Subtherapeutic treatment or 'underdosing' is considered a common problem in the control of parasitic helminths of animals and people and can hasten the emergence of anthelmintic resistance. Increasing reliance on the long-acting macrocyclic lactone, moxidectin, in both veterinary and medical settings may increase exposure of incoming helminth populations to subtherapeutic drug concentrations due to its extended half-life. However, we lack genetic markers to monitor emerging resistance as the mechanism(s) underlying resistance to the macrocyclic lactones are unresolved in parasitic helminths. Furthermore, the impact of prior ivermectin exposure on the evolution of moxidectin resistance is unclear. To test the impact of subtherapeutic selection on the emergence of macrocyclic lactone resistance, we exposed a fully drug susceptible isolate of an economically important parasitic helminth of livestock, Haemonchus contortus, to low but increasing doses of ivermectin or moxidectin in vivo for phenotypic, genomic, and transcriptomic analyses. After a single subtherapeutic dose of ivermectin or moxidectin, we find evidence of selection at a shared genetic locus on Chromosome V, with the signal of selection increasing with subsequent doses. After only three subtherapeutic treatments, ivermectin-selected lines were resistant to a full standard (label) dose of ivermectin. However, moxidectin selected lines remained susceptible to a half dose of moxidectin. This was despite showing higher resistance to ivermectin in vitro and a stronger signal of selection at the Chromosome V locus than the equivalent ivermectin-selected lines. Our findings highlight the rapid selection for anthelmintic resistance with subtherapeutic treatment and implicate the pre-existence of ivermectin and moxidectin resistance haplotypes in a drug-naïve population. We demonstrate that ivermectin selected lines show emerging moxidectin resistance, underpinned by a shared genetic locus of resistance. Finally, we speculate that key differences in the resistance phenotype between ivermectin and moxidectin selected lines relate to differences in the inheritance of resistance within this shared locus, with ivermectin resistance manifesting as dominant trait while moxidectin resistance appears to be recessive.

Pathogens have developed multiple strategies to modulate host immune defense mechanisms. Understanding how this is achieved has potential to inform novel therapeutics for diseases caused by immune dysfunction. Parasitic helminths are masters of immune evasion, via release of secreted products, resulting in chronic infection. Helminths secrete small regulatory microRNA (miRNAs), which can interact with host cells. Here we show that a single parasite miRNA (miR-5352), conserved across gastrointestinal (GI) nematodes, suppresses IL-13-induced GI epithelial cell differentiation and cytokine responses, and promotes stem cell maintenance. Mechanistically, this is achieved through targeted repression of critical host factors, including Klf-4 and the IL-22 receptor, together with modulation of Wnt and Notch signalling pathways. Nematode miR-5352 shows seed sequence conservation with mammalian miR-92a family members, indicating that through convergent evolution, GI nematodes exploit a host miRNA regulatory network to suppress host innate responses, promote tissue regeneration and establish a favourable environment for chronic infection.

The parasitic nematode Teladorsagia circumcincta is one of the most important pathogens of sheep and goats in temperate climates worldwide and can rapidly evolve resistance to drugs used to control it. To understand the genetics of drug resistance, we have generated a highly contiguous genome assembly for the UK T. circumcincta isolate, MTci2. Assembly using PacBio long-reads and Hi-C long-molecule scaffolding together with manual curation resulted in a 573 Mb assembly (N50 = 84 Mb, total scaffolds = 1,286) with five autosomal and one sex-linked chromosomal-scale scaffolds consistent with its karyotype. The genome resource was further improved via annotation of 22,948 genes, with manual curation of over 3,200 of these, resulting in a robust and near complete resource (96.3% complete protein BUSCOs) to support basic and applied research on this important veterinary pathogen. Genome-wide analyses of drug resistance, combining evidence from three distinct experiments, identified selection around known candidate genes for benzimidazole, levamisole and ivermectin resistance, as well as novel regions associated with ivermectin and moxidectin resistance. These insights into contemporary and historic genetic selection further emphasise the importance of contiguous genome assemblies in interpreting genome-wide genetic variation associated with drug resistance and identifying key loci to prioritise in developing diagnostic markers of anthelmintic resistance to support parasite control.

Intestinal helminth parasites trigger the host immune response through epithelial sensory tuft cells, but helminth-derived molecules that may activate tuft cells are poorly characterized. The study aimed to identify small molecules released in vitro by two nematode parasites, that infect rodents (Nippostrongylus brasiliensis) and ruminants (Haemonchus contortus), and to test candidate ligands in an in vivo model of tuft cell differentiation. Small molecules were analyzed by hydrophilic interaction liquid chromatography (HILIC) of material released by adult parasites incubated in serum-free media, followed by mass spectrometry; selected molecules were administered to mice and tuft cell expansion enumerated after 5 days. A range of different conditions (culture media, timing, oxygenation) were tested, and comparisons made between the conditions, and between the two nematode species at selected points. Common products across the conditions and species included carboxylic acids (malate, succinate), medium chain fatty acids (such as decanoic and undecanoic acids), purines (guanine, xanthine and their derivatives), and phosphocholine compounds. We selected 19 of the prominent molecules for in vivo testing by oral administration, including succinate, a known activator of tuft cell differentiation. Malate elicited a low but significant level of tuft cell expansion, while undecanoic acids with or without a bromine substitution were also able to induce significant differentiation comparable to succinate. Other molecules including phosphorylcholine had no effect. Multiple molecular species including decanoic and undecanoic acids released by helminths may contribute to activation of tuft cells in vivo.

Upon parasitic helminth infection, activated intestinal tuft cells secrete interleukin-25 (IL-25), which initiates a type 2 immune response during which lamina propria type 2 innate lymphoid cells (ILC2s) produce IL-13. This causes epithelial remodeling, including tuft cell hyperplasia, the function of which is unknown. We identified a cholinergic effector function of tuft cells, which are the only epithelial cells that expressed choline acetyltransferase (ChAT). During parasite infection, mice with epithelial-specific deletion of ChAT had increased worm burden, fitness, and fecal egg counts, even though type 2 immune responses were comparable. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. Finally, we demonstrated a direct effect of ACh on worms, which reduced their fecundity via helminth-expressed muscarinic ACh receptors. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infection provides an additional type 2 immune response effector function.

How parasites develop and survive, and how they stimulate or modulate host immune responses are important in understanding disease pathology and for the design of new control strategies. Microarray analysis and bulk RNA sequencing have provided a wealth of data on gene expression as parasites develop through different life-cycle stages and on host cell responses to infection. These techniques have enabled gene expression in the whole organism or host tissue to be detailed, but do not take account of the heterogeneity between cells of different types or developmental stages, nor the spatial organisation of these cells. Single-cell RNA-seq (scRNA-seq) adds a new dimension to studying parasite biology and host immunity by enabling gene profiling at the individual cell level. Here we review the application of scRNA-seq to establish gene expression cell atlases for multicellular helminths and to explore the expansion and molecular profile of individual host cell types involved in parasite immunity and tissue repair. Studying host-parasite interactions in vivo is challenging and we conclude this review by briefly discussing the applications of organoids (stem-cell derived mini-tissues) to examine host-parasite interactions at the local level, and as a potential system to study parasite development in vitro. Organoid technology and its applications have developed rapidly, and the elegant studies performed to date support the use of organoids as an alternative in vitro system for research on helminth parasites.

Abstract Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross‐species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well‐recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth‐derived EVs in these processes and highlights EVs as an important participant in cross‐phylum communication. While the mammalian EV field is guided by a community‐agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan‐helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth‐derived EVs to complement the MISEV guidelines. We summarise community‐agreed standards for studying EVs derived from this broad set of non‐model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Haemonchus contortus is a parasitic haematophagous nematode that primarily affects small ruminants and causes significant economic loss to the global livestock industry. Treatment of haemonchosis typically relies on broad-spectrum anthelmintics, resistance to which is an important cause of treatment failure. Resistance to levamisole remains less widespread than to other major anthelmintic classes, prompting the need for more effective and accurate surveillance to maintain its efficacy. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) is a recently developed diagnostic method that facilitates multiplex target detection with single nucleotide polymorphism (SNP) specificity and portable onsite testing. In this study, we designed a new LEC-LAMP assay and applied it to detect the levamisole resistance marker S168T in H. contortus. We explored multiplexing probes for both the resistant S168T and the susceptible S168 alleles in a single-tube assay. We then included a generic probe to detect the acr-8 gene in the multiplex assay, which could facilitate the quantification of both resistance markers and overall genetic material from H. contortus in a single step. Our results showed promising application of these technologies, demonstrating a proof-of-concept assay which is amenable to detection of resistance alleles within the parasite population, with the potential for multiplex detection, and point-of-care application enabled by lateral flow end-point detection. However, further optimisation and validation is necessary.

Fires in grassy ecosystems consume vegetation and initiate high-quality regrowth, which results in pyric herbivory when mammalian grazers concentrate feeding in recent burns. For environmentally transmitted parasites with transmission mechanisms linked to vegetation structure, fire should exert direct effects on parasites, as well as indirect effects resulting from subsequent enhanced herbivory, which can affect parasite input and exposure to environmental conditions.We combined an experimental manipulation with observational data in the Serengeti National Park to investigate the direct and indirect effects of fire on parasites. We assessed the direct effects of fire by measuring changes in parasitic nematode larvae in the grass layer before and after fire on paired experimental burned and control plots. To investigate indirect effects linked to pyric herbivory, we sampled herbivore dung, grass biomass, ground temperature and larval densities every month for 5 months following fire in seven pairs of burned and unburned monitoring plots. Finally, to assess if fire-driven changes to larval densities affected host infection burdens, we collected faecal samples from a key host, Grant's gazelle (Nanger granti), each month for 5 months to estimate within-host parasite burdens.Fire killed all larvae and increased grazer dung inputs by 40% for 2 months following fire. Dung inputs after fire led to larval parasite recolonization of burned patches, but intense herbivory kept grass short and larval densities were associated with changes in ground temperature linked to grass biomass and ambient temperature. Grant's gazelles had lower parasite burdens when sampled in areas with higher compared to lower burned area fraction.Fire and pyric herbivory change the densities of larval parasites in the environment and divide the landscape into burned and unburned regions with distinct infection risks for local herbivores.The indirect effects quantified here represent a novel finding with major implications for all grazing systems impacted by fire. Mioto katika mifumo ikolojia yenye nyasi nyingi humaliza mimea na kuanzisha uotaji upya wa malisho yenye virutubisho jambo ambalo husababisha ulaji mwingi wa nyasi baada ya moto wakati mamalia walao nyasi wanapokusanyika katika sehemu zilizoungua hivi karibuni. Kwa vimelea vinavyosambazwa katika mazingira vilivyo na njia za uambukizaji zinazohusishwa na muundo wa uotaji wa mimea, moto unapaswa kuwa na athari za moja kwa moja kwa vimelea, pamoja na athari zisizo za moja kwa moja zinazotokana na wanyama kula mimea, ambapo inaweza kuathiri uingizaji na mfichuo wa vimelea katika mazingira.Tuliunganisha majaribio na takwimu za uchunguzi katika Hifadhi ya Taifa ya Serengeti ili kuchunguza athari za moja kwa moja na zisizo za moja kwa moja za moto kwa vimelea. Tulitathmini athari za moja kwa moja za moto kwa kupima mabadiliko ya idadi ya mabuu ya vimelea vya minyoo kwenye safu ya nyasi kabla na baada ya moto katika jozi ya viwanja vilivyochomwa na visivyochomwa. Ili kuchunguza athari zisizo za moja kwa moja zinazohusishwa na ulaji wa mimea baada ya moto, tulichukua sampuli za vinyesi vya wanyama walao nyasi, uzito wa nyasi, jotoridi la ardhini, na msongamano wa mabuu ya vimelea kila mwezi kwa muda wa miezi mitano kufuatia moto katika jozi saba za maeneo ya ufuatiliaji yaliyoungua na ambayo hayajachomwa. Hatimaye, ili kutathmini kama mabadiliko yanayotokana na moto kwa msongamano wa mabuu ya vimelea yaliathiri kiwango cha maambukizi kwa wanyama, tulikusanya sampuli za vinyesi kutoka kwa Swala Granti (Nanger granti), kila mwezi kwa miezi mitano ili kukadiria kiwango cha vimelea ndani ya mnyama.Moto uliua mabuu yote ya vimelea na kuongeza kiwango cha vinyesi vya wanyama kwa 40% kwa muda wa miezi miwili baada ya moto. Ongezeko la vinyesi baada ya moto lilisababisha kurudi upya kwa mabuu ya vimelea kwenye sehemu zilizoungua, lakini ulaji wa nyasi kwa wingi ulipelekea nyasi kuwa fupi na msongamano wa mabuu ulihusishwa na mabadiliko ya jotoridi la ardhini yanayohusishwa na kiwango cha nyasi na halijoto iliyopo. Swala Granti walikuwa na kiwango cha chini cha vimelea walipochukuliwa sampuli katika maeneo yaliyoungua sana ukilinganisha na maeneo yaliyoungua kidogo.Moto na ulaji wa nyasi baada ya moto hubadilisha msongamano wa mabuu ya vimelea katika mazingira na kugawanya mandhari katika maeneo yaliyochomwa moto na yasiyochomwa moto yenye hatari tofauti za maambukizi kwa wanyama walao nyasi wanaopatikana katika eneo husika.Athari zisizo za moja kwa moja zilizohesabiwa hapa zinawakilisha matokeo mapya yenye athari kubwa kwa mifumo yote ya malisho iliyoathiriwa na moto. Previous work on fire effects on parasites have focused on the direct impact of fire on parasite survival. Here, we show that concentrated herbivory following fire can lengthen the effects of fire on free-living parasites, and create divided landscapes with burned and unburned areas that have distinct infection risks.image

SUMMARY Upon parasitic helminth infection, activated intestinal tuft cells secrete IL-25, which initiates a type 2 immune response during which lamina propria ILC2s produce IL-13. This causes epithelial remodelling, including tuft cell hyperplasia with an unknown function. We describe a novel cholinergic effector function of tuft cells, which we show are the only epithelial cells expressing Choline Acetyltransferase (ChAT). During parasite infections, mice with epithelial-specific deletion of ChAT have increased worm burden and faecal egg counts although they are able to mount a comparable type 2 immune response. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. We demonstrate a direct effect of ACh on worms, reducing their viability and fecundity via helminth muscarinic ACh receptors, with effects promoted by inhibition of acetylcholinesterase, an helminth-secreted enzyme. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infections serves an additional type 2 immune response effector function.

Tuft cells have recently emerged as the focus of intense interest following the discovery of their chemosensory role in the intestinal tract, and their ability to activate Type 2 immune responses to helminth parasites. Moreover, they populate a wide range of mucosal tissues and are intimately connected to immune and neuronal cells, either directly or through the release of pharmacologically active mediators. They are now recognised to fulfil both homeostatic roles, in metabolism and tissue integrity, as well as acting as the first sensors of parasite infection, immunity to which is lost in their absence. In this review we focus primarily on the importance of tuft cells in the intestinal niche, but also link to their more generalised physiological role and discuss their potential as targets for the treatment of gastrointestinal disorders.

Haemonchus contortus is a haematophagous parasitic nematode that infects small ruminants and causes significant animal health concerns and economic losses within the livestock industry on a global scale. Treatment primarily depends on broad-spectrum anthelmintics, however, resistance is established or rapidly emerging against all major drug classes. Levamisole (LEV) remains an important treatment option for parasite control, as resistance to LEV is less prevalent than to members of other major classes of anthelmintics. LEV is an acetylcholine receptor (AChR) agonist that, when bound, results in paralysis of the worm. Numerous studies implicated the AChR sub-unit, ACR-8, in LEV sensitivity and in particular, the presence of a truncated acr-8 transcript or a deletion in the acr-8 locus in some resistant isolates. Recently, a single non-synonymous SNP in acr-8 conferring a serine-to-threonine substitution (S168T) was identified that was strongly associated with LEV resistance. Here, we investigate the role of genetic variation at the acr-8 locus in a controlled genetic cross between the LEV susceptible MHco3(ISE) and LEV resistant MHco18(UGA 2004) isolates of H. contortus . Using single worm PCR assays, we found that the presence of S168T was strongly associated with LEV resistance in the parental isolates and F3 progeny of the genetic cross surviving LEV treatment. We developed and optimised an allele-specific PCR assay for the detection of S168T and validated the assay using laboratory isolates and field samples that were phenotyped for LEV resistance. In the LEV-resistant field population, a high proportion (>75%) of L 3 encoded the S168T variant, whereas the variant was absent in the susceptible isolates studied. These data further support the potential role of acr-8 S168T in LEV resistance, with the allele-specific PCR providing an important step towards establishing a sensitive molecular diagnostic test for LEV resistance.

Like other pathogens, parasitic helminths can rapidly evolve resistance to drug treatment. Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to tracking its spread and improving the efficacy and sustainability of parasite control. Here, we use an in vivo genetic cross between drug-susceptible and multi-drug-resistant strains of Haemonchus contortus in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies new alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor cky-1 in ivermectin resistance. This gene is within a locus under selection in ivermectin-resistant populations worldwide; expression analyses and functional validation using knockdown experiments support that cky-1 is associated with ivermectin survival. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode and identifies variants for the development of molecular diagnostics to combat drug resistance in the field.

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and response to treatment in parasitic helminths.

Helminth parasite infections of humans and livestock are a global health and economic problem. Resistance of helminths to current drug treatment is an increasing problem and alternative control approaches, including vaccines, are needed. Effective vaccine design requires knowledge of host immune mechanisms and how these are stimulated. Mouse models of helminth infection indicate that tuft cells, an unusual type of epithelial cell, may ‘sense’ infection in the small intestine and trigger a type 2 immune response. Currently nothing is known of tuft cells in immunity in other host species and in other compartments of the gastrointestinal (GI) tract. Here we address this gap and use immunohistochemistry and single cell RNA-sequencing to detail the presence and gene expression profile of tuft cells in sheep following nematode infections. We identify and characterize tuft cells in the ovine abomasum (true stomach of ruminants) and show that they increase significantly in number following infection with the globally important nematodes Teladorsagia circumcincta and Haemonchus contortus . Ovine abomasal tuft cells show enriched expression of tuft cell markers POU2F3, GFI1B, TRPM5 and genes involved in signaling and inflammatory pathways. However succinate receptor SUCNR1 and free fatty acid receptor FFAR3 , proposed as ‘sensing’ receptors in murine tuft cells, are not expressed, and instead ovine tuft cells are enriched for taste receptor TAS2R16 and mechanosensory receptor ADGRG6. We also identify tuft cell sub-clusters at potentially different stages of maturation, suggesting a dynamic process not apparent from mouse models of infection. Our findings reveal a tuft cell response to economically important parasite infections and show that while tuft cell effector functions have been retained during mammalian evolution, receptor specificity has diverged. Our data advance knowledge of host-parasite interactions in the GI mucosa and identify receptors that may potentiate type 2 immunity for optimized control of parasitic nematodes.

Haemonchus contortus is a globally distributed and economically important gastrointestinal pathogen of small ruminants and has become a key nematode model for studying anthelmintic resistance and other parasite-specific traits among a wider group of parasites including major human pathogens. Here, we report using PacBio long-read and OpGen and 10X Genomics long-molecule methods to generate a highly contiguous 283.4 Mbp chromosome-scale genome assembly including a resolved sex chromosome for the MHco3(ISE).N1 isolate. We show a remarkable pattern of conservation of chromosome content with Caenorhabditis elegans , but almost no conservation of gene order. Short and long-read transcriptome sequencing allowed us to define coordinated transcriptional regulation throughout the parasite’s life cycle and refine our understanding of cis- and trans- splicing. Finally, we provide a comprehensive picture of chromosome-wide genetic diversity both within a single isolate and globally. These data provide a high-quality comparison for understanding the evolution and genomics of Caenorhabditis and other nematodes and extend the experimental tractability of this model parasitic nematode in understanding helminth biology, drug discovery and vaccine development, as well as important adaptive traits such as drug resistance.

Differential expression analysis between parasitic nematode strains is commonly used to implicate candidate genes in anthelmintic resistance or other biological functions. We have tested the hypothesis that the high genetic diversity of an organism such as Haemonchus contortus could complicate such analyses. First, we investigated the extent to which sequence polymorphism affects the reliability of differential expression analysis between the genetically divergent H. contortus strains MHco3(ISE), MHco4(WRS) and MHco10(CAVR). Using triplicates of 20 adult female worms from each population isolated under parallel experimental conditions, we found that high rates of sequence polymorphism in RNAseq reads were associated with lower efficiency read mapping to gene models under default TopHat2 parameters, leading to biased estimates of inter-strain differential expression. We then showed it is possible to largely compensate for this bias by optimising the read mapping single nucleotide polymorphism (SNP) allowance and filtering out genes with particularly high single nucleotide polymorphism rates. Once the sequence polymorphism biases were removed, we then assessed the genuine transcriptional diversity between the strains, finding ≥824 differentially expressed genes across all three pairwise strain comparisons. This high level of inter-strain transcriptional diversity not only suggests substantive inter-strain phenotypic variation but also highlights the difficulty in reliably associating differential expression of specific genes with phenotypic differences. To provide a practical example, we analysed two gene families of potential relevance to ivermectin drug resistance; the ABC transporters and the ligand-gated ion channels (LGICs). Over half of genes identified as differentially expressed using default TopHat2 parameters were shown to be an artifact of sequence polymorphism differences. This work illustrates the need to account for sequence polymorphism in differential expression analysis. It also demonstrates that a large number of genuine transcriptional differences can occur between H. contortus strains and these must be considered before associating the differential expression of specific genes with phenotypic differences between strains.

Small RNAs are important regulators of gene expression. They were first identified inCaenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes - microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) - and their proposed functions. miRNAs regulate development inC. elegansand the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodesin vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host-parasite interface.

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.