Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and response to treatment in a parasitic nematode.

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.

Real-time single-base specific detection of the Haemonchus contortus S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification.

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.

Whole genome amplification and sequencing of individual Dirofilaria immitis microfilariae.

Dirofilaria immitis is a filarial parasitic nematode of veterinary significance. With the emergence of drug-resistant isolates in the USA, it is imperative to determine the likelihood of resistance occurring in other regions of the world. One approach is to conduct population genetic studies across an extensive geographical range, and to sequence the genomes of individual worms to understand genome-wide genetic variation associated with resistance. The immature life stages of D. immitis found in the host blood are more accessible and less invasive to sample compared to extracting adult stages from the host heart. To assess the use of immature life stages for population genetic analyses, we have performed whole genome amplification and whole-genome sequencing on nine (n = 9) individual D. immitis microfilaria samples isolated from dog blood. On average, less than 1% of mapped reads aligned to each D. immitis genome (nuclear, mitochondrial, and Wolbachia endosymbiont). For the dog genome, an average of over 99% of mapped reads aligned to the nuclear genome and less than 1% aligned to the mitochondrial genome. The average coverage for all D. immitis genomes and the dog nuclear genome was less than 1, while the dog mitochondrial genome had an average coverage of 2.87. The overwhelming proportion of sequencing reads mapping to the dog host genome can be attributed to residual dog blood cells in the microfilariae samples. These results demonstrate the challenges of conducting genome-wide studies on individual immature parasite life stages, particularly in the presence of extraneous host DNA.

Complete representation of a tapeworm genome reveals chromosomes capped by centromeres, necessitating a dual role in segregation and protection.

BACKGROUND: Chromosome-level assemblies are indispensable for accurate gene prediction, synteny assessment, and understanding higher-order genome architecture. Reference and draft genomes of key helminth species have been published, but little is yet known about the biology of their chromosomes. Here, we present the complete genome of the tapeworm Hymenolepis microstoma, providing a reference quality, end-to-end assembly that represents the first fully assembled genome of a spiralian/lophotrochozoan, revealing new insights into chromosome evolution. RESULTS: Long-read sequencing and optical mapping data were added to previous short-read data enabling complete re-assembly into six chromosomes, consistent with karyology. Small genome size (169 Mb) and lack of haploid variation (1 SNP/3.2 Mb) contributed to exceptionally high contiguity with only 85 gaps remaining in regions of low complexity sequence. Resolution of repeat regions reveals novel gene expansions, micro-exon genes, and spliced leader trans-splicing, and illuminates the landscape of transposable elements, explaining observed length differences in sister chromatids. Syntenic comparison with other parasitic flatworms shows conserved ancestral linkage groups indicating that the H. microstoma karyotype evolved through fusion events. Strikingly, the assembly reveals that the chromosomes terminate in centromeric arrays, indicating that these motifs play a role not only in segregation, but also in protecting the linear integrity and full lengths of chromosomes. CONCLUSIONS: Despite strong conservation of canonical telomeres, our results show that they can be substituted by more complex, species-specific sequences, as represented by centromeres. The assembly provides a robust platform for investigations that require complete genome representation.

Genomic introgression mapping of field-derived multiple-anthelmintic resistance in Teladorsagia circumcincta.

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans.

Population genomic and evolutionary modelling analyses reveal a single major QTL for ivermectin drug resistance in the pathogenic nematode, Haemonchus contortus.

BACKGROUND: 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. RESULTS: Here, we present a genome-wide analysis of two genetic crosses between ivermectin resistant and sensitive isolates of the parasitic nematode Haemonchus contortus, an economically important gastrointestinal parasite of small ruminants and a model for anthelmintic research. Whole genome sequencing of parental populations, and key stages throughout the crosses, identified extensive genomic diversity that differentiates populations, but after backcrossing and selection, a single genomic quantitative trait locus (QTL) localised on chromosome V was revealed to be associated with ivermectin resistance. This QTL was common between the two geographically and genetically divergent resistant populations and did not include any leading candidate genes, suggestive of a previously uncharacterised mechanism and/or driver of resistance. Despite limited resolution due to low recombination in this region, population genetic analyses and novel evolutionary models supported strong selection at this QTL, driven by at least partial dominance of the resistant allele, and that large resistance-associated haplotype blocks were enriched in response to selection. CONCLUSIONS: We have described the genetic architecture and mode of ivermectin selection, revealing a major genomic locus associated with ivermectin resistance, the most conclusive evidence to date in any parasitic nematode. This study highlights a novel genome-wide approach to the analysis of a genetic cross in non-model organisms with extreme genetic diversity, and the importance of a high-quality reference genome in interpreting the signals of selection so identified.

Screening the Saccharomyces cerevisiae Nonessential Gene Deletion Library Reveals Diverse Mechanisms of Action for Antifungal Plant Defensins.

Plant defensins are a large family of proteins, most of which have antifungal activity against a broad spectrum of fungi. However, little is known about how they exert their activity. The mechanisms of action of only a few members of the family have been investigated and, in most cases, there are still a number of unknowns. To gain a better understanding of the antifungal mechanisms of a set of four defensins, NaD1, DmAMP1, NbD6, and SBI6, we screened a pooled collection of the nonessential gene deletion set of Saccharomyces cerevisiae Strains with increased or decreased ability to survive defensin treatment were identified based on the relative abundance of the strain-specific barcode as determined by MiSeq next-generation sequencing. Analysis of the functions of genes that are deleted in strains with differential growth in the presence of defensin provides insight into the mechanism of action. The screen identified a novel role for the vacuole in the mechanisms of action for defensins NbD6 and SBI6. The effect of these defensins on vacuoles was further confirmed by using confocal microscopy in both S. cerevisiae and the cereal pathogen Fusarium graminearum These results demonstrate the utility of this screening method to identify novel mechanisms of action for plant defensins.

MethPat: a tool for the analysis and visualisation of complex methylation patterns obtained by massively parallel sequencing.

BACKGROUND: DNA methylation at a gene promoter region has the potential to regulate gene transcription. Patterns of methylation over multiple CpG sites in a region are often complex and cell type specific, with the region showing multiple allelic patterns in a sample. This complexity is commonly obscured when DNA methylation data is summarised as an average percentage value for each CpG site (or aggregated across CpG sites). True representation of methylation patterns can only be fully characterised by clonal analysis. Deep sequencing provides the ability to investigate clonal DNA methylation patterns in unprecedented detail and scale, enabling the proper characterisation of the heterogeneity of methylation patterns. However, the sheer amount and complexity of sequencing data requires new synoptic approaches to visualise the distribution of allelic patterns. RESULTS: We have developed a new analysis and visualisation software tool "Methpat", that extracts and displays clonal DNA methylation patterns from massively parallel sequencing data aligned using Bismark. Methpat was used to analyse multiplex bisulfite amplicon sequencing on a range of CpG island targets across a panel of human cell lines and primary tissues. Methpat was able to represent the clonal diversity of epialleles analysed at specific gene promoter regions. We also used Methpat to describe epiallelic DNA methylation within the mitochondrial genome. CONCLUSIONS: Methpat can summarise and visualise epiallelic DNA methylation results from targeted amplicon, massively parallel sequencing of bisulfite converted DNA in a compact and interpretable format. Unlike currently available tools, Methpat can visualise the diversity of epiallelic DNA methylation patterns in a sample.

Functional validation of novel levamisole resistance marker S168T in Haemonchus contortus.

Recently, a S168T variant in the acetylcholine receptor subunit ACR-8 was associated with levamisole resistance in the parasitic helminth Haemonchus contortus. Here, we used the Xenopus laevis oocyte expression system and two-electrode voltage-clamp electrophysiology to measure the functional impact of this S168T variant on the H. contortus levamisole-sensitive acetylcholine receptor, L-AChR-1.1. Expression of the ACR-8 S168T variant significantly reduced the current amplitude elicited by levamisole compared to acetylcholine, with levamisole changing from a full to partial agonist on the recombinant L-AChR. Functional validation of the S168T mutation on modulating levamisole activity at the receptor level highlights its critical importance as both a mechanism and a marker of levamisole resistance.

Genome structure and population genomics of the canine heartworm Dirofilaria immitis.

The heartworm, Dirofilaria immitis, is a filarial parasitic nematode responsible for significant morbidity and mortality in wild and domesticated canids. Resistance to macrocyclic lactone drug prevention represents a significant threat to parasite control and has prompted investigations to understand the genetic determinants of resistance. This study aimed to improve the genomic resources of D. immitis to enable a more precise understanding of how genetic variation is distributed within and between parasite populations worldwide, which will inform the likelihood and rate by which parasites, and in turn, resistant alleles, might spread. We have guided the scaffolding of a recently published genome assembly for D. immitis (ICBAS_JMDir_1.0) using the chromosomal-scale reference genomes of Brugia malayi and Onchocerca volvulus, resulting in an 89.5 Mb assembly composed of four autosomal- and one sex-linked chromosomal-scale scaffolds representing 99.7% of the genome. Publicly available and new whole-genome sequencing data from 32 D. immitis samples from Australia, Italy and the USA were assessed using principal component analysis, nucleotide diversity (Pi) and absolute genetic divergence (Dxy) to characterise the global genetic structure and measure within- and between-population diversity. These population genetic analyses revealed broad-scale genetic structure among globally diverse samples and differences in genetic diversity between populations; however, fine-scale subpopulation analysis was limited and biased by differences between sample types. Finally, we mapped single nucleotide polymorphisms previously associated with macrocyclic lactone resistance in the new genome assembly, revealing the physical linkage of high-priority variants on chromosome 3, and determined their frequency in the studied populations. This new chromosomal assembly for D. immitis now allows for a more precise investigation of selection on genome-wide genetic variation and will enhance our understanding of parasite transmission and the spread of genetic variants responsible for resistance to treatment.

Genome-based tools for onchocerciasis elimination: utility of the mitochondrial genome for delineating Onchocerca volvulus transmission zones.

National programs in Africa have expanded their objectives from control of onchocerciasis (river blindness) as a public health problem to elimination of parasite transmission, motivated by the reduction of Onchocerca volvulus infection prevalence in many African meso- and hyperendemic areas due to mass drug administration of ivermectin (MDAi). Given the large, contiguous hypo-, meso-, and hyperendemic areas, sustainable elimination of onchocerciasis in sub-Saharan Africa requires delineation of geographic boundaries for parasite transmission zones, so that programs can consider the risk of parasite re-introduction through vector or human migration from areas with ongoing transmission when making decisions to stop MDAi. We propose that transmission zone boundaries can be delineated by characterising the parasite genetic population structure within and between potential zones. We analysed whole mitochondrial genome sequences of 189 O. volvulus adults to determine the pattern of genetic similarity across three West African countries: Ghana, Mali, and Côte d'Ivoire. Population genetic structure indicates that parasites from villages near the Pru, Daka, and Black Volta rivers in central Ghana belong to one parasite population, indicating that the assumption that river basins constitute individual transmission zones is not supported by the data. Parasites from Mali and Côte d'Ivoire are genetically distinct from those from Ghana. This research provides the basis for developing tools for elimination programs to delineate transmission zones, to estimate the risk of parasite re-introduction via vector or human movement when intervention is stopped in one area while transmission is ongoing in others, to identify the origin of infections detected post-treatment cessation, and to investigate whether persisting prevalence despite ongoing interventions in one area is due to parasites imported from others.

The stage- and sex-specific transcriptome of the human parasite Schistosoma mansoni.

The flatworm Schistosoma mansoni is an important but neglected pathogen that causes the disease schistosomiasis in millions of people worldwide. The parasite has a complex life cycle, undergoing sexual reproduction in a mammalian host and asexual replication in a snail host. Understanding the molecular mechanisms that the parasite uses to transition between hosts and develop into dimorphic reproductively competent adults may reveal new strategies for control. We present the first comprehensive transcriptomic analysis of S. mansoni, from eggs to sexually naïve worms. Focusing on eight life stages spanning free-living water-borne and parasitic stages from both intermediate and definitive hosts, we have generated deep RNA-seq data for five replicates per group for a total of 75 data sets. The data were produced using a single approach to increase the accuracy of stage-to-stage comparisons and made accessible via a user-friendly tool to visualise and explore gene expression ( https://lifecycle.schisto.xyz/ ). These data are valuable for understanding the biology and sex-specific development of schistosomes and the interpretation of complementary genomic and functional genetics studies.

Trophic ecology of Octopus vulgaris paralarvae along the Iberian Canary current eastern boundary upwelling system.

Our knowledge of the diet of wild octopus paralarvae, Octopus vulgaris, is restricted to the first 2 weeks of its planktonic phase when they are selective hunters found near the coastline. These small paralarvae, bearing only three suckers per arm, are transported by oceanic currents from the coast towards offshore waters, where they complete the planktonic phase over 2 months. Here, we have investigated the trophic ecology of O. vulgaris paralarvae in two contrasting upwelling sub-regions of the Iberian Canary current (ICC) eastern boundary upwelling system and have evaluated dietary change as paralarvae develop (inferred by counting the number of suckers per arm, ranging from three to 15) along the coastal-oceanic gradient during their planktonic phase. Using high-throughput amplicon sequencing, we have characterised the diet of 100 paralarvae collected along the Northwest Iberian Peninsula (n = 65, three to five suckers per arm) and off the west coast of Morocco (n = 35, three to 15 suckers per arm), identifying up to 87 different prey species. The diet of paralarvae varied along the ICC, with crabs (53.4%), siphonophores (12.2%), copepods (12.3%), cnidarians (8.4%) and pteropods (3.7%) accounting for 90% of the variability detected off NW Iberian Peninsula, whereas off W Morocco, crabs (46.2%), copepods (23.1%), cnidarians (12.9%), krill (9.3%) and fishes (4.2%) explained 95.6% of the variability observed using frequency of observance (FOO%) data. Ontogenetic changes in the diet based on groups of paralarvae with similar numbers per arm were evidenced by the decreasing contribution of coastal meroplankton and an increase in oceanic holoplankton, including siphonophores, copepods, pteropods and krill. Trophic niche breadth values ranged from 0.06 to 0.67, with averaged values ranging from 0.23 to 0.33 (generalist = 1 and specialist = 0), suggesting that O. vulgaris paralarvae are selective predators through their ontogenetic transition between coastal and oceanic environments.

Evaluation of genome skimming to detect and characterise human and livestock helminths.

The identification of gastrointestinal helminth infections of humans and livestock almost exclusively relies on the detection of eggs or larvae in faeces, followed by manual counting and morphological characterisation to differentiate species using microscopy-based techniques. However, molecular approaches based on the detection and quantification of parasite DNA are becoming more prevalent, increasing the sensitivity, specificity and throughput of diagnostic assays. High-throughput sequencing, from single PCR targets through to the analysis of whole genomes, offers significant promise towards providing information-rich data that may add value beyond traditional and conventional molecular approaches; however, thus far, its utility has not been fully explored to detect helminths in faecal samples. In this study, low-depth whole genome sequencing, i.e. genome skimming, has been applied to detect and characterise helminth diversity in a set of helminth-infected human and livestock faecal material. The strengths and limitations of this approach are evaluated using three methods to characterise and differentiate metagenomic sequencing data based on (i) mapping to whole mitochondrial genomes, (ii) whole genome assemblies, and (iii) a comprehensive internal transcribed spacer 2 (ITS2) database, together with validation using quantitative PCR (qPCR). Our analyses suggest that genome skimming can successfully identify most single and multi-species infections reported by qPCR and can provide sufficient coverage within some samples to resolve consensus mitochondrial genomes, thus facilitating phylogenetic analyses of selected genera, e.g. Ascaris spp. Key to this approach is both the availability and integrity of helminth reference genomes, some of which are currently contaminated with bacterial and host sequences. The success of genome skimming of faecal DNA is dependent on the availability of vouchered sequences of helminths spanning both taxonomic and geographic diversity, together with methods to detect or amplify minute quantities of parasite nucleic acids in mixed samples.

Improving helminth genome resources in the post-genomic era.

Rapid advancement in high-throughput sequencing and analytical approaches has seen a steady increase in the generation of genomic resources for helminth parasites. Now, helminth genomes and their annotations are a cornerstone of numerous efforts to compare genetic and transcriptomic variation, from single cells to populations of globally distributed parasites, to genome modifications to understand gene function. Our understanding of helminths is increasingly reliant on these genomic resources, which are primarily static once published and vary widely in quality and completeness between species. This article seeks to highlight the cause and effect of this variation and argues for the continued improvement of these genomic resources - even after their publication - which is necessary to provide a more accurate and complete understanding of the biology of these important pathogens.

A genetic TRP down the channel to praziquantel resistance.

The anthelmintic praziquantel (PZQ) is an essential tool in controlling schistosomiasis, so reports of reduced PZQ efficacy are of great public health concern. Le Clec'h et al. recently identified a gene responsible for PZQ resistance in experimentally selected resistant Schistosoma mansoni. The importance of this locus in natural infections remains to be established.

Genome-wide analysis of the response to ivermectin treatment by a Swedish field population of Haemonchus contortus.

Haemonchus contortus is a pathogenic gastrointestinal nematode of small ruminants and, in part due to its capacity to develop resistance to drugs, contributes to significant losses in the animal production sector worldwide. Despite decades of research, comparatively little is known about the specific mechanism(s) driving resistance to drugs such as ivermectin in this species. Here we describe a genome-wide approach to detect evidence of selection by ivermectin treatment in a field population of H. contortus from Sweden, using parasites sampled from the same animals before and seven days after ivermectin exposure followed by whole-genome sequencing. Despite an 89% reduction in parasites recovered after treatment measured by the fecal egg count reduction test, the surviving population was highly genetically similar to the population before treatment, suggesting that resistance has likely evolved over time and that resistance alleles are present on diverse haplotypes. Pairwise gene and SNP frequency comparisons indicated the highest degree of differentiation was found at the terminal end of chromosome 4, whereas the most striking difference in nucleotide diversity was observed in a region on chromosome 5 previously reported to harbor a major quantitative trait locus involved in ivermectin resistance. These data provide novel insight into the genome-wide effect of ivermectin selection in a field population as well as confirm the importance of the previously established quantitative trait locus in the development of resistance to ivermectin.

Worms and bugs of the gut: the search for diagnostic signatures using barcoding, and metagenomics-metabolomics.

Gastrointestinal (GI) helminth infections cause significant morbidity in both humans and animals worldwide. Specific and sensitive diagnosis is central to the surveillance of such infections and to determine the effectiveness of treatment strategies used to control them. In this article, we: (i) assess the strengths and limitations of existing methods applied to the diagnosis of GI helminth infections of humans and livestock; (ii) examine high-throughput sequencing approaches, such as targeted molecular barcoding and shotgun sequencing, as tools to define the taxonomic composition of helminth infections; and (iii) discuss the current understanding of the interactions between helminths and microbiota in the host gut. Stool-based diagnostics are likely to serve as an important tool well into the future; improved diagnostics of helminths and their environment in the gut may assist the identification of biomarkers with the potential to define the health/disease status of individuals and populations, and to identify existing or emerging anthelmintic resistance.

Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus.

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.

Faecal egg counts and nemabiome metabarcoding highlight the genomic complexity of equine cyathostomin communities and provide insight into their dynamics in a Scottish native pony herd.

Understanding the composition of gastrointestinal nematode communities may help to mitigate or exploit parasite adaptations within their host. We have used nemabiome deep amplicon sequencing of internal transcribed spacer-2 (ITS-2) ribosomal DNA to describe the temporal and host species composition of gastrointestinal nematode communities following sampling of six Scottish ponies across 57 months. In the absence of parasite control, each horse showed seasonal trends of increases and decreases in faecal egg counts, consistent with the epidemiology of equine strongylid parasites, however, the composition of parasites within individuals changed over time. Sixteen presumptive strongylid species were identified in each of the horses, 13 of which were distributed in a complex clade together with small numbers of amplicon sequences which could not be classified beyond the Cyathostominae subfamily level. Egg shedding of seven trichostrongylid species, which had previously been identified in co-grazed Soay sheep, was identified during the early spring. Faecal egg counts and the percentage of amplicon sequences assigned to each gastrointestinal nematode species were combined to describe their relative abundance across both host and time. Significant differences in species diversity between horses and between months were observed, being greatest from March to May and least from October to December. The magnitude of the individual horse effect varied between months and, conversely, the magnitude of the seasonal effect varied between individual horses. The most abundant gastrointestinal nematode in each of the horses was Cylicostephanus longibursatus (46.6% overall), while the abundance of the other strongylid species varied between horses and relative to each other. Patent C. longibursatus infections over the winter months might represent a genetic adaptation towards longer adult worm survival, or a lower rate of developmental arrest in the autumn. This study provides insight into highly complex phylogenetic relationships between closely related cyathostomin species; and describes the dynamics of egg shedding and pasture contamination of co-infecting equine gastrointestinal nematode communities. The results could be applied to determine how climatic and management factors affect the equilibrium between hosts and their parasites, and to inform the development of sustainable gastrointestinal nematode control strategies for different host species.