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(UGA2004) 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 L3 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.

The small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development and anthelmintic resistance research. Its remarkable propensity to develop resistance threatens the viability of the sheep industry in many regions of the world and provides a cautionary example of the effect of mass drug administration to control parasitic nematodes. Its phylogenetic position makes it particularly well placed for comparison with the free-living nematode Caenorhabditis elegans and the most economically important parasites of livestock and humans.

MicroRNAs (miRNAs) play key roles in regulating post-transcriptional gene expression and are essential for development in the free-living nematode Caenorhabditis elegans and in higher organisms. Whether microRNAs are involved in regulating developmental programs of parasitic nematodes is currently unknown. Here we describe the the miRNA repertoire of two important parasitic nematodes as an essential first step in addressing this question.

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.

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.

Sheep farmers in the UK rely on strategic anthelmintic use to treat and control gastrointestinal roundworms in their flocks. However, resistance to these drugs is now widespread and threatens the sustainability of sheep production. The mechanisms underlying resistance to the most commonly used class, the macrocyclic lactones, are not known and sensitive diagnostic tools based on molecular markers are not currently available. This prohibits accurate surveillance of resistance or assessment of strategies aimed at controlling its spread. In this study, we examined four UK field populations of Haemonchus contortus, differing in macrocyclic lactone treatment history, for evidence of selection at 'candidate gene' loci identified as determining macrocyclic lactone resistance in previously published research. Individual worms were genotyped at Hc-lgc-37, Hc-glc-5, Hc-avr-14 and Hc-dyf-7, and four microsatellite loci. High levels of polymorphism were identified at the first three candidate gene loci with remarkably little polymorphism at Hc-dyf-7. While some between-population comparisons of individual farms with and without long-term macrocyclic lactone use identified statistically significant differences in allele frequency and/or fixation index at the Hc-lgc-37, Hc-glc-5 or Hc-avr-14 loci, we found no consistent evidence of selection in other equivalent comparisons. While it is possible that different mechanisms are important in different populations or that resistance may be conferred by small changes at multiple loci, our findings suggest that these are unlikely to be major loci conferring macrocyclic lactone resistance on UK farms or suitable for diagnostic marker development. More powerful approaches, using genome-wide or whole genome sequencing, may be required to define macrocyclic lactone resistance loci in such genetically variable populations.

microRNAs are small non-coding RNAs that are important regulators of gene expression in a range of animals, including nematodes. We have analysed a cluster of four miRNAs from the pathogenic nematode species Haemonchus contortus that are closely linked in the genome. We find that the cluster is conserved only in clade V parasitic nematodes and in some ascarids, but not in other clade III species nor in clade V free-living nematodes. Members of the cluster are present in parasite excretory-secretory products and can be detected in the abomasum and draining lymph nodes of infected sheep, indicating their release in vitro and in vivo. As observed for other parasitic nematodes, H. contortus adult worms release extracellular vesicles (EV). Small RNA libraries were prepared from vesicle-enriched and vesicle-depleted supernatants from both adult worms and L4 stage larvae. Comparison of the miRNA species in the different fractions indicated that specific miRNAs are packaged within vesicles, while others are more abundant in vesicle-depleted supernatant. Hierarchical clustering analysis indicated that the gut is the likely source of vesicle-associated miRNAs in the L4 stage, but not in the adult worm. These findings add to the growing body of work demonstrating that miRNAs released from parasitic helminths may play an important role in host-parasite interactions.

Infections with helminths cause an enormous disease burden in billions of animals and plants worldwide. Large scale use of anthelmintics has driven the evolution of resistance in a number of species that infect livestock and companion animals, and there are growing concerns regarding the reduced efficacy in some human-infective helminths. Understanding the mechanisms by which resistance evolves is the focus of increasing interest; robust genetic analysis of helminths is challenging, and although many candidate genes have been proposed, the genetic basis of resistance remains poorly resolved.

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.

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.

Whole-genome sequencing is being rapidly applied to the study of helminth genomes, including de novo genome assembly, population genetics, and diagnostic applications. Although late-stage juvenile and adult parasites typically produce sufficient DNA for molecular analyses, these parasitic stages are almost always inaccessible in the live host; immature life stages found in the environment for which samples can be collected non-invasively offer a potential alternative; however, these samples typically yield very low quantities of DNA, can be environmentally resistant, and are susceptible to contamination, often from bacterial or host DNA. Here, we have tested five low-input DNA extraction protocols together with a low-input sequencing library protocol to assess the feasibility of whole-genome sequencing of individual immature helminth samples. These approaches do not use whole-genome amplification, a common but costly approach to increase the yield of low-input samples. We first tested individual parasites from two species spotted onto FTA cards-egg and L1 stages of Haemonchus contortus and miracidia of Schistosoma mansoni-before further testing on an additional five species-Ancylostoma caninum, Ascaridia dissimilis, Dirofilaria immitis, Strongyloides stercoralis, and Trichuris muris-with an optimal protocol. A sixth species-Dracunculus medinensis-was included for comparison. Whole-genome sequencing followed by analyses to determine the proportion of on- and off-target mapping revealed successful sample preparations for six of the eight species tested with variation both between species and between different life stages from some species described. These results demonstrate the feasibility of whole-genome sequencing of individual parasites, and highlight a new avenue toward generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

Heat shock protein 90 (Hsp-90) is a highly conserved essential protein in eukaryotes. Here we describe the molecular characterisation of hsp-90 from three nematodes, the free-living Caenorhabditis elegans (Ce) and the parasitic worms Brugia pahangi (Bp) and Haemonchus contortus (Hc). These molecules were functionally characterised by rescue of a Ce-daf-21 (hsp-90) null mutant. Our results show a gradient of rescue: the C. elegans endogenous gene provided full rescue of the daf-21 mutant, while Hc-hsp-90 provided partial rescue. In contrast, no rescue could be obtained using a variety of Bp-hsp-90 constructs, despite the fact that Bp-hsp-90 was transcribed and translated in the mutant worms. daf-21 RNA interference (RNAi) experiments were carried out to determine whether knock-down of the endogenous daf-21 mRNA in N2 worms could be complemented by expression of either parasite gene. However neither parasite gene could rescue the daf-21 (RNAi) phenotypes. These results indicate that factors other than the level of sequence identity are important for determining whether parasite genes can functionally complement in C. elegans.

Some nematode species are economically important parasites of livestock, while others are important human pathogens causing some of the most important neglected tropical diseases. In both humans and animals, anthelmintic drug administration is the main control strategy, but the emergence of drug-resistant worms has stimulated the development of alternative control approaches. Among these, vaccination is considered to be a sustainable and cost effective strategy. Currently, Barbervax® for the ruminant strongylid Haemonchus contortus is the only registered subunit vaccine for a nematode parasite, although a vaccine for the human hookworm Necator americanus is undergoing clinical trials (HOOKVAC consortium). As both these vaccines comprise a limited number of proteins, there is potential for selection of nematodes with altered sequences or expression of the vaccine antigens. Here we compared the transcriptome of H. contortus populations from sheep vaccinated with Barbervax® with worms from control animals. Barbervax® antigens are native integral membrane proteins isolated from the brush border of the intestinal cells of the adult parasite and many of those are proteases. Our findings provide no evidence for changes in expression of genes encoding Barbervax® antigens in the surviving parasite populations. However, surviving parasites from vaccinated animals showed increased expression of other proteases and regulators of lysosome trafficking, and displayed up-regulated lipid storage and defecation abilities that may have circumvented the effect of the vaccine. Implications for other potential vaccines for human and veterinary nematodes are discussed.

The parasitic nematode Haemonchus contortus is an economically and clinically important pathogen of small ruminants, and a model system for understanding the mechanisms and evolution of traits such as anthelmintic resistance. Anthelmintic resistance is widespread and is a major threat to the sustainability of livestock agriculture globally; however, little is known about the genome architecture and parameters such as recombination that will ultimately influence the rate at which resistance may evolve and spread. Here, we performed a genetic cross between two divergent strains of H. contortus, and subsequently used whole-genome resequencing of a female worm and her brood to identify the distribution of genome-wide variation that characterizes these strains. Using a novel bioinformatic approach to identify variants that segregate as expected in a pseudotestcross, we characterized linkage groups and estimated genetic distances between markers to generate a chromosome-scale F1 genetic map. We exploited this map to reveal the recombination landscape, the first for any helminth species, demonstrating extensive variation in recombination rate within and between chromosomes. Analyses of these data also revealed the extent of polyandry, whereby at least eight males were found to have contributed to the genetic variation of the progeny analyzed. Triploid offspring were also identified, which we hypothesize are the result of nondisjunction during female meiosis or polyspermy. These results expand our knowledge of the genetics of parasitic helminths and the unusual life-history of H. contortus, and enhance ongoing efforts to understand the genetic basis of resistance to the drugs used to control these worms and for related species that infect livestock and humans throughout the world. This study also demonstrates the feasibility of using whole-genome resequencing data to directly construct a genetic map in a single generation cross from a noninbred nonmodel organism with a complex lifecycle.

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.

Resistance to anthelmintic drugs is a major problem in the global fight against parasitic nematodes infecting humans and animals. While previous studies have identified mutations in drug target genes in resistant parasites, changes in the expression levels of both targets and transporters have also been reported. The mechanisms underlying these changes in gene expression are unresolved. Here, we take a novel approach to this problem by investigating the role of small regulatory RNAs in drug resistant strains of the important parasite Haemonchus contortus. microRNAs (miRNAs) are small (22 nt) non-coding RNAs that regulate gene expression by binding predominantly to the 3' UTR of mRNAs. Changes in miRNA expression have been implicated in drug resistance in a variety of tumor cells. In this study, we focused on two geographically distinct ivermectin resistant strains of H. contortus and two lines generated by multiple rounds of backcrossing between susceptible and resistant parents, with ivermectin selection. All four resistant strains showed significantly increased expression of a single miRNA, hco-miR-9551, compared to the susceptible strain. This same miRNA is also upregulated in a multi-drug-resistant strain of the related nematode Teladorsagia circumcincta. hco-miR-9551 is enriched in female worms, is likely to be located on the X chromosome and is restricted to clade V parasitic nematodes. Genes containing predicted binding sites for hco-miR-9551 were identified computationally and refined based on differential expression in a transcriptomic dataset prepared from the same drug resistant and susceptible strains. This analysis identified three putative target mRNAs, one of which, a CHAC domain containing protein, is located in a region of the H. contortus genome introgressed from the resistant parent. hco-miR-9551 was shown to interact with the 3' UTR of this gene by dual luciferase assay. This study is the first to suggest a role for miRNAs and the genes they regulate in drug resistant parasitic nematodes. miR-9551 also has potential as a biomarker of resistance in different nematode species.

Recent reports of monepantel (MPTL) resistance in UK field isolates of Teladorsagia circumcincta has highlighted the need for a better understanding of the mechanism of MPTL-resistance in order to preserve its anthelmintic efficacy in this economically important species. Nine discrete populations of T. circumcincta were genotypically characterised; three MPTL-susceptible isolates, three experimentally selected MPTL-resistant strains and three field derived populations. Full-length Tci-mptl-1 gene sequences were generated and comparisons between the MPTL-susceptible isolates, MPTL-resistant strains and one field isolate, showed that different putative MPTL-resistance conferring mutations were present in different resistant isolates. Truncated forms of the Tci-mptl-1 gene were also observed. The genetic variability of individual larvae, within and between populations, was examined using microsatellite analyses at 10 'neutral' loci (presumed to be unaffected by MPTL). Results confirmed that there was little background genetic variation between the populations, global FST <0.038. Polymorphisms present in exons 7 and 8 of Tci-mptl-1 enabled genotyping of individual larvae. A reduction in the number of genotypes was observed in all MPTL-resistant strains compared to the MPTL-susceptible strains that they were derived from, suggesting there was purifying selection at Tci-mptl-1 as a result of MPTL-treatment. The potential link between benzimidazole (BZ)-resistance and MPTL-resistance was examined by screening individual larvae for the presence of three SNPs associated with BZ-resistance in the β-tubulin isotype-1 gene. The majority of larvae were BZ-susceptible homozygotes at positions 167 and 198. Increased heterozygosity at position 200 was observed in the MPTL-resistant strains compared to their respective MPTL-susceptible population. There was no decrease in the occurrence of BZ-resistant genotypes in larvae from each population. These differences, in light of the purifying selection at this locus in all MPTL-resistant isolates, suggests that Tci-mptl-1 confers MPTL-resistance in T. circumcincta, as in Haemonchus contortus, but that different mutations in Tci-mptl-1 can confer resistance in different populations.

Hsp-90 from the free-living nematode Caenorhabditis elegans is unique in that it fails to bind to the specific Hsp-90 inhibitor, geldanamycin (GA). Here we surveyed 24 different free-living or parasitic nematodes with the aim of determining whether C. elegans Hsp-90 was the exception or the norm amongst the nematodes. We combined these data with codon evolution models in an attempt to identify whether hsp-90 from GA-binding and non-binding species has evolved under different evolutionary constraints.

Filarial nematodes are important pathogens in the tropics transmitted to humans via the bite of blood sucking arthropod vectors. The molecular mechanisms underpinning survival and differentiation of these parasites following transmission are poorly understood. microRNAs are small non-coding RNA molecules that regulate target mRNAs and we set out to investigate whether they play a role in the infection event.