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Profiling microRNAs through development of the parasitic nematode Haemonchus identifies nematode-specific miRNAs that suppress larval development

Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.


Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms.
Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.
For parasitic nematodes, identifying the mechanisms regulating co-ordinated expression of mRNA transcripts is important in understanding parasite development in response to varying host or environmental conditions. Here we focus on changes in microRNA (miRNA) levels during development of the pathogenic gastrointestinal nematode Haemonchus contortus. miRNAs are small (21-23 nucleotides) non-coding RNAs found in animals, plants and viruses that regulate gene expression post-transcriptionally. miRNAs bind with partial sequence complementarity to sites, most often in the 3′ untranslated region (UTR) of their target mRNAs 1 , resulting in translational repression and mRNA degradation 2 . The association of specific miRNAs with physiological and pathological conditions has led to their exploitation as biomarkers of disease 3 and development of miRNA mimics and inhibitors as novel therapeutics 4,5 .
miRNAs were first identified in the free-living nematode Caenorhabditis elegans, with lin-4 and let-7 shown to be essential regulators of development 6,7 . Individual loss of most C. elegans miRNAs leads to no obvious phenotype under laboratory conditions 8 . Neverthless, miRNAs have subtle roles in specific processes or under stress conditions, as identified using specific assays [9][10][11][12] . While there is a wealth of data on C. elegans miRNA expression and function, little is known of miRNA function in parasitic nematodes. The availability of genome sequence data for a number of helminth species has enabled identification of parasite miRNAs by deep sequencing and computational approaches (reviewed in 13 ). We previously identified 192 miRNAs in H. contortus by deep sequencing of small RNAs expressed in infective L3 and mixed sex adult worms 14 . Identifying in more detail when and where these are expressed, and the genes they target, will help reveal the regulatory mechanisms controlling parasite development and adaptation to the host environment.
For many parasitic nematodes, infection of vertebrate hosts is initiated by the infective L3 stage, considered analogous to the developmentally arrested dauer larvae of C. elegans that form in response to unfavourable conditions (starvation and crowding) 15 . In C. elegans, development is promoted by the insulin/IGF-1 and TGF-β signaling pathways, that ultimately lead to binding of steroid hormones to the nuclear hormone receptor DAF-12 16 . While DAF-12 and components of the insulin signaling pathway, daf-2 and daf-16, are conserved in parasitic nematodes [17][18][19] , the mechanisms that regulate parasite larval activation and development have yet to be identified 20 .
The aim of this work was to profile the expression of miRNAs through different developmental stages of a gastrointestinal parasitic nematode, and determine the potential targets and functions of some of these. We focused on H. contortus, a highly pathogenic, blood-feeding parasite of small ruminants with a global distribution. Genome and transcriptome information is well-advanced for H. contortus 21,22 , with the genome assembled in chromosomes 23 , making it an important model for related nematodes, including human hookworms 24 . As with other parasitic nematodes, control of H. contortus infection relies largely on treatment with broad-spectrum anthelmintic drugs, but the efficacy of these is under serious threat from drug resistance 25 . Identifying the regulatory molecules and pathways essential for nematode development has the potential to lead to novel therapeutics for nematode control.

Results
Microarray profiling of H. contortus miRNAs. A custom microarray containing probes to H. contortus and C. elegans miRNAs (Methods) was screened with RNA from five H. contortus life-cycle stages: sheathed infective L3, exsheathed L3 (exsheathed in 5% sodium hypochlorite solution and cultured in vitro at 37 °C for 24 h), L4 larvae collected from the abomasum of infected sheep 7 days post-infection, and adult male and egg-producing female worms collected 28 days post-infection. In addition, the array was probed with RNA from gut tissue dissected from adult female worms. Patterns of expression for hco-let-7 and hco-lin-4 were similar to that reported in C. elegans 26 supporting the reliability of the data ( Fig. 1 and Supplementary Table S1). From normalised data, ANOVA identified 55 miRNAs showing significant variation in expression (p < 0.05; microarray fluorescent signal >500) between at least two life-cycle stages or between adult whole worm extract and gut tissue. No difference was found for any probe between sheathed versus exsheathed L3, consistent with the reported paucity of changes in H. contortus mRNA expression following L3 exsheathment by the same method 21 ; only data for sheathed L3 were examined further. Hierarchical clustering ( Fig. 1) identified five major miRNA groups based upon their enriched expression in each life-cycle stage or in adult female gut tissue. Based on our criteria, most differentially expressed miRNAs were conserved in other nematodes (47%) or in different phyla (30%), with only 23% unique to H. contortus (miRBase release 21) ( Supplementary Fig. 1). Of the lowest expressed miRNAs on the array (fluorescent signal <500), most (91%) have been found to date only in H. contortus. These miRNAs may not yet have developed significant functional roles or, alternatively, may be expressed in specific cells or tissues, and therefore of low abundance.

H. contortus miRNAs are developmentally and spatially regulated. Pairwise t-test comparisons
(p-values < 0.05; Log 2 fold change >2) identified four miRNAs with significantly greater abundance in infective L3 relative to L4 stage (volcano plot Fig. 2a and Supplementary Table S3). Of these, expression of hco-mir-228-5p and hco-mir-235-3p peaked in L3 (Fig. 1), while most H. contortus miRNAs were not abundantly expressed in L3 larvae. Fourteen miRNAs were significantly more abundant in L4 relative to L3 stage (Fig. 2a and Supplementary  Table S3), while 11 and 16 miRNAs were significantly upregulated in adult male or female worms, respectively, relative to L4 (Fig. 2b,c, Supplementary Table S3), suggesting possible roles in reproduction or in adult worm survival/lifespan. Five miRNAs were more abundant in adult female gut tissue relative to adult female whole worm extract (see Fig. 2d and Supplementary Table S3), two of which were enriched in L4 stage, suggesting possible roles in gut development (hco-mir-60-3p and hco-mir-236-3p). Blood-feeding begins in L4 larvae and development from L3 to L4 is marked by enlargement of the gut lumen and a switch in metabolic pathways 21 . In C. elegans, mir-60 and mir-236 are also gut expressed 27 , suggesting similar regulatory functions across nematode species. Only a single miRNA, hco-mir-5948-3p, was enriched in adult males relative to adult female worms (Fig. 2e). qPCR of selected miRNAs showed good correlation with the microarray data (Fig. 3). For hco-mir-228-5p and hco-mir-235-3p (peak in L3), both were expressed in the pre-infective L1 stage, but at a lower level than in L3 (57% and 49% of L3 level, respectively) ( Supplementary Fig. 2).
Some of the miRNAs profiled here by microarray were also identified in a recent study by Ma et al. 28 using small RNA library sequencing to characterise the miRNAome of L3, exsheathed L3 and in vitro generated L4 of H. contortus. Fewer miRNAs were found (88) but for those identified in both studies, the developmental expression patterns were similar. In the present study, in addition to confirming upregulation of hco-mir-5976-5p in L4 versus L3 stage 28 , we also show that this miRNA is abundant in gut tissue (Fig. 1). www.nature.com/scientificreports www.nature.com/scientificreports/ L4, after 48-72 h, relative to EBSS medium/37 °C/5% CO 2 alone ( Fig. 4a; P = 0.0039 at 1 μM DA). L4 larvae were identified by their open buccal cavity and enlarged gut lumen (Fig. 4b). This is similar to recent findings from Ma et al. 30 who achieved L3 to L4 development with exogenous DA and also identified endogenous Δ7-DA in H. contortus L3 larvae. Consistent with these observations, we identified expression of a homologue of C. elegans daf-12 in H. contortus L3 by RT-PCR (GenBank Accession number MN017114).
Addition of mimics of hco-mir-228 and hco-mir-235 in combination, either in the presence or absence of DA, did not significantly reduce the rate or number of L3 developing to L4 in vitro, relative to cultures treated with a control miRNA mimic (up to 2 μM) (not shown). Conversely, in the presence of inhibitors for both hco-mir-228 and hco-mir-235 there was a slight, statistically significant (P = 0.038) increase in the percentage of L3 developing to L4 after five days in culture, but not with control inhibitor (concentration 2 μM) ( Fig. 4c; 87% L4 with inhibitors; 77% L4 with control inhibitor). Albeit slight, this is the first report, as far as we are aware, of a phenotypic effect of miRNA inhibition in a parasitic nematode and suggests that hco-mir-228 and hco-mir-235 have an inhibitory effect on larval development that can be observed in vitro. mir-228 and mir-235 regulate development in C. elegans. Stable gene knockout is not currently feasible in H. contortus or most other parasitic nematodes. We adopted a comparative approach and examined effects of genetic deletion of mir-228 and mir-235 on dauer formation in the related clade V nematode C. elegans. No defects in dauer arrest or development have been reported for these miRNAs, however cel-mir-235 is required for maintaining L1 arrest, when eggs hatch in the absence of food 12 , while dietary restriction of C. elegans adult worms induces expression of mir-235 31 , mir-228 and mir-71 32 .
Genetic deletion of cel-mir-228 (strain MT14446) or cel-mir-235 (strain MT17997) individually caused an insignificant reduction in dauer formation relative to C. elegans wild-type N2 strain (Fig. 5a). We generated a cel-mir-228 and cel-mir-235 double mutant strain (CLB061) and this showed a significant decrease in dauer arrest (46% reduction, P = 0.012), indicating that these miRNAs function together to regulate development. In contrast, deletion of the gut-expressed cel-mir-60 (strain MT16471) had no effect on number of dauers (Fig. 5a). Increasing the concentration of pheromone to 3% did not significantly increase the number of dauers formed in the mutant strains, and the double mutant (cel-mir-235; cel-mir-228) showed normal dye filling with the lipophilic dye Dil, indicating normal amphid structure (data not shown). www.nature.com/scientificreports www.nature.com/scientificreports/

Loss of mir-228 and mir-235 can suppress daf-2 loss of function effect. C. elegans development
to reproductive stage requires activation of the DAF-2 insulin receptor and daf-2 loss of function (lf) mutants are dauer constitutive (daf-c) at restrictive temperature 33 . Daf-2(lf) effects are dependent on DAF-16 FOXO transcription factor, which regulates many genes involved in developmental arrest, reproduction and longevity 34,35 . We investigated whether cel-mir-228 and cel-mir-235 may synergise with DAF-16 by determining if these miR-NAs were required for daf-2(lf) effects. At 25 °C, daf-2(e1370) forms 100% dauers; additional loss of mir-228 and mir-235 (strain CLB065 (mir-235(n4504)I; daf-2(e1370)III; mir-228(n4382)IV) showed no reduction in the highly penetrant daf-c phenotype at 25 °C. However, at 22.5 °C, daf-2(e1370) develop beyond dauer stage and become sterile adults or adults producing embryonic lethal progeny 36,37 . At 22.5 °C, triple mutant strain CLB065 produced a significantly greater number of viable progeny relative to the daf-2 mutant strain; CLB065 produced 69% of the number of progeny produced by wild-type N2 or daf-16:daf-2 mutant, significantly more than daf-2 mutant which produced only 9% of the number of progeny produced by N2 or daf-16; daf-2 mutant (p = 0.005 and p = 0.000018 for CLB065 lines 1 and 2, respectively) (Fig. 5b). In addition, loss of mir-228 and mir-235 in strain CLB065 suppressed the protruding vulva phenotype observed in daf-2(e1370) adults at 22.5 °C (Fig. 5c). Therefore, mir-228 and mir-235 synergise with DAF-16 activity and may act in parallel or downstream of DAF-16 to suppress development. The differences observed at 25 °C and 22.5 °C suggest that mir-228 and mir-235 function at a threshold level of DAF-16: these miRNAs can enhance the effects of DAF-16 and add robustness to DAF-16 www.nature.com/scientificreports www.nature.com/scientificreports/ activity at non-saturating levels (22.5 °C) but not at saturating, high levels (25 °C), consistent with a role of miR-NAs in fine-tuning gene function.

Predicted targets of mir-228 and mir-235 regulate nematode metabolism and development.
Target prediction was carried out using 3′UTR databases for C. elegans and H. contortus to identify possible mechanisms by which mir-228 and mir-235 may regulate development. For cel-mir-228, multiple target sites were predicted by PITA 38 and TargetScan 39 , 56 of which were identified by both algorithms (Supplementary Table S4). These predictions were refined to a subset of 13 high confidence genes (see Methods) ( Table 1) and included genes encoding enzymes that function in metabolic pathways, particularly lipid, amino acid and carbohydrate metabolism. For C. elegans mir-235, again multiple targets were predicted using PITA and TargetScan, 87 of which were common to both algorithms (Supplementary Table S5). Five targets satisfied the high confidence criteria (see Methods) and these encoded genes involved in development and lipid storage/metabolism (Table 1). Interestingly, transcription factor ETS-4 is involved in lipid catabolism 40 and is also a proposed antagonist of DAF-16 function in C. elegans longevity 41 . This suggests a potential mechanism for mir-235 to enhance DAF-16 activity, by suppressing ETS-4.
GO analysis of the target gene lists for cel-mir-228 showed significant enrichment for KEGG pathways relating to metabolism and development, including valine, leucine and isoleucine degradation ( Table 2 and  Supplementary Table S6); these are branched chain amino acids (BCAAs) that accumulate in different long-lived mutants of C. elegans 42 . A more limited range of GO terms were enriched for targets predicted for cel-mir-235, including locomotion and alternative splicing (Tables 2 and S7).
GO analysis of the hco-mir-228 and hco-mir-235 full target gene list was carried out with C. elegans putative homologs of H. contortus target genes as input, where these could be identified. Among the KEGG terms enriched for hco-mir-228 MiRanda-predicted targets were valine, leucine and isoleucine degradation, metabolic pathways, propanoate metabolism and carbon metabolism (Supplementary Table S11), similar to the GO terms enriched for cel-mir-228 (Table 2). For hco-mir-235, a number of GO terms were enriched for target genes, including locomotion, nematode larval development, oxidoreductase activity, metalloendopeptidase activity, mTOR signaling and aminoacyl tRNA biosynthesis (Supplementary Table S12). These terms are consistent with reduced BCAA degradation, protein translation and TOR signaling during C. elegans arrest 42 and suggest that mir-228 and mir-235 in C. elegans and H. contortus regulate some of the same metabolic pathways to suppress reproductive development.
Validation of miRNA-target mRNA interactions. miRNA:mRNA regulation of targets predicted in C. elegans was examined using three approaches: qPCR, dual luciferase reporter assay and, for ets-4, use of a GFP reporter construct. Target gene expression was compared by qPCR using RNA from synchronized C. elegans L4 stage of CLB061 (mir-235; mir-228) and wild-type N2. Expression levels of two predicted targets of cel-mir-235 were increased in CLB061 relative to N2 worms (ets-4; 4 fold (Log 2 2.05)) and F33D4.6; 16 fold (Log 2 4.2)) (Fig. 6a). There was no detectable increase in expression of two targets of cel-mir-228 tested, mboa-2 and T04A8.7, in CLB061 relative to N2 worms. www.nature.com/scientificreports www.nature.com/scientificreports/ Interaction between cel-mir-228 or cel-mir-235 and the 3′UTR of predicted targets was then examined using a dual luciferase reporter assay, as described previously 45 . A significant reduction in reporter expression was observed with the 3′UTRs of two targets tested in the presence of cel-mir-235 (F33D4.6, 27% reduction, P = 0.045; ets-4, 22% reduction, P = 0.044 (Fig. 6b). For the 3′UTRs tested in the presence of cel-mir-228, no detectable reductions in luciferase levels were observed relative to the control (data not shown).
Transcription factor Cel-ETS-4 is a proposed antagonist of DAF-16 41 and promotes lipid catabolism 40 . To further examine negative regulation of ets-4 by cel-mir-235, expression of a GFP reporter under the control of ets-4 3′UTR (C. elegans strain RAF1713) 40 was compared in the presence and absence of cel-mir-235. This was achieved by genetic crossing of RAF1713 with strain MT17997(mir-235(n4504)) to generate strain CLB072. In both RAF1713 and CLB072, GFP expression was observed in gut cell nuclei, but was observed down the length of the gut only in the mir-235 mutant background (CLB072). In the mir-235 mutant background, GFP expression was stronger and significantly more adult worms showed posterior gut nuclei expression (67% versus 31% in RAF1713; t-test P = 0.022 and p = 0.047 for two independent lines CLB072(1) and CLB072(2)) (Fig. 6c,d).  www.nature.com/scientificreports www.nature.com/scientificreports/ Together, the above data indicate that cel-mir-235 can suppress ets-4 via its 3′UTR, and therefore can potentially enhance DAF-16 activity and also repress lipid catabolism associated with development.

Discussion
For optimal survival, nematodes must respond to environmental cues and co-ordinate developmental programs accordingly, particularly during life-cycle transitions. The importance of epigenetic mechanisms, including DNA methylation 46 , histone acetylation/methylation [47][48][49] and post-transcriptional regulation by microRNAs (reviewed in 13 ), are beginning to be revealed, but how these modulate parasite development is not known. miRNAs often function together to regulate gene networks to produce a co-ordinated and rapid change in gene expression that ensures robust developmental progression 32 . In this study we profiled the expression of all known miRNAs of H. contortus and identified those significantly up-or down-regulated during development from infective L3 stage to reproducing adult worms. Pan-nematode or more widely conserved miRNAs were enriched in the L3 and L4 larval stages, suggesting regulation of evolutionary conserved targets. In contrast, almost half of the miRNAs abundantly expressed in adult worms, or in adult gut tissue, are unique to H. contortus (from miRBase Release 21), which may indicate roles in adaptation and survival within the host. We focused on two miRNAs, mir-228 and mir-235, significantly enriched in H. contortus L3 stage that may function to maintain L3 arrest and prevent  [71] or strain CLB072 (two independent lines 1 and 2), generated by crossing RAF1713 with MT17997(mir-235(n4504)I. Error bars indicate standard deviation (two-tailed t-test, P = 0.022 and P = 0.047 for CLB072 lines 1 and 2, respectively). (d) ets-4::GFP expression in posterior gut nuclei in CLB072 but not in RAF1713, indicating enhanced GFP expression in the absence of cel-mir-235. Images taken at same exposure at x40 magnification. To enable visualisation of GFP expression, RNAi of ets-4 suppressor rege-1 was required 38 and was carried out by RNAi feeding of both RAF1713 and CLB072 strains, in an identical manner. Arrow indicates rectum, arrowheads indicate GFP + gut nuclei. Smaller autofluorescent gut granules can also been seen. developmental progression. By generating a C. elegans mutant of the homologous miRNAs, we showed that they are required together for larval arrest, with formation of significantly fewer dauer larvae. Our data suggest that mir-228 and mir-235 re-inforce activity of FOXO-transcription factor DAF-16 to suppress metabolic pathways required for development.
Sequences with identity or near identity to mir-228 are present in other nematodes, including the filarial parasite B. malayi and pig roundworm Ascaris suum (http://www.mirbase.org) but not in other organisms. mir-235 is restricted to clade V nematodes, however a related miRNA, mir-92, is expressed in nematode clades III and IV, and is similar to the mammalian mir-17-92 cluster. Wang et al. 50 profiled miRNA expression during early development of A. suum and showed that mir-92 is most abundant in day 21 larvae, at which time larvae are arrested L3 stage. This suggests that mir-235 plays a conserved role in parasitic nematode quiescence prior to host infection. In C. elegans, mir-235 12 and mir-71 51 are essential for maintaining L1 arrest, while mir-71 together with mir-34 are required for L1 and dauer arrest, and during adult worm ageing, consistent with roles in stress resistance 52 . Cel-mir-235 was also recently shown to be required for longevity of dietary restricted eat-2 mutants by suppressing Wnt signaling, leading to the proposal that mir-235 is a major sensor of different dietary restriction regimens 31 . Our data show that mir-235 is important in suppressing development and maintaining arrest, under starvation or stress conditions, and that this function is conserved in free-living and parasitic nematode species.
Mimics and inhibitors of mir-228 and mir-235 were tested directly on H. contortus cultured in vitro. A significant, albeit slight, increase in L3 to L4 development was observed in the presence of inhibitors, supporting a role for hco-mir-228 and hco-mir-235 in suppressing development, not only in vivo but also during in vitro conditions. Consistent with this, Ma et al. 28 observed a decrease in mir-235 transcript level in in vitro derived L4 relative to L3 stage. Although small, this is the first report of a phenotypic effect in a parasitic nematode using miRNA inhibitors. The limited effect may reflect inefficient uptake and/or distribution of inhibitor within the worms. It will be important in future studies to determine effects of mir-228 and mir-235 inhibitors and mimics on parasite development and reproduction within the host, as we did previously for RNAi silencing of H. contortus vaccine antigen aminopeptidase H11 53 .
Effects on ovarian structure of adult liver fluke Schistosoma japonicum have been reported following in vitro delivery of miRNA inhibitors by electroporation 54 , but we found that this delivery method led to high mortality in H. contortus larvae. The use of viral vectors to deliver and express parasite genes 55 and the demonstration of gene knockout in S. stercoralis using CRISPR/Cas9 56 should facilitate greater manipulation of parasite miRNA and mRNA levels for more detailed functional analysis. We also demonstrate here that the steroid hormone Δ7-DA can enhance H. contortus larval development in vitro, consistent with recent findings 30 . Improvements to in vitro culture conditions, such as this, should help in future studies of small RNA uptake for gene silencing and subsequent phenotypic analysis.
Epistasis studies using mutants of the C. elegans daf-2 insulin receptor gene demonstrated interaction between the activities of mir-228, mir-235 and the insulin signaling pathway. Loss of mir-228 and mir-235 had no effect on the daf-c phenotype of daf-2(e1370) mutants at the restrictive temperature of 25 °C. However, at semi-permissive temperature (22.5 °C), daf-2(e1370) mutants develop to sterile or near-sterile adults 36,37 and we show here that this requires mir-228 and mir-235, as triple mutants develop to reproductive adults. Importantly, the differences observed at 25 °C and 22.5 °C suggest that these miRNAs modulate the effects of transcription factor DAF-16 and reinforce DAF-16 function at a threshold level to ensure robustness of stress response programs. This is consistent with analysis of daf-2 mosaics that show an all or nothing tissue response 37 , suggesting there is a quantitative threshold of signal required to commit to developmental progression or maintain arrest. We propose that mir-228 and mir-235, and possibly other miRNAs, function with DAF-16 to inhibit progression to the commitment threshold required for development. DAF-2 and DAF-16 are functionally conserved in parasitic nematode species [17][18][19] . Interestingly, no DAF-2 agonistic ligands have been identified to date in the genome of H. contortus 57 , suggesting that host signals may drive post-infection development and miRNAs may be important in co-ordinating developmental progression in response to such signals.
Target genes for mir-228 and mir-235 identified computationally were involved in metabolic regulation, in particular lipid metabolism and BCAA degradation. In the C. elegans dauer stage, lipid stores are abundant initially, while lipid and amino acid metabolism are reduced, while a converse pattern occurs during reproductive growth, characterized by aerobic lipid and carbohydrate metabolism 58 . Transcriptome data for H. contortus also indicate a developmental switch in metabolic activity, from gluconeogenesis in the L3 stage to oxidative metabolism of carbohydrate and lipid in L4 and adult worms 21 . Lipid metabolism can be modulated by addition of DA, which promotes H. contortus L3 to L4 development 30 . Analysis of gene clusters differentially expressed during C. elegans dauer and reproductive development also showed downregulation of genes involved in BCAA and fatty acid degradation, carbon metabolism and glyoxylate and dicarboxylate metabolism after dauer commitment, while genes associated with splicing, ribosome, RNA polymerase were upregulated in L4 stage 59 . The target prediction data presented here suggest that mir-228 and mir-235 are involved in regulating these metabolic switches to suppress development, while, in contrast, DA promotes metabolic and signaling pathways associated with development 30 .
Using different experimental approaches we show that mir-235 suppresses expression of target gene ets-4 and interacts with ets-4 3′UTR. ETS-4 is a transcription factor that promotes lipid catabolism by increasing expression of lipid metabolic genes 40 . We propose that one function of mir-235 is to suppress ets-4 levels, leading to a reduction in multiple lipid catabolic pathways. In addition, ETS-4 is a putative antagonist of DAF-16 41 and therefore, by reducing ETS-4, mir-235 may also enhance DAF-16 activity in suppressing development.
In conclusion, we profiled the expression of miRNAs during development of H. contortus parasitic stages, determined predicted targets of two miRNAs and identified their potential synergism with DAF-16 in regulating nematode metabolism and development. With resistance to current anthelmintics, which predominantly target neurotransmission, becoming an increasing concern, drug repurposing and omics-based approaches are being www.nature.com/scientificreports www.nature.com/scientificreports/ used to identify small molecules that target metabolic pathway enzymes, as alternative therapeutics 60,61 . Our findings from miRNA expression and target analysis help identify multiple metabolic pathways and switches required for development, some of which may be novel nematode-specific targets for blocking parasite survival and ameliorating disease. Total RNA extraction and miRNA microarray. Parasites were ground in liquid nitrogen and total RNA extracted using Trizol (Invitrogen), with RNA for miRNA microarray prepared with an additional 75% ethanol wash. RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Three biological replicate samples (parasites isolated from different animals) were prepared, except for L4, where two replicates were used. Microarrays were performed by LC Sciences (Texas, USA), using a custom array containing probes to 609 H. contortus miRNA sequences (all mature and star miRNA sequences we identified previously 14 ) and all C. elegans sequences in miR-Base release 16. Microarray data are deposited in NCBI's Gene Expression Omnibus (GEO Series accession number GSE101501; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101501).

Methods
Normalised microarray data was corrected as described 63 , using a microarray threshold signal of >500 fluorescent units and P-value < 0.05. Data was subjected to hierarchical clustering using the matrix visualization and analysis platform GENE-E, version 3.0.240 (http://www.broadinstitute.org/cancer/software/GENE-E/ index.html). Row distance was calculated using a one-minus Pearson correlator metric and complete linkage (farthest-neighbour approach). All other settings were default. A rooted dendrogram and three-colour heat-map were generated. Groups were labeled according the life-cycle stage or gut tissue with highest level of expression. Pairwise t-test comparisons were carried using GenePattern software (http://software.broadinstitute.org/cancer/software/genepattern/gene-expression-analysis); t-test P-values of <0.05 and Log 2 fold changes of >2 were retained. Volcano plots were generated using Multiplot (v2) (http://software.broadinstitute.org/cancer/software/ genepattern/modules/docs/Multiplot/2). Conservation of miRNAs was carried out by interrogation of miRBase 21 using the miRNA precursor sequences for all known H. contortus miRNAs. miRNA and mRNA qPCR. miRNA cDNA Synthesis and qPCR Master Mix protocols were followed (Agilent)) using 1 μg DNase I-treated (Ambion) total RNA. RNA samples for L3, adult male and adult female qPCR were independent of those used on microarray. Two independent L1 samples were used in qPCR and compared with L3. Results were normalized to hco-miR-5899-3p, a constitutively expressed miRNA. mRNA was prepared from 1 μg of DNase I-treated total RNA, using SuperScript II (Invitrogen) with oligo(dT) primer and qPCR carried out using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent) with C. elegans actin (act-3) as a normalising gene 64  and incubated in 120 μl Earle's Balanced Salt Solution (EBSS) containing penicillin (250 U/ml), streptomycin (50 μg/ml) and fungizone (1.25 μg/ml) at 37 °C, 5% CO 2 in 96 well plates (1.5 L3/ μl). miRIDIAN double-stranded miRNA mimics for hco-mir-228 and hco-mir-235 were designed by Dharmacon (http://dharmacon.gelifesciences.com) and added together at a final concentration of 2 μM to cultures containing 2.5 μM Δ7-DA. Miridian control mimic #1 (Dharmacon) was added at the same final concentration. miRCURY LNA Power Inhibitors of hco-mir-228 and hco-mir-235 were designed by Exiqon (http://www.exiqon.com/) and tested together at a final concentration of 2 μM, and compared to wells containing miRCURY LNA Power Inhibitor Negative control A at the same final concentration. All experiments were carried out at least three times and statistical analysis performed using two-sample t-test. Δ7-dafachronic acid (Δ7-DA) in ethanol, was added at final concentrations of 1, 2.5, 5 and 10 μM. Control wells contained the same volume of ethanol. Larvae were examined microscopically every 24 h and number of larvae developing from L3 to L4 were counted from triplicate experiments, with total sample size between 246-367 larvae.
For H. contortus target gene prediction, two custom databases were generated exactly as described 65 and when combined, contained either 3′UTR or downstream sequences for approximately 95% of H. contortus genes. The combined databases were interrogated for putative hco-mir-228 or hco-mir-235 binding sites using three target prediction programs: miRanda 43 , PITA 38 and RNAhybrid 44 that allow input of query 3′UTR sequence. Predictions were performed using the Unbuntu (14.04 LTS) operating system. Results were filtered using the following criteria: miRanda: Score >145 and energy <−10; PITA: seed sequences = 8 and ΔΔG < −10; RNAhybrid: P-value < 0.1 and energy <−22, as described 66 .
Predicted targets of cel-miR-228 and cel-mir-235 were used in pathway analysis using DAVID 67 (https://david. ncifcrf.gov/) and converted into associated biological pathways based on gene-annotation enrichment analysis. P-values were adjusted for multiple testing to control the false discovery rate 63 and pathways with P < 0.05 were considered significant. DAVID Gene Ontology (GO) analysis was also performed for hco-mir-228 and hco-mir-235 target genes predicted by each program individually and genes common to all three prediction programs. It was necessary to use the putative C. elegans homologous genes as input sequence, where available, identified using BLASTp (query coverage and identity >40% and best match by reciprocal BLASTp). P-value was set to 0.1 to include all potentially relevant biological pathways.
Generation of C. elegans crossed strains and production of synchronized larvae. C. elegans strains used are listed in Supplementary Table 14. A double mutant strain for mir-235 and mir-228 (CLB061) was generated by crossing strains MT17997(mir-235(n4504)I) and MT14446 (mir-228(n4382)IV). Seventy F1 heterozygous progeny were picked to single plates and DNA extracted and PCR amplified from F2 worms to identify those carrying homozygous deletions at both loci.
Strain CLB061 was crossed with CB1370(daf-2 (e1370)III) to generate strain CLB065, which was maintained at 15 °C and homozygous deletion at each locus confirmed by PCR. Effects of mir-228 and mir-235 loss on daf-2(e1370) development were examined by placing eight L4 on NGM agar plates seeded with OP50 E. coli and allowing to egg lay overnight at 25 °C or 22.5 °C. Development of progeny to dauer or reproducing adults was examined daily. To score number of progeny in the F1 generation, eight L4 were placed on NGM/OP50 plates and progeny counted 48 hours after the start of egg laying. Triplicate plates were scored, with total sample size between 140-206.
For qPCR, sixty adult C. elegans N2 hermaphrodites were allowed to egg lay overnight on NGM/OP50 plates. After 48 h, synchronized L4 were washed from plates in M9 buffer, collected by centrifugation at 6000 rpm and frozen at −80 °C. Total RNA was extracted and used in qPCR as described above.
Quantifying dauer formation in C. elegans. Dauer arrest was assessed in C. elegans mutant strains by allowing 20 gravid adult worms to lay eggs on NGM agar plates containing 2% (v/v) crude dauer pheromone extract, prepared as described 68 . After 4-6 hours, adults were removed and plates incubated at 27 °C for 72 hours. Temperature and pheromone concentration were optimised to induce high levels of dauer formation in N2 wild-type strain, as described 68 , enabling detection of any reduction in dauer formation in mutant strains. A temperature of 27 °C was applied to obtain high numbers of dauers; this was previously used to identify novel genes involved in dauer arrest and did not lead to non-specific effects 69,70 . The total number of worms on each plate was counted before plates were flooded with 1% SDS and incubated at 20 °C for 20 minutes. Worms that survived SDS-exposure, indicated by motility and possessing a dauer-like appearance, were scored as dauers. The mutant strains tested were: MT14446 (mir-228(n4382)IV); MT17997 (mir-235(n4504)I); CBL061 (mir-235(n4504)I; mir-228(n4382)IV); MT16471 (mir-60(n4947)II). Statistical significance was measured using one-way ANOVA with corrected P-value < 0.05. Triplicate plates were scored with a total sample size between 136-338 larvae.

Data availability
All microarray files are available from the NCBI GEO database (Accession Number GSE101501). Gene sequence data is available from GenBank (Accession Number MN017114).