Experimental tests of the effects of host heterogeneities on parasite transmission are rare; previous studies focus on a single host trait over one generation of transmission. Thus, the long-term epidemiological consequences of interacting host heterogeneities remain unclear. We used a laboratory-based experimental system comprising the red flour beetle (Tribolium castaneum) infected with the eugregarine parasite, Gregarina cloptoni, to show, firstly, that two colonies of the beetle are heterogeneous in their susceptibility and infectiousness to the eugregarine. We then constructed experimental populations that differed in population-level heterogeneity in susceptibility and infectiousness by varying proportions of the colonies in the populations and tracked parasite transmission over eight weeks. We found that differences in parasite transmission among populations were explained by population mean susceptibility and infectiousness, rather than heterogeneity. Finally, to test the effects of heterogeneity on equilibrium parasite transmission we developed an agent-based model (ABM) for our host-parasite system. Results from our model showed that populations with the highest level of heterogeneity had the highest between-simulation variability in epidemiological measures. This demonstrated that even with the same starting conditions, host heterogeneities can drive high levels of uncertainty in epidemiological predictability. Our work presents a rare experimental assessment of how heterogeneities in susceptibility and infectiousness affect parasite transmission, showing that while these heterogeneities may not affect transmission directly, they can still affect epidemic variability, and hence predictability. This variability has considerable consequences for outbreak management strategies yet remains understudied, so further tests of the effects of host heterogeneity on epidemic variability will be important.

Abstract Soil-transmitted helminths (STHs) pose significant challenges to public health in endemic areas, necessitating reliable methods for their detection. Shotgun metagenomics enables simultaneous detection of STHs and microbes in a sample without prior knowledge of what is present. However, validation of shotgun metagenomics with known infection intensity or across different sequencing platforms has not been carried out for eukaryote parasites including STHs, and false positives remain a pervasive issue. We validated shotgun metagenomics as a method of STH detection in faecal samples. Using the Strongyloides ratti laboratory model of a STH infection we investigated how analytical methods (nucleotide-nucleotide matching, nucleotide-protein matching, marker gene detection, mitochondrial mapping), infection intensity and sequencing technology (short-read vs. long-read) affects sensitivity and specificity of detection. S. ratti was accurately detected at a standard laboratory dose, but low intensity infections were more difficult to detect. Only mitochondrial sequence mapping was 100% accurate at identifying S. ratti with no false positives. Overall, short-read outperformed long-read sequencing methods. We applied the same analytical methods to human faecal samples with confirmed infections for at least one of four STHs. Mitochondrial sequence mapping was also the most effective method for detecting STHs in human faecal samples, detecting 100% of Necator americanus and 92% of Ascaris spp. infections, but could not reliably detect STHs where DNA levels are expected to be low or variable. In conclusion, mitochondrial mapping was the most effective method of detection for sensitivity and specificity in both the laboratory system and human faecal samples. Our findings indicate that shotgun metagenomics should be approached cautiously using validated methods, particularly when infection intensity or DNA levels are expected to be low. Author Summary Soil-transmitted helminths (STH) such as the parasite Strongyloides , are important gastrointestinal parasites of humans and livestock. Accurate methods of detection for diagnostics and monitoring are important to implement suitable control and treatment strategies. Here we validate a shotgun metagenomics approach, where all DNA in a sample is sequenced, for detecting STH in faecal samples using a Strongyloides laboratory model for infection. Strongyloides was reliability detected in faecal samples at higher infection levels, but mitochondrial genome mapping of the sequences was the only analytical method that reliably detected Strongyloides at lower infections levels. These results were reflected in stool samples from humans infected with STH, where mitochondrial mapping was also the most reliable method. However, species that were associated with low levels of parasite material or DNA in the faeces including Strongyloides stercoralis, were more difficult to detect. We compared two sequencing methods: short-read Illumina and long-read Oxford Nanopore Technologies, but short-read outperformed long-read shotgun metagenomics. Contamination of bacteria sequences in parasite genome assemblies was problematic for analysis and contributed to false positive results. Future work should focus on specific targeting of eukaryote DNA either at the laboratory or bioinformatic stage to improve STH detection further.

Host heterogeneities in susceptibility and infectiousness can affect parasite transmission, but standard analyses typically consider a limited range of epidemiological scenarios. We propose two contrasting phenomenological scenarios that capture a wide range of processes by which host infectiousness is determined and explore how these scenarios affect the impact of host heterogeneities on parasite transmission. Specifically, we contrast two limiting cases for how host infectiousness may be determined: 'Recipient dependence' (RD), where a host's infectiousness is a fixed characteristic of the recipient host being infected, and 'Donor dependence' (DD), where a host's infectiousness is determined at the time of infection by properties of the donor host that infected them. Contrasting susceptible-infected models of these two phenomenological scenarios show that under RD, R0 is driven by population-level covariance between susceptibility and infectiousness, but for DD scenarios, it is driven by the maximum infectiousness present in the population. Our results show that the RD and DD scenarios generate distinctive epidemiological signatures. Although many host-parasite systems are likely to lie somewhere along a RD-DD continuum, with infectiousness influenced by both characteristics of the recipient and donor host, understanding these limiting cases provides a valuable framework for interpreting how host heterogeneity influences transmission in more complex systems.

Wild animals live in a pathogen-rich environment, and are normally infected with a wide range of micro- and macro-parasites. Wild animals’ T cells are central to the effectiveness of their adaptive immune response in ameliorating the effect of these infections. Here we have investigated the T-cell receptor (TCR) repertoire of wild mice to investigate how it varies in animals of different ages and sex, and from different sites. We sequenced the TCR alpha and beta chains of CD4 + and CD8 + T-cells of 65 wild Mus musculus domesticus from two UK sites. We analysed repertoire richness and diversity finding that wild mice have large TCR repertoires. Repertoire richness, which measures the breadth of the repertoire, was not significantly affected by mouse age or sex, suggesting that wild mice maintain the capacity to respond to novel antigens throughout their lives. In contrast, repertoire diversity (measured by Shannon’s index) was affected by a mouse sex-by-age interaction. This low diversity, coupled with constant richness, points to older mice having comparatively more highly abundant clones in their repertoires, perhaps due to chronic exposure to persistent pathogens in their environment. These findings provide a novel description of the wild mouse TCR, revealing an immune system that balances maintaining a broad response capacity with developing strong, lasting responses to infections in the natural environment.

Abstract It is well established that the microbiome can have major effects on animal biology. Here we have investigated the composition of the bacterial and eukaryotic gut microbiome of wild mice from three sample sites and sought to understand what affects its composition. We find that the bacterial and eukaryotic microbiome differs among mice from different sites. Among mouse traits, we found that only gut inflammation and the concentration of faecal immunoglobulin A affected the microbiome diversity. However, the microbiome diversity was more commonly affected by the microbiome composition itself, both within-bacterial and within-eukaryotic, but also by cross bacterial-eukaryotic effects. We found that most hosts produce IgA that binds some of their gut bacteria, though mice are largely idiosyncratic in which taxa they bind with IgA, with a few taxa commonly IgA-bound. The eukaryotic microbiome was dominated by fungal taxa, and included Eimeria infection that was particularly common at one of the sites. At the high Eimeria prevalence site, mice had comparatively marked caecal inflammation and significantly greater IgA responses. Our results emphasise the substantial among-individual mouse differences in gut microbiome composition, gut physiology and immunology, and the biological significance of the bacterial-eukaryotic effects that we suggest requires further study.

Abstract Soil-transmitted helminth (STH) infections are a major public health burden, and there are programmes of mass drug administration that attempt to ameliorate the harm that they cause. There has been increasing use of genomics to study STH infections and other parasitic nematodes, with particular interest in whole genome sequencing (WGS). For such studies, samples are commonly stored frozen, but in settings where these infections are endemic this can be difficult, and so there would be advantages to having ambient temperature storage methods. We investigated two ambient temperature storage methods – FTA cards and DESS buffer – for infective larvae of the rat parasites Nippostrongylus brasiliensis and Strongyloides ratti , prior to DNA extraction and then WGS. Our results showed that for individual larvae stored on FTA cards or in DESS buffer, this resulted in a lower proportion of sequence reads that mapped to the reference genomes, compared to the frozen control samples. Generally, for individual larvae, DESS-storage resulted in better sequencing results than FTA-storage. However, for pools of 10 or 50 larvae, then these ambient temperature storage methods generally resulted in comparable sequence read mapping to the frozen control samples.

The depletion of ribosomal RNA (rRNA) is a critical step in RNA-sequence analyses, used to enhance the detection of non-rRNA molecules, such as messenger RNAs and non-coding RNAs. However, the efficiency and potential biases introduced by different rRNA depletion methods remain poorly characterized. Here, we evaluated three commercially available rRNA depletion kits - QIAseq FastSelect, riboPOOL, Zymo-Seq RiboFree - for their performance with the parasitic nematode Strongyloides ratti. We assessed the kits' efficiency in rRNA removal, the recovery of expressed genes and transposable elements, and the detection of spliced leader sequences and genes' operonic organization. Zymo-Seq demonstrated the highest sensitivity and minimal bias in a measure of gene expression, while QIAseq showed the least rRNA depletion and significant differential expression biases. Our findings underscore the importance of empirical validation of rRNA depletion methods, particularly for parasites and non-model organisms, and we suggest that Zymo-Seq as the optimal choice for S. ratti and related nematodes.

The depletion of ribosomal RNA (rRNA) is a critical step in RNA-sequence analyses, used to enhance the detection of non-rRNA molecules, such as messenger RNAs and non-coding RNAs. However, the efficiency and potential biases introduced by different rRNA depletion methods remain poorly characterized. Here, we evaluated three commercially available rRNA depletion kits – QIAseq FastSelect, riboPOOL, Zymo-Seq RiboFree – for their performance with the parasitic nematode Strongyloides ratti . We assessed the kits’ efficiency in rRNA removal, the recovery of expressed genes and transposable elements, and the detection of spliced leader sequences and genes’ operonic organization. Zymo-Seq demonstrated the highest sensitivity and minimal bias in a measure of gene expression, while QIAseq showed the least rRNA depletion and significant differential expression biases. Our findings underscore the importance of empirical validation of rRNA depletion methods, particularly for parasites and non-model organisms, and we suggest that Zymo-Seq as the optimal choice for S. ratti and related nematodes. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/X008673/1

Gut nematode worms are important parasites of people and other animals. The parasitic nematode Strongyloides stercoralis infects an estimated 600 million people worldwide and is one of the soil-transmitted helminthiases, a WHO-defined neglected tropical disease. It has long been suggested that human S. stercoralis infection may be a zoonosis from dogs. We investigated this by whole genome sequence analysis of S. stercoralis from sympatric human and dog populations in Asia. We find that human- and dog-derived S. stercoralis have genetically distinct nuclear genomes, but we also find evidence of rare cross-infection. Analysis of the S. stercoralis mitochondrial genome reveals evidence of historical introgression between human- and dog-derived parasites. Based on these data, we suggest that S. stercoralis was originally a parasite of canids, that began to infect humans when people domesticated dogs, since when human- and dog-derived parasites have differentiated, but have not become separate species.

Gut nematode worms are important parasites of people and other animals. The parasitic nematode Strongyloides stercoralis infects an estimated 600 million people worldwide, and is one of the soil-transmitted helminthiases, a WHO-defined neglected tropical disease. It has long been suggested that human S. stercoralis infection may be a zoonosis from dogs. We investigated this by whole genome sequence analysis of S. stercoralis from sympatric human and dog populations in Asia. We find that human- and dog-derived S. stercoralis have genetically distinct nuclear genomes, but we also find evidence of rare cross infection. Analysis of the S. stercoralis mitochondrial genome reveals evidence of historical introgression between human- and dog-derived parasites. Based on these data we suggest that S. stercoralis was originally a parasite of canids, that began to infect humans when people domesticated dogs, since when human- and dog-derived parasites have differentiated, but have not become separate species. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Background Strongyloides nematodes are livestock parasites, and Strongyloides papillosus infecting ruminant livestock can cause disease. Recent genomic analysis of several Strongyloides species is now facilitating population genomic analyses of natural Strongyloides infections, for example finding that Strongyloides ratti in wild UK rats exists as an assemblage of long-lived, asexual lineages. Methods Here we have initiated an investigation into the population genomics of S. papillosus in goats in Pakistan. We sampled Strongyloides from goat faeces and then whole genome sequenced individual larvae. Results We find that S. papillosus is common, with a prevalence of 28%; that the population is genetically diverse and that individual goats commonly have mixed-genotype infections, and that there is evidence of admixture in only ca. 20% of worms. Conclusions These results now provoke further questions about the host range of different S. papillosus genotypes that can be investigated by further population genomic analyses in the future. Graphical Abstract

It is well established that heterogeneities in host susceptibility and infectiousness can affect population-level parasite transmission. However, the role played by the underlying ‘infectiousness determination scenario’, or how infectious a newly infected host (the ‘recipient’) becomes upon infection, in mediating the effects of host heterogeneity on parasite transmission is unknown. Broadly, host infectiousness may be an inherent, pre-determined trait of the recipient (which we call ‘Recipient-Dependent’ (RD)), or it may be determined by the infectiousness of the ‘donor’ host that infected them (which we call ‘Donor-Dependent’ (DD)). To investigate how these alternative infectiousness determination scenarios mediate the epidemiological consequences of host heterogeneity we developed two Susceptible-Infected compartmental models which incorporate host heterogeneity in both susceptibility and infectiousness, under the contrasting RD and DD scenarios. We then quantified how changing initial population-level heterogeneity affected three epidemiological measures in each case: the basic reproduction number ( R 0 ), temporal changes in heterogeneity, and equilibrium host abundance. We found that the primary driver of R 0 differs between the two scenarios – covariance between host susceptibility and infectiousness drives R 0 in the RD scenario, versus maximum infectiousness in the DD scenario. Heterogeneity stayed the same over time in both scenarios, though there were differences between the scenarios in the consequences of host heterogeneity on equilibrium host abundance. Overall, the findings we present demonstrate that the infectiousness determination scenario can change epidemiological outcomes considerably and should be accounted for when modelling host heterogeneities in parasite transmission. ### Competing Interest Statement The authors have declared no competing interest.

Protozoa are well-known inhabitants of the mammalian gut and so of the gut microbiome. While there has been extensive study of a number of species of gut protozoa in laboratory animals, particularly rodents, the biology of the gut protozoa of wild rodents is much less well-known. Here we have systematically searched the published literature to describe the gut protozoa of wild rodents, in total finding records of 44 genera of protozoa infecting 228 rodent host species. We then undertook meta-analyses that estimated the overall prevalence of gut protozoa in wild rodents to be 24%, with significant variation in prevalence among some host species. We investigated how host traits may affect protozoa prevalence, finding that for some host lifestyles some protozoa differed in their prevalence. This synthesis of existing data on wild rodent gut protozoa provides a better understanding of the biology of these common gut inhabitants and suggests directions for their future study.

Wild animals are under constant threat from a wide range of micro- and macroparasites in their environment. Animals make immune responses against parasites, and these are important in affecting the dynamics of parasite populations. Individual animals vary in their anti-parasite immune responses. Genetic polymorphism of immune-related loci contributes to inter-individual differences in immune responses, but most of what we know in this regard comes from studies of humans or laboratory animals; there are very few such studies of wild animals naturally infected with parasites. Here we have investigated the effect of single nucleotide polymorphisms (SNPs) in immune-related loci (the major histocompatibility complex [MHC], and loci coding for cytokines and Toll-like receptors) on a wide range of immune and infection phenotypes in UK wild house mice, Mus musculus domesticus. We found strong associations between SNPs in various MHC and cytokine-coding loci on both immune measures (antibody concentration and cytokine production) and on infection phenotypes (infection with mites, worms and viruses). Our study provides a comprehensive view of how polymorphism of immune-related loci affects immune and infection phenotypes in naturally infected wild rodent populations.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue ‘ Strongyloides : omics to worm-free populations’.

Wild animals are naturally infected with a range of viruses, some of which may be zoonotic. During the human COVID pandemic there was also the possibility of rodents acquiring SARS-CoV-2 from people, so-called reverse zoonoses. To investigate this, we sampled rats (Rattus norvegicus) and mice (Apodemus sylvaticus) from urban environments in 2020 during the human COVID-19 pandemic. We metagenomically sequenced lung and gut tissue and faeces for viruses, PCR screened for SARS-CoV-2, and serologically surveyed for anti-SARS-CoV-2 Spike antibodies. We describe the range of viruses that we found in these two rodent species. We found no molecular evidence of SARS-CoV-2 infection, though in rats we found lung antibody responses and evidence of neutralization ability that are consistent with rats being exposed to SARS-CoV-2 and/or exposed to other viruses that result in cross-reactive antibodies.

Nematodes are important parasites of people and animals, and in natural ecosystems they are a major ecological force. Strongyloides ratti is a common parasitic nematode of wild rats and we have investigated its population genetics using single-worm, whole-genome sequencing. We find that S. ratti populations in the UK consist of mixtures of mainly asexual lineages that are widely dispersed across a host population. These parasite lineages are likely very old and may have originated in Asia from where rats originated. Genes that underly the parasitic phase of the parasite's life cycle are hyperdiverse compared with the rest of the genome, and this may allow the parasites to maximise their fitness in a diverse host population. These patterns of parasitic nematode population genetics have not been found before and may also apply to Strongyloides spp. that infect people, which will affect how we should approach their control.

Summary The gut microbiome is an assemblage of microbes that have profound effects on their hosts. The composition of the microbiome is affected by bottom-up, among-taxa interactions and by top-down, host effects, which includes the host immune response. While the high-level composition of the microbiome is generally stable over time, component strains and genotypes will constantly be evolving, with both bottom-up and top-down effects acting as selection pressures, driving microbial evolution. Secretory IgA is a major feature of the gut’s adaptive immune response, and a substantial proportion of gut bacteria are coated with IgA, though the effect of this on bacteria is unclear. Here we hypothesize that IgA binding to gut bacteria is a selection pressure that will drive the evolution of IgA-bound bacteria, so that they will have a different evolutionary trajectory than those bacteria not bound by IgA. We know very little about the microbiome of wild animals and even less about their gut immune responses, but it must be a priority to investigate this hypothesis to understand if and how host immune responses contribute to microbiome evolution.

Strongyloides' developmental switch between direct, parasitic and indirect, free-living development has intrigued, confused, and fascinated biologists since it was first discovered more than 100 years ago. Proximately, the switch is controlled by environmental conditions that developing larvae are exposed to, but genotypes differ in their sensitivity to these cues. Ultimately, selection will act on this switch to generate a direct vs. indirect phenotype that maximises a genotype's fitness, but we have a poor understanding of the relative fitness advantages of these different routes of development. Mechanistically, the switch senses and transduces environmental cues, integrates signals that are then used to make a developmental decision which is then enacted. Seeking to understand the molecular form of this process has focussed on the C. elegans dauer hypothesis, but this has been found to be wanting. So, we argue that the time has come to move beyond the dauer hypothesis and better refine our question to ask: What is it that controls the variation in developmental switching among Strongyloides genotypes? We discuss approaches to achieve this research aim that now lies within our grasp.