with 14 shared between the two genders—Fig 1B and Table 1), we called these “high confidence targets”. By performing a hypergeometric test, we calculated the probability that these overlaps are significant, using as background the 1,476 protein-coding genes that possess at least the core sma-miR-277 6mer GCATTT. Our results show that there is significant enrichment in the overlaps between TargetScan+Miranda vs. Male Sylamer genes (p-value < 0.002) and also between TargetScan+Miranda vs. Male (p-value < 1e-50) while the overlap between TargetScan+Miranda vs. Female Sylamer genes was not significant.

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Table 1. High confidence targets of the sma-miR-277 family identified with a combined approach (Sylamer, miRanda and TargetScan with conservation).

F = female; M = male.

https://doi.org/10.1371/journal.pntd.0005559.t001

Gene Ontology (Biological Processes) enrichment analysis of the male and female high confidence targets shows significant enrichment (p-value < 0.01) for “chromatin assembly or disassembly” (four genes: Smp_155060, Smp_041760, Smp_158610, Smp_079650), “regulation of transcription, DNA-templated” (11 genes: Smp_042300, Smp_009630, Smp_147950, Smp_139530, Smp_041760, Smp_158610, Smp_079650, Smp_021340, Smp_163240, Smp_100090, Smp_008830) and “protein dephosphorylation” (three genes: Smp_173900, Smp_169320, Smp_149000) categories (Supplementary S5 Table).

2.2. Genomic localisation, structural characterisation of sma-miR-277 and identification of sma-miR-4989

Using Drosophila melanogaster dme-miR-277 as a query in the miRBase database (Release 21, [56]) we found full-length sequence similarity and seed sequence (nucleotides 2–7) identity with sja-miR-277 from S. japonicum [28] as well as with two Echinococcus spp miRNA sequences namely egr- or emu–miR-277 and miR-4989 [74].

Sma-miR-277 has previously been found in vesicles derived from cultured larval stages of S. mansoni [75] as well as circulating in naturally infected patients and experimentally infected animals [76]. However, in both reports the genomic location of this miRNA was not described. In tapeworms, miR-277 is located within a cluster with miR-4989 [77], i.e. they are very close together in the genome (less that 10 Kb) and miRNAs encoded in clusters are likely to be transcribed as a single precursor unit [78]. A search for the genome locus of miR-277 in S. mansoni showed that it is found in Chromosome 4 (21600667–21600688—reverse). We extended our analysis to include alignments of previously generated small RNA-seq libraries resulting in the identification of another miR-277 family member located in the vicinity of sma-miR-277. Sma-miR-4989 (named by us “novel255”) is located within ~200 bp of sma-miR-277 suggesting that, as is the case in tapeworms, these two miRNAs form a miRNA cluster. Other sequencing reads found in the same region suggested the presence of passenger strands or miRNA* (Fig 2A). We found that sma-miR-4989 (novel255) folds into a stable structure with its passenger strand (novel2620), the two miRNAs matching to form the lower part in a precursor with an extended stem. The miRNAs sma-miR-277 and its passenger (novel3014) match up similarly, again forming the lower part of an extended stem (Fig 2B). These two extended stem-loop structures were consistently identified among different excisions of the region. They persist when folding the whole (1,700 nt) genomic loci (Fig 2C), where the region folds into a highly stable stem-rich structure with low minimum free energy. To assess the significance of this fold, we randomly shuffled its sequence while preserving dinucleotide frequency. An ensemble of 1,000 randomly shuffled sequences was folded. This yielded a distribution of minimum free energy scores, leading to a Z-score of 3.5 for minimum free energy of the genomic sequence fold, confirming the stability of the stem-rich structure. FASTA sequences for the hairpins, mature and passenger strands are presented in Supplementary S2 Dataset and an image comparing the folds obtained for different S. mansoni predicted miRNAs is presented in Supplementary S4 Fig.

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Fig 2. Sma-miR-277 and sma-miR-4989 belong to a gene cluster.

(A) The genomic locus in Chromosome 4 of sma-miR-277 and sma-miR-4989 suggests they belong to a gene cluster. The average distance between genes (represented by coloured boxes) is 109 bases. Here the mature miRNAs (sma-miR2-277 and sma-miR-4989) and passenger miRNAs are represented with coverage plot and aligned reads from one of the small RNA libraries. (B) Predicted stem-loop structures for sma-miR-277 and sma-miR-4989 –individual cases. Mature miRNAs are located in the 3’-end of the hairpin. (C) Due to the cluster organisation of sma-miR-277/sma-miR-4989, it is likely that they are transcribed as one precursor RNA molecule. This figure represents the predicted stem-loop structure for sma-miR-277/sma-miR-4989 when arising from a larger transcript.

https://doi.org/10.1371/journal.pntd.0005559.g002

2.3. Targets of sma-miR-277/sma-miR-4989 are down regulated in paired females compared with unpaired females

Female worms only achieve sexual maturity when they are coupled with a male worm. Although highly unlikely in natural infections, it is possible to obtain unpaired “virgin” females and unpaired males by infecting mice with single-sex cercariae. Using RNASeq data from paired and unpaired males and females, we found that 16 out of the 34-female high-confidence targets of sma-miR-277 family had significantly (adjusted p-value <0.01) lower expression in paired females compared to unpaired “virgin” females (Fig 3B), while the male targets were not differentially expressed between paired and unpaired males (Fig 3A). Furthermore, the time-course expression analysis indicated that the expression of these targets was gradually reduced as female maturation progressed. Notably, this was not observed in males.

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Fig 3. Sma-miR-277 family predicted targets downregulated in developing female parasites.

Fold change expression (Log₂) of high confidence targets of sma-miR-277 family during the development of male and female worms in two conditions: paired (solid line red) and unpaired (dashed green). Black lines represent the mean expression of genes in paired (solid black line) and unpaired (dashed black line) worms.

https://doi.org/10.1371/journal.pntd.0005559.g003

2.4. Sma-miR-4989 is up-regulated during juvenile to adult transition

Tentative miR-277 targets were down-regulated during the juvenile to adult transition. If the miRNA effect is that of causing the decay of its targets, it is expected that the expression of miRNAs from that family would show an opposite pattern to that of its targets, i.e. while the targets of the miR-277 family are down-regulated, sma-miR-277 and sma-miR-4989 should increase their expression.

The expression sma-miR-4989 was quantified by RT-qPCR during juvenile to adult development. This miRNA showed significant up-regulated (28 vs. 49 d.p.i., males t-test p-value = 0.0097; females t-test p-value = 0.01) expression in developing worms (Fig 4) suggesting an association between increased expression in miRNA levels and decreased expression of their targets. Furthermore, sma-miR-277 is also up-regulated in a similar manner, as well as sma-miR-4989 passenger miRNA novel2620 (Supplementary S3 Fig).

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Fig 4. Sma-miR-4989 is significantly up-regulated during male and female maturation.

Fold change expression of sma-miR-4989 during development of juvenile to adult worms in male (blue bars) and females (red bars) as measured by RT-qPCR. Samples were collected at the time points (days post infection) indicated in the x-axis from murine hosts infected with pooled (mixed sex) cercariae. Each barplot represents the mean of three biological replicates. T-tests were performed between 49 d.p.i. and 28 d.p.i. and were both significant with p-value ≤ 0.01. Error bars show the standard error of the mean, based on three biological replicates.

https://doi.org/10.1371/journal.pntd.0005559.g004

2.5. Sma-miR-4989 mRNA is localised in the male anterior oesophagus gland

In order to gain insight into the potential functions of sma-miR-4989, we used both whole mount in situ hybridisation (WISH), as well as fluorescence in situ hybridization (FISH) in adult male and female worms. Given the small size of miRNAs we employed anti-sense Locked Nucleic Acid (LNA) probes to detect the cells expressing these RNAs. To determine the specificity of this approach we performed control experiments with LNA probes targeting a 22nt long sequence of the CathepsinB messenger RNA (Smp_103610), which is detected in the intestine by WISH [68,79]. Consistent with previous reports, we robustly detected cathepsinB in the intestine (Fig 5A). To further assess the feasibility to detect Schistosome miRNAs using LNA probes we also examined sma-miR-124a-3p, whose closely related C. elegans homolog (miR-124a) is expressed in the nervous system [80]. Consistent with the localization of miR-124a in C. elegans, we detected the expression of sma-miR-124a-3p in the schistosome cephalic ganglia and in the nerve chords by WISH (Fig 5B). To determine if this miRNA was broadly expressed in the cephalic ganglia or in subsets of cells in the cephalic ganglia, we performed double FISH with prohormone convertase 2 (pc2), that is expressed in the large number of cells in the schistosome nervous system. Schistosomes possess a pair of cephalic ganglia whose neuronal cell bodies surround a neuropil composed of a network of neural projections. The individual ganglia are connected via a commissure of neural projections that extend across the midline. We observed that sma-miR-124a-3p and pc2 were co-expressed in cell bodies that comprise both the cephalic ganglia and the nerve chords (Supplementary S5A Fig). Interestingly, we detected sma-miR-124a-3p not just in pc2⁺ cell bodies but also broadly throughout the neuropil of the cephalic ganglia (Supplementary S5B Fig). Since the neuropil is comprised of neural projections (axons and dendrites) these data suggest that sma-miR-124a-3p may have functions to regulate its target mRNAs outside the cell bodies.

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Fig 5. Sma-miR-4989 is expressed in the cells surrounding the oesophagus and cells of the tegument in adult worms.

Whole mount in situ hybridisation for (A) cathepsin B, (B) sma-miR-124a-3p (124a), and (C) sma-miR-4989. (D) Fluorescence in situ hybridisation showing the colocalization of sma-miR-4989 with four co-expressed tegument-specific mRNAs (calpain, npp-5, annexin and gtp-4). Nuclei are stained with DAPI and shown in blue. Anterior of worms is to the left in A-C. Scale Bars: A-C 100 μm; D 10 μm.

https://doi.org/10.1371/journal.pntd.0005559.g005

Given our success in detecting sma-miR-124a-3p, we set out to determine the localization of sma-miR-4989. By WISH, we failed to detect reproducible signal in female parasites. However, we strongly detected sma-miR-4989 in cells surrounding the male oesophagus (Fig 5C) as well as cells throughout in the parenchyma. Since many of the sma-miR-4989-expressing cells in the parenchyma appeared to be quite superficial, just beneath the parasites muscle layer, we explored the possibility these cells comprise the schistosome syncytial epidermis, a structure known as the tegument. To test this, we utilized a mix of probes targeting the mRNAs of four well-characterized tegumental factors: calpain, npp-5, annexin and gtp-4 (ref. [69–73]). By FISH we observed that the sma-miR-4989 RNA was broadly expressed in cells that make up the schistosome tegument (Fig 5D).