International Journal for Parasitology

The endocannabinoid signalling (ECS) system is a complex lipid signalling pathway that modulates diverse physiological processes in both vertebrate and invertebrate systems. In nematodes, knowledge of endocannabinoid (EC) biology is derived primarily from the free-living model species Caenorhabditis elegans, where ECS has been linked to key aspects of nematode biology. The conservation and complexity of nematode ECS beyond C. elegans is largely uncharacterised, undermining the understanding of ECS biology in nematodes including species with key importance to human, veterinary and plant health. In this study we exploited publicly available omics datasets, in silico bioinformatics and phylogenetic analyses to examine the presence, conservation and life-stage expression profiles of EC-effectors across phylum Nematoda. Our data demonstrate that: (i) ECS is broadly conserved across phylum Nematoda, including in therapeutically and agriculturally relevant species; (ii) EC-effectors appear to display clade and lifestyle-specific conservation patterns; (iii) filarial species possess a reduced EC-effector complement; (iv) there are key differences between nematode and vertebrate EC-effectors; (v) life stage-, tissue- and sex-specific EC-effector expression profiles suggest a role for ECS in therapeutically relevant parasitic nematodes. These data also highlight putative novel targets for anthelmintic therapies. To our knowledge, this study represents the most comprehensive characterisation of ECS pathways in phylum Nematoda and inform our understanding of nematode ECS complexity. Fundamental knowledge of nematode ECS systems will seed follow-on functional studies in key nematode parasites to underpin novel drug target discovery efforts. CONTRIBUTION TO THE FIELDThis manuscript reports the in silico characterisation of endocannabinoid (EC) signalling pathways across the nematode phylum. The physiological relevance and therapeutic potential of EC signalling in higher organisms has received significant attention. In contrast much of our knowledge on EC signalling in nematodes has been derived from the free-living nematode Caenorhabditis elegans where the EC signalling system appears to play key roles in nematode biology and features GPCRs distinct from vertebrate cannabinoid receptors. Unfortunately, the configuration and broader biological significance of EC signalling pathways across the nematode phylum, including in parasites of agricultural, veterinary and medical significance, remains unknown. The in silico exploration of the nematode EC signalling system reported here will provide baseline data on novel neuronal signalling pathways to seed future drug target discovery pipelines for parasites.

Identifying key drivers of pathogen infection prevalence and intensity in wildlife is essential to understand disease dispersal and transmission. Calodium hepatica (syn. Capillaria hepatica) is a generalist nematode that infects liver parenchyma of mammals worldwide and is capable of human infections. Prevalence ranges from 0-100% in wildlife, often varying across small geographic areas, making it an ideal parasite for understanding ecological drivers of variation. Here, we quantify prevalence of Calodium hepatica and present initial surveys of synanthropic small mammals in four villages representing differing land cover. Cross-sectional rodent trapping was conducted within and around households over consecutive dry seasons in Eastern Uganda. 18s rRNA gene of C. hepatica was amplified and a sub-set of PCR products sequenced to confirm presence of C. hepatica. Landscape structural diversity was classified by tree crown density and mean canopy height derived from 30m LiDAR data within a 0.5km buffer. Multivariable binomial generalised linear models were fit to C. hepatica prevalence. C. hepatica infection was common (overall 34.5%, CI95% 27.9-41.0) and found in rodent and shrew species inside and outside residences. We observe village-level differences in prevalence (18.2%-75.0%), with higher C. hepatica prevalence associated with higher relative proportion of Rattus rattus to other species (aOR=2.22 CI95% 1.30-3.85). Host diversity appears to be protective against parasite prevalence. Differences in molecular and macroscopy identification highlight challenges in diagnosis and a need for more specialized molecular tools. Further investigation is required to understand individual host and community variation in pathogen infection intensity and implications for zoonotic risk. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/674808v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@e9cfdaorg.highwire.dtl.DTLVardef@4d0f6dorg.highwire.dtl.DTLVardef@12fe43dorg.highwire.dtl.DTLVardef@1318d83_HPS_FORMAT_FIGEXP M_FIG C_FIG Key FindingsO_LIZoonotic reservoirs, including Rattus rattus, Mastomys erythroleucus and Crocidura olivieri, are abundant in human-modified habitats and show elevated levels of household incursion C_LIO_LIHigh prevalence of Calodium hepatica, a nematode endoparasite, was identified in a range of small mammal species C_LIO_LIHigher proportion of native small mammal species relative to Rattus rattus appears protective against zoonotic pathogen load, with higher village-level C. hepatica prevalence in ecologically depleted sites C_LIO_LISmall-holder agriculture may provide a dilution effect through secondary wildlife support and small mammal competition, while dense village settings potentiate C. hepatica prevalence C_LI

BackgroundOnchocerca volvulus infection is linked to onchocerciasis-associated epilepsy (OAE) in humans, but the role of Onchocerca ochengi in epilepsy development remains unexplored. This study aimed to investigate whether O. ochengi infection contributes to epilepsy development. Methodology/Principal FindingsGerbils were implanted with O. ochengi worm masses (test group) or underwent sham surgery (control group). Behavioral and physical assessments were performed between days 15-19 using multiple tests, including the elevated plus maze, open-field, object recognition, and hanging wire tests. On day 21, gerbils were sacrificed, and body/organ weights were recorded, along with worm mass survival. Implantation of 15 worm masses resulted in 100% mortality in the test group, while implantation of 10 worm masses resulted in 53.3% mortality, with all control animals surviving. At day 21, worm mass survival averaged 1.4 out of 10, with a viability score of 93.3%. Test animals showed significant reductions in body weight and increased spleen weight compared to controls, but no significant behavioral differences were observed. Conclusions/SignificanceWhile O. ochengi infection caused notable physical effects, including high mortality and changes in body/organ weights, no behavioral evidence of epilepsy was observed. The high mortality rate and limited observation period restrict the interpretation of these findings. Further studies with larger cohorts and longer observation periods are needed to assess the potential role of Onchocerca spp. in epilepsy development. To the best of our knowledge, this study represents the first attempt to establish an animal model for OAE. Author SummaryOnchocerca volvulus infection is linked to onchocerciasis-associated epilepsy (OAE) in humans, but we know less about whether other types of Onchocerca, such as Onchocerca ochengi, might also contribute to epilepsy. Our study aimed to investigate this question by infecting gerbils with O. ochengi worm masses and observing the impact on their behavior and physical health. We found that while the infection caused significant physical changes, including high mortality rates and changes in body and organ weights, there were no signs of behavioral changes typical of epilepsy. In particular, we did not see any of the usual neurological symptoms that might indicate epilepsy. These results suggest that while O. ochengi can affect animal health in some ways, it might not be directly involved in causing epilepsy. However, the high mortality rate in the infected gerbils and the relatively short duration of the study mean that we cannot draw firm conclusions. Future research with more animals and a longer time frame will be important to better understand whether Onchocerca worms contribute to epilepsy development in humans.

Schistosoma mansoni is a parasitic helminth that is vectored through freshwater snails. While the anti-schistosome defense of the South American snail, Biomphalaria glabrata, is well studied, little is known about the immune response of the African snail, Biomphalaria sudanica. We measured expression of five candidate immune genes in B. sudanica 8, 24, and 72 hours post-exposure to S. mansoni using reverse transcription quantitative PCR. Expression patterns of resistant snails were compared to susceptible snails and those sham exposed. We also assessed how diet (lettuce vs. pellet) affected expression of three genes, given prior findings that pellet-fed snails were more susceptible to S. mansoni. Results indicated that resistant snails constitutively expressed higher levels of superoxide dismutase 1 (SOD1) than susceptible snails, consistent with expression patterns of resistant B. glabrata. Parasite-induced expression occurred at 8 hours in SOD1, biomphalysin, thioester protein 1 (TEP1), and granulin (GRN); however, for biomphalysin and TEP1, induced expression was only detected for susceptible snails. At 24 hours, biomphalysin expression increased in exposed resistant snails, and at 72 hours, all exposed snails decreased biomphalysin expression compared to controls. Parasite-induced expression of SOD1, biomphalysin, TEP1, and GRN supports the hypothesis that these genes play a role in B. sudanica anti-schistosome defense, however increased expression does not necessarily yield clearance of S. mansoni. SOD1 expression was higher in lettuce-fed snails at 8 and 24 hours, consistent with their greater resistance. Together, these results demonstrate the conserved and unique aspects of the B. sudanica anti-schistosome response.

BackgroundParasitic nematodes significantly undermine global human and animal health and productivity. Parasite control is reliant on anthelmintic administration however over-use of a limited number of drugs has resulted in escalating parasitic nematode resistance, threatening the sustainability of parasite control and underscoring an urgent need for the development of novel therapeutics. FMRFamide-like peptides (FLPs), the largest family of nematode neuropeptides, modulate nematode behaviours including those important for parasite survival, highlighting FLP receptors (FLP-GPCRs) as appealing putative novel anthelmintic targets. Advances in omics resources have enabled the identification of FLPs and neuropeptide-GPCRs in some parasitic nematodes, but remaining gaps in FLP-ligand libraries hinder the characterisation of receptor-ligand interactions, which are required to drive the development of novel control approaches. ResultsIn this study we exploited recent expansions in nematode genome data to identify 2143 flp-genes in >100 nematode species across free-living, entomopathogenic, plant, animal and human lifestyles and representing 7 of the 12 major nematode clades (1). Our data reveal that: (i) the phylum-spanning flps, flp-1, -8, -14, and -18, may be representative of the flp profile of the last common ancestor of nematodes; (ii) the majority of parasitic nematodes have a reduced flp complement relative to free-living species; (iii) FLP prepropeptide architecture is variable within and between flp-genes and across nematode species; (iv) FLP prepropeptide signatures facilitate flp-gene discrimination; (v) FLP motifs display variable length, amino acid sequence, and conservation; (vi) CLANS analysis provides insight into the evolutionary history of flp-gene sequelogues and reveals putative flp-gene paralogues and, (viii) flp expression is upregulated in the infective larval stage of several nematode parasites. ConclusionsThese data provide the foundation required for phylum-spanning FLP-GPCR deorphanisation screens in nematodes to seed the discovery and development of novel parasite control approaches.

Infections with soil transmitted helminths (STHs) are highly prevalent in humans living in the intertropical region. While, in most cases, STHs can establish chronic infections, the dynamics of the infection can be altered when other parasites exploit the same host. These changes can have consequences in terms of the health of the host, the epidemiology of the disease (e.g., the duration of the infection and the inter-host transmission success) and the fitness of the parasite. Here, we investigated if the coinfection with Plasmodium yoelii alters the dynamics and the fitness of the murine nematode Heligmosomoides polygyrus. We found that, compared to single infected mice, coinfection produced an increase in the number of excreted eggs, while the biomass of adult worms in the intestine did not differ between single infected and coinfected mice. Moreover, the increase in egg excretion was also observed when Plasmodium infected hosts that had been harboring the nematode during the past four weeks (i.e., when the population size of adult worms can only decrease due to mortality). Therefore, the enhanced shedding of eggs reflects a plastic adjustment of worm fecundity to the environment provided by a coinfected host. This plastic response was modulated by the host Th2 immunity, as coinfection inhibited IL-4 and IL-13 gene expression, plasma levels of IL-5 and IL-13, and the expansion of GATA-3+ CD4+ T cells in the spleen. In agreement with this, experimentally inhibiting IL-13 with neutralizing antibodies reproduced the results observed in coinfected mice (an increase in egg excretion), while the administration of recombinant IL-13 reduced egg shedding. Interestingly, coinfection had a net positive effect on parasite fitness as shown by a longer persistence within the host and higher cumulative number of eggs excreted up to 99 days post-infection. Although the gene expression of Th2 cytokines was lower at day 99 p.i., coinfected mice still had a downregulated expression compared to single infected hosts. These results show that coinfection with Plasmodium has the potential to affect the epidemiology of STHs by increasing the number of eggs excreted over the whole infectious period and maintaining a larger environmental reservoir of transmissible stages. Author SummaryCoinfection between soil-transmitted helminths and malaria is common in several countries of the intertropical region, especially among the most vulnerable populations. Coinfection has the potential to worsen the symptoms caused by malaria, therefore it is important to understand what are the epidemiological and ecological factors that promote the occurrence of coinfection. Transmission of soil-transmitted helminths usually requires human contact with transmissible stages (parasitic eggs or larvae) in the environment; therefore, high egg excretion in the feces of infected people is a key factor contributing to maintain a reservoir of infective stages from which humans can get infected. In this study, we experimentally investigated whether coinfection with malaria alters the dynamics (egg excretion, infection persistence) of a murine intestinal nematode. We found that hosts infected with malaria and subsequently infected with the nematode, excreted more nematode eggs for a longer period, compared to single infected hosts. These changes were mediated by an impaired Th2 immune response in coinfected hosts. These results suggest that malaria coinfection produces positive feedback on key epidemiological traits of the nematode that can further enhance the risk of malaria/helminths cooccurrence.

BackgroundNatural interspecific hybridization between the human parasite (Schistosoma haematobium [Sh]) and bovine parasites (S. bovis [Sb], S. curassoni [Sc]) is increasingly reported in Africa. We developed a multi-locus PCR DNA-Seq strategy that amplifies two unlinked nuclear (transITS, BF) and two linked organellar genome markers (CO1, NAD5) to genotype S. haematobium eggs collected from infected people in Ile Oluji/Oke Igbo, Ondo State (an agrarian community) and Kachi, Jigawa State (a pastoral community) in Southwestern and Northern Nigeria, respectively. Principal FindingsWe applied this methodology against 57 isolates collected from a total of 219 participants. All patients from Jigawa state were infected with just one of two haplotypes of an S. haematobium x S. bovis hybrid based on sequences obtained at CO1, NAD5, transITS and BF markers. Whereas samples collected from Ondo state were varied. Mitonuclear discordance was observed in all 17 patients, worms possessed an Sb mitochondrial genome but one of four different haplotypes at the nuclear markers, either admixed (heterozygous between Sh x Sc or Sh x Sb) at both markers (n=10), Sh at BF and admixed at transITS (Sh x Sc) (n=5), admixed (Sh x Sc) at BF and homozygous Sc at transITS (n=1) or homozygous Sh at BF and homozygous Sc at transITS (n=1). SignificancePrevious work suggested that zoonotic transmission of S. bovis in pastoral communities, where humans and animals share a common water source, is a driving factor facilitating interspecific hybridization. However, our data showed that all isolates were hybrids, with greater diversity identified in Southwestern Nigeria, a non-pastoral site. Further, one patient possessed an S. bovis mitochondrial genome but was homozygous for S. haematobium at BF and homozygous for S. curassoni at transITS supporting at least two separate backcrosses in its origin, suggesting that interspecific hybridization may be an ongoing process. Author SummaryInterspecific hybridization between trematode parasites poses serious health risks to humans. Many systems have shown possible hybridization between different schistosome species. As evidence of natural hybridization between human S. haematobium and animal S. bovis or S. curassoni has grown in recent years, epidemiological surveys across potential hybrid zones are required, particularly in endemic African regions. According to several reports, indiscriminate human-animal water contact is a major factor contributing to hybridization of human and animal schistosomes. We collected and genotyped 57 parasite isolates from pastoral and non-pastoral communities in Kachi, Jigawa state, and Ile Oluji/Oke Igbo, Ondo state, Nigeria to screen for hybrids. In both sites, we found Schistosoma hybrids with mitonuclear discordance and repeated backcrossing between S. haematobium, S. bovis, and S. curassoni. Contrary to previous reports, Schistosoma hybrids appear to be widespread and not solely dependent on human-animal water interactions.

Hybrids between Schistosoma haematobium and S. bovis are linked to both human and animal infections, highlighting the complex interspecies interactions that contribute to the spread of schistosomiasis. Additionally, S. bovis can infect multiple ruminant hosts, facilitating cross- species transmission and increasing the risk of zoonotic outbreaks. In this study, we investigated transcriptomic plasticity as a potential mechanism enabling hybrid schistosomes to adapt to alternative definitive hosts. We focused on two contexts: 1) introgressed S. haematobium x S. bovis hybrids, which demonstrated higher virulence in sheep compared to parental S. bovis, and 2) S. bovis infecting different host species. Our analysis uncovered 366 differentially expressed genes (DEGs), representing 4% of the total protein-coding genes, between introgressed hybrids and parental S. bovis in sheep. We also identified transcriptomic changes in S. bovis across different mammalian hosts (hamster and sheep), with around 30% of the total genes differentially expressed, demonstrating that S. bovis parasites display a high transcriptomic plasticity, allowing them to infect different definitive hosts. Shared enriched biological processes during introgression and host change include nuclear-transcribed mRNA catabolic processes, inner mitochondrial membrane organization, microtubule-based movement, response to endoplasmic reticulum stress, and sensory perception. These findings suggest that transcriptomic plasticity in S. bovis and hybrid worms enhance their ability to adapt and infect diverse host species, potentially increasing their zoonotic potential. This raises concerns for schistosomiasis epidemiology, as this plasticity could expand the parasites transmission capacity and complicate control efforts.

Anthelmintic resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We described a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV) resistant single nucleotide polymorphism (SNP) within arc-8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ) resistance SNPs within {beta}-tubulin isotype-1 and ITS-2 nemabiome. This constitutes the first multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing of LEV resistant H. contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an important anthelmintic class for small ruminants in New South Wales. The mixed amplicon metabarcoding approach described herein, paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector.

Helminths can modulate the immune systems of their hosts through the release of soluble molecules known as excretory-secretory products (ESPs). To better understand the extent and mechanisms behind helminth-mediated immunosuppression, we used an emerging model of host-parasite interactions, threespine stickleback, Gasterosteus aculeatus, and its tapeworm parasite Schistocephalus solidus. We examined the impacts of exposure to ESPs of S. solidus (SsESPs) originating from four Canadian lakes on the viability and function of threespine stickleback splenocytes. We found that 24 hours of exposure to low concentrations of SsESPs significantly increased overall splenocyte viability with the greatest impact on the lymphocyte population. While SsESP exposure did not alter baseline splenocyte respiratory burst activity, SsESP-treated splenic cultures demonstrated significant increases in ROS production in response to phorbol 12-myristate 13-acetate (PMA) stimulation, suggesting that SsESPs may lower the threshold for activation of the respiratory burst. These results in splenocytes contrast with previous studies demonstrating that SsESPs suppress head kidney leukocytes (HKL) viability and function, suggest that S. solidus-derived excretory secretory products may have cell-or tissue-specific immunomodulatory effects, and highlight the importance of studying host-parasite interactions across diverse immune tissues.

The liver fluke, Fasciola hepatica, is a major parasitic threat to ruminant health and productivity worldwide, with important implications for food security, animal welfare, and zoonotic risk. This study developed and validated a multiplex deep amplicon sequencing assay targeting the mitochondrial NADH dehydrogenase 1 (mt-ND1) and cytochrome c oxidase subunit 1 (mt-COX1) loci for high-throughput genotyping of F. hepatica. DNA was extracted from eggs sedimented from sheep and cattle faeces (n = 78) received from farms and from adult worm pools (n = 12) isolated at abattoirs from diverse regions across the UK. Following high-throughput sequencing, bioinformatics analysis was performed to demultiplex Illumina sequence reads and extract amplicon sequence variants (ASVs). A total of 11 ASVs were identified at each locus (mt-ND1: 264-279 bp; mt-COX1: 312-319 bp), with two or three predominant ASVs per locus, along with rare variants. Network and PCA analyses revealed two distinct clusters at the mt-ND1 locus: one primarily associated with sheep and another shared between sheep and cattle. In contrast, mt-COX1 sequence reads formed a single dominant cluster. Population analyses revealed extensive ASV sharing across regions, indicating high gene flow, likely facilitated by livestock movement and parasite adaptation.

The neglected tropical disease schistosomiasis impacts the lives of over 700 million people globally. Schistosoma mansoni, the trematode parasite that causes the most common type of schistosomiasis, requires planorbid pond snails of the genus Biomphalaria to support its larval development and transformation to the form that can infect humans. A greater understanding of neural signaling systems that are specific to the Biomphalaria intermediate host could lead to novel strategies for parasite or snail control. This study characterized a Biomphalaria glabrata neural receptor that is gated by the molluscan neuropeptide FMRF-NH2. The Biomphalaria glabrata FMRF-NH2 gated sodium channel (Bgl-FaNaC) amino acid sequence was highly conserved with FaNaCs found in related gastropods, especially the planorbid Planorbella trivolvis (91% sequence identity). In common with the P. trivolvis FaNaC, the B. glabrata receptor exhibited a low affinity (EC50: 3 x 10-4 M) and high specificity for the FMRF-NH2 agonist. Its expression in the central nervous system, detected by immunohistochemistry and in situ hybridization, was widespread, with the protein localized mainly to neuronal fibers and the mRNA confined to cell bodies. Colocalization was observed with the FMRF-NH2 tetrapeptide precursor in some neurons associated with male mating behavior. At the mRNA level, Bgl-FaNaC expression in the visceral and left parietal ganglia decreased at 20 days post infection by S. mansoni and in the shedding phase. Altered FMRF-NH2 signaling could be vital for parasite survival and proliferation in its snail intermediate host.

In 2012, the reduction in Onchocerca volvulus infection prevalence through long-term mass ivermectin distribution in African meso- and hyperendemic areas motivated expanding control of onchocerciasis (river blindness) as a public health problem to elimination of parasite transmission. Given the large contiguous hypo-, meso- and hyperendemic areas with an estimated population of 204 million, sustainable elimination requires an understanding of the geographic, and in turn genetic, boundaries of different parasite populations to ensure interventions are only stopped where the risk of re-introduction of the parasite through vector or human migration from areas with ongoing transmission is acceptable. These boundaries, which define the transmission zones of the parasite, may 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 Cote dIvoire. Population structure measures indicate that parasites from the Pru, Daka and Black Volta/Tombe river basins in central Ghana belong to one parasite population, showing that different river basins cannot be assumed to constitute independent transmission zones. This research forms 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 mass ivermectin administration 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 migration contributes to persisting prevalence levels during interventions.

Changes in climate and land use can alter risk of transmission of parasites between domestic hosts and wildlife, particularly when mediated by vectors that can travel between populations. Here we focused on tsetse flies (genus Glossina), the cyclical vectors for both Human African Trypanosomiasis (HAT) and Animal African Trypanosomiasis (AAT). The aims of this study were to investigate: 1) the diversity of vertebrate hosts that flies fed on; 2) whether host feeding patterns varied in relation to type of hosts, tsetse feeding behaviour, site or tsetse age and sex; and 3) if there was a relationship between trypanosome detection and host feeding behaviours or host types. Sources of blood meals of Glossina pallidipes were identified by sequencing of the mitochondrial cytochrome b gene and analyzed in relationship with previously determined trypanosome detection in the same flies. In an area dominated by wildlife but with seasonal presence of livestock (Nguruman), 98% of tsetse fed on single wild host species, whereas in an area including a mixture of resident domesticated animals, humans and wildlife (Shimba Hills), 52% of flies fed on more than one host species. Multiple Correspondence Analysis revealed strong correlations between feeding pattern, host type and site but these were resolved along a different dimension than trypanosome status, sex and age of the flies. Our results suggest that individual G. pallidipes in interface areas may show higher feeding success on wild hosts when available but often feed on both wild and domesticated hosts. This illustrates the importance of G. pallidipes as a vector connecting the sylvatic and domestic cycles of African trypanosomes.

In wildlife research, wild-caught vertebrates are often given anti-parasitic drugs when brought into captivity to protect animal health and reduce confounding effects of parasitic infection on research outcomes. However, the impacts of antiparasitic drugs on non-target parasite taxa are understudied, especially regarding host sex. To help address this gap, we investigated the impact of an anthelmintic medication on a protozoan gut parasite by quantifying coccidia oocyst burden in the feces of wild-caught dark-eyed juncos (Junco hyemalis) before and after two oral doses of ivermectin. Prior to treatment, males showed higher oocyst burdens than females. Following treatment, ivermectin-treated males showed larger increases in oocyst burden compared to both control males and ivermectin-treated females, while there was no difference in baseline and post-treatment oocyst burden in treated females. Our results suggest that wild-caught songbirds should be housed separately by sex during ivermectin treatment and that male enclosures should be cleaned at a relatively higher frequency due to excessive oocyst shedding. It is unclear whether increases in oocyst burden among males were attributable to helminth removal or a direct impact of ivermectin on host immune function. Further work investigating sex differences in the impact of antiparasitic drugs on non-target parasite taxa is warranted.

Seasonality is a ubiquitous feature of wildlife disease ecology, but is determined by a complex interplay of environmental, parasitological and host factors. Gastrointestinal parasites often exhibit strong seasonal dynamics in wild vertebrate populations due to, for example, environmental influences on free-living or vectored life stages, and variation in the physiological and immune status of hosts across their annual cycle. At the same time, wild populations are typically infected with multiple parasites. The seasonal dynamics of co-infecting parasites may differ depending on age and reproductive status, and associations among parasites may be driven by short-term within-individual changes or longer-term interactions that are consistent among hosts. Here, we used faecal samples and egg counts collected repeatedly from individually marked and monitored wild Soay sheep that were part of a long-term study to investigate seasonal dynamics of six gastrointestinal parasite groups (strongyle nematodes, coccidian protozoa, Capillaria, Strongyloides, Nematodirus, and Moniezia). Prevalence and abundance generally tended to be higher spring and summer, and burdens were higher in lambs than adults. Within the highly prevalent strongyle nematode group, we found differences in seasonality of egg counts depending on adult reproductive status. Reproductive ewes had increased counts in spring around the time of birth followed by a drop in abundance in summer, while barren ewes showed little evidence of seasonality. Males showed a sustained rise in egg counts through spring and summer, and sex differences were only strongly apparent in summer. In contrast, in similarly prevalent coccidia we found a peak in faecal oocyst counts in spring but no differences in seasonality among males, barren and pregnant ewes. Using multivariate mixed-effects models, we went on to show that both strongyle and coccidia counts are moderately repeatable across seasons among individuals. We further show that apparent positive correlation between strongyle and coccidia counts was driven by short-term within-individual changes in both parasite burdens rather than long-term among-individual covariation. Overall, our results demonstrate that seasonality varies across demographic and parasite groups and highlight the value of investigating fluctuating susceptibility and exposure over time for understanding epidemiology of a population.

Human infection with the intestinal nematode Strongyloides stercoralis is persistent unless effectively treated, and potentially fatal in immunosuppressed individuals. Epidemiological data are lacking due to inadequate diagnosis. A rapid antigen detection test is a priority for population surveillance, validating cure after treatment, and for screening prior to immunosuppression. We analysed open access omics data sets and used online predictors to identify S. stercoralis proteins that are likely to be present in infected stool, Strongyloides-specific, and antigenic. Transcriptomic data from gut and non-gut dwelling life cycle stages of S. stercoralis revealed 328 proteins that are differentially expressed. Strongyloides ratti proteomic data for excreted and secreted (E/S) proteins were matched to S. stercoralis, giving 1,057 orthologues. Five parasitism-associated protein families (SCP/TAPS, prolyl oligopeptidase, transthyretin-like, aspartic peptidase, acetylcholinesterase) were compared phylogenetically between S. stercoralis and outgroups, and proteins with least homology to the outgroups were selected. Proteins that overlapped between the transcriptomic and proteomic datasets were analysed by multiple sequence alignment, epitope prediction and 3D structure modelling to reveal S. stercoralis candidate peptide/protein coproantigens. We describe 22 candidates from seven genes, across all five protein families for further investigation as potential S. stercoralis diagnostic coproantigens, identified using open access data and freely-available protein analysis tools. This powerful approach can be applied to many parasitic infections with omic data to accelerate development of specific diagnostic assays for laboratory or point-of-care field application. Author summaryThe worm Strongyloides stercoralis causes infectious disease in people throughout tropical and sub-tropical regions, leading to an extensive reduction in quality of life and even death. Millions of people are at risk of infection with this parasite and improved diagnostic and control methods and technologies are urgently required. Currently, most diagnosis is carried out through methods involving visual inspection of patients faeces, which has a number of drawbacks, particularly its poor sensitivity. This paper presents a new method to develop improved diagnostic tests for S. stercoralis, by computational analysis of publicly available gene and protein sequences to predict proteins that may be detectable in faeces. This would enable the development of rapid diagnostic tests in the form of lateral flows or dipsticks, with better predictive ability and fewer drawbacks than current diagnostic methods. A number of potential proteins, predicted to have all the desired characteristics for use in such tests were found through the new method and have been presented in this paper. With validation, new diagnostic tests for S. stercoralis could be developed from these results and the computational approach could be used to target other parasitic diseases.

BackgroundThere is growing recognition that mass drug administration must be complemented with environmental interventions to interrupt schistosomiasis transmission. Accurate mapping of snails and schistosome parasite distribution is critical to identify foci of human exposure and prioritize sites for interventions. MethodologyWe conducted longitudinal environmental surveys of snails and schistosomes in 467 waterbodies across 86 villages in northwestern Tanzania to describe spatial and temporal variation in snail and schistosome parasites presence and identify relevant underlying predictors. We conducted time constrained net sampling of Bulinus snails from vegetation, sediments, and floating objects and then examined all collected snails for patent infections. Principal findingsA total of 43,272 Bulinus snails were collected across the three visits to each waterbody spanning November 2020 - August 2021, and we conducted statistical analyses on the latter two visits with more in-depth surveys (25,052 snails). We found patent schistosome infections in 0.87% of snails, 9.8% of waterbodies, and 31% of villages in all six districts. Variance decomposition indicated that variation among waterbodies was associated with variation in snail presence and the prevalence of schistosomes and nonschistosome parasites, whereas variation among villages and districts was not. Snail presence was highest in March-May a period of heavy rains, but otherwise not associated with waterbody characteristics. Waterbodies permitting cattle use had significantly higher prevalence of schistosomes than those permitting only human use. Nonschistosome parasites were more prevalent in June to September the dry season but were not associated with other waterbody characteristics. Waterbody permanence and distance to the nearest primary school were not associated with snails or parasites. Conclusions/significanceThis study revealed substantial variation in snails, schistosome and nonschistosome abundance at local (waterbody) scales, and it suggests links between community-driven water use decisions and schistosome transmission. The identification of local drivers of snail and schistosome abundance level and transmission factors at waterbody scale can complement studies across larger scales to shed light on transmission hotspots and guide the development of targeted interventions for schistosomiasis control. Author summaryCurrently there is increasing need to supplement mass drug administration with environmental interventions by identifying potential sites for human exposure to disrupt schistosomiasis transmission. Here we focus on environmental surveys to determine leading factors for presence and sequential variation in snails and schistosomes. Using a timed sampling strategy, snail vectors were collected and examined for infections. We screened snails collected from November 2020 to August 2021 and found variation among waterbodies in snail presence and infections of schistosomes and nonschistosome parasites. This variation was not observed at village and district levels. High abundance of snails was observed in March to May during heavy rainfall but not connected with waterbody distinctiveness. Waterbodies used by cattle had higher prevalence of schistosomes compared to waterbodies used by humans alone. Nonschistosomes were higher in June to September the dry season but not related to waterbody characteristics. Although chemotherapy reduces schistosomiasis burden, our results suggest that identification of transmission sites at waterbody scale could aid development of targeted interventions for schistosomiasis control.

Understanding the genes and molecular pathways that shape host responses to infection is essential for advancing our knowledge of host-parasite interactions and their ecological and evolutionary implications. Such insights are especially valuable for conserving endangered species that may be vulnerable to emerging or novel pathogens. Here, we investigated the impact of the recently introduced gastrointestinal nematode Ashworthius sidemi on gene expression in its novel host, the European bison (Bison bonasus). We analyzed abomasal transcriptomes (n = 45) from individuals characterized by a wide range of infection intensities (0-8,620 parasites per host). Despite substantial variation in parasite burden, differential expression analyses detected no individual genes significantly associated with infection intensity. However, gene set enrichment analyses based on p-value distributions revealed multiple immune-related gene ontology categories, including B and T cell activation, neutrophil chemotaxis, inflammatory responses, and regulation of IL-6 production. These findings indicate that European bison mount a clear yet subtle transcriptional response to A. sidemi infection and highlight molecular pathways potentially involved in mediating host defense against this emerging parasite.

Root-knot nematodes (RKNs) of the genus Meloidogyne are important pests in agriculture. RKNs are generalist herbivores with a wide host range including crop and wild plants. The latter are an important source of defensive metabolites that may be helpful for RKN management. Milkweeds (Asclepias spp.) produce toxic cardenolides that protect them from herbivory. However, it is unclear if cardenolides may defend milkweeds against RKNs. Here, we tested this directly through herbivory assays with the RKN M. incognita on milkweed species that produce negligible and high levels of cardenolides, A. tuberosa and A. curassavica, respectively. We found that M. incognita induces fewer galls on A. curassavica than A. tuberosa and fails to reproduce after reaching maturity on the former but not the latter species. This suggests that the predominantly polar cardenolides in A. curassavica may engender long-term reproductive toxicity. Further toxicity assays with the polar cardenolide ouabain showed that cardenolides can also have more immediate toxic effects on M. incognita at higher concentrations. Ouabain caused a coiled, paralytic phenotype in juvenile RKNs, a sign of neurotoxicity, leading to lethality in a subset of RKNs. Some nematodes recovered upon ouabain removal, confirming that neurotoxic cardenolides have a nematostatic effect that results in death when exposure persists. Taken together, our results provide further evidence that cardenolides may function as anti-herbivore defenses against RKNs. The dead end host plant A. curassaviva appears to possess useful properties that may be leveraged for control of RKNs in agriculture.