Strongyloides questions-a research agenda for the future.

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'.

The parasitic nematode Strongyloides ratti exists predominantly as populations of long-lived asexual lineages.

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.

The genetics of immune and infection phenotypes in wild mice, Mus musculus domesticus.

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 ecology of viruses in urban rodents with a focus on SARS-CoV-2.

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.

Gut immune responses and evolution of the gut microbiome-a hypothesis.

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.

piRNA-like small RNAs target transposable elements in a Clade IV parasitic nematode.

The small RNA (sRNA) pathways identified in the model organism Caenorhabditis elegans are not widely conserved across nematodes. For example, the PIWI pathway and PIWI-interacting RNAs (piRNAs) are involved in regulating and silencing transposable elements (TE) in most animals but have been lost in nematodes outside of the C. elegans group (Clade V), and little is known about how nematodes regulate TEs in the absence of the PIWI pathway. Here, we investigated the role of sRNAs in the Clade IV parasitic nematode Strongyloides ratti by comparing two genetically identical adult stages (the parasitic female and free-living female). We identified putative small-interfering RNAs, microRNAs and tRNA-derived sRNA fragments that are differentially expressed between the two adult stages. Two classes of sRNAs were predicted to regulate TE activity including (i) a parasite-associated class of 21-22 nt long sRNAs with a 5' uridine (21-22Us) and a 5' monophosphate, and (ii) 27 nt long sRNAs with a 5' guanine/adenine (27GAs) and a 5' modification. The 21-22Us show striking resemblance to the 21U PIWI-interacting RNAs found in C. elegans, including an AT rich upstream sequence, overlapping loci and physical clustering in the genome. Overall, we have shown that an alternative class of sRNAs compensate for the loss of piRNAs and regulate TE activity in nematodes outside of Clade V.

Approaches to studying the developmental switch of Strongyloides - Moving beyond the dauer hypothesis.

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.

A One Health Approach to Child Stunting: Evidence and Research Agenda.

Stunting (low height for age) affects approximately one-quarter of children aged < 5 years worldwide. Given the limited impact of current interventions for stunting, new multisectoral evidence-based approaches are needed to decrease the burden of stunting in low- and middle-income countries (LMICs). Recognizing that the health of people, animals, and the environment are connected, we present the rationale and research agenda for considering a One Health approach to child stunting. We contend that a One Health strategy may uncover new approaches to tackling child stunting by addressing several interdependent factors that prevent children from thriving in LMICs, and that coordinated interventions among human health, animal health, and environmental health sectors may have a synergistic effect in stunting reduction.

Network analysis of the immune state of mice.

The mammalian immune system protects individuals from infection and disease. It is a complex system of interacting cells and molecules, which has been studied extensively to investigate its detailed function, principally using laboratory mice. Despite the complexity of the immune system, it is often analysed using a restricted set of immunological parameters. Here we have sought to generate a system-wide view of the murine immune response, which we have done by undertaking a network analysis of 120 immune measures. To date, there has only been limited network analyses of the immune system. Our network analysis identified a relatively low number of communities of immune measure nodes. Some of these communities recapitulate the well-known T helper 1 vs. T helper 2 cytokine polarisation (where ordination analyses failed to do so), which validates the utility of our approach. Other communities we detected show apparently novel juxtapositions of immune nodes. We suggest that the structure of these other communities might represent functional immunological units, which may require further empirical investigation. These results show the utility of network analysis in understanding the functioning of the mammalian immune system.

Toward genetic modification of plant-parasitic nematodes: delivery of macromolecules to adults and expression of exogenous mRNA in second stage juveniles.

Plant-parasitic nematodes are a continuing threat to food security, causing an estimated 100 billion USD in crop losses each year. The most problematic are the obligate sedentary endoparasites (primarily root knot nematodes and cyst nematodes). Progress in understanding their biology is held back by a lack of tools for functional genetics: forward genetics is largely restricted to studies of natural variation in populations and reverse genetics is entirely reliant on RNA interference. There is an expectation that the development of functional genetic tools would accelerate the progress of research on plant-parasitic nematodes, and hence the development of novel control solutions. Here, we develop some of the foundational biology required to deliver a functional genetic tool kit in plant-parasitic nematodes. We characterize the gonads of male Heterodera schachtii and Meloidogyne hapla in the context of spermatogenesis. We test and optimize various methods for the delivery, expression, and/or detection of exogenous nucleic acids in plant-parasitic nematodes. We demonstrate that delivery of macromolecules to cyst and root knot nematode male germlines is difficult, but possible. Similarly, we demonstrate the delivery of oligonucleotides to root knot nematode gametes. Finally, we develop a transient expression system in plant-parasitic nematodes by demonstrating the delivery and expression of exogenous mRNA encoding various reporter genes throughout the body of H. schachtii juveniles using lipofectamine-based transfection. We anticipate these developments to be independently useful, will expedite the development of genetic modification tools for plant-parasitic nematodes, and ultimately catalyze research on a group of nematodes that threaten global food security.

Correction to: The population genetics of parasitic nematodes of wild animals.

Unfortunately, the original version of this article [1] contains an error. In the section entitled "Influence of anthropogenic disruption on parasitic nematode population genetics", the passage.

The gut microbiota of wild rodents: Challenges and opportunities.

The gut microbiota can have important, wide-ranging effects on its host. To date, laboratory animals, particularly mice, have been the major study system for microbiota research. It is now becoming increasingly clear that laboratory animals often poorly model aspects of the biology of wild animals, and this concern extends to the study of the gut microbiota. Here, the relatively few studies of the microbiota of wild rodents are reviewed, including a critical assessment of how the gut microbiota differs between laboratory and wild rodents. Finally, the many potential advantages and opportunities of wild-animal systems for research into the gut microbiota are considered.

The population genetics of parasitic nematodes of wild animals.

Parasitic nematodes are highly diverse and common, infecting virtually all animal species, and the importance of their roles in natural ecosystems is increasingly becoming apparent. How genes flow within and among populations of these parasites - their population genetics - has profound implications for the epidemiology of host infection and disease, and for the response of parasite populations to selection pressures. The population genetics of nematode parasites of wild animals may have consequences for host conservation, or influence the risk of zoonotic disease. Host movement has long been recognised as an important determinant of parasitic nematode population genetic structure, and recent research has also highlighted the importance of nematode life histories, environmental conditions, and other aspects of host ecology. Commonly, factors influencing parasitic nematode population genetics have been studied in isolation, such that an integrated view of the drivers of population genetic structure of parasitic nematodes is still lacking. Here, we seek to provide a comprehensive, broad, and integrative picture of these factors in parasitic nematodes of wild animals that will be a useful resource for investigators studying non-model parasitic nematodes in natural ecosystems. Increasingly, new methods of analysing the population genetics of nematodes are becoming available, and we consider the opportunities that these afford in resolving hitherto inaccessible questions of the population genetics of these important animals.

The ecology of immune state in a wild mammal, Mus musculus domesticus.

The immune state of wild animals is largely unknown. Knowing this and what affects it is important in understanding how infection and disease affects wild animals. The immune state of wild animals is also important in understanding the biology of their pathogens, which is directly relevant to explaining pathogen spillover among species, including to humans. The paucity of knowledge about wild animals' immune state is in stark contrast to our exquisitely detailed understanding of the immunobiology of laboratory animals. Making an immune response is costly, and many factors (such as age, sex, infection status, and body condition) have individually been shown to constrain or promote immune responses. But, whether or not these factors affect immune responses and immune state in wild animals, their relative importance, and how they interact (or do not) are unknown. Here, we have investigated the immune ecology of wild house mice-the same species as the laboratory mouse-as an example of a wild mammal, characterising their adaptive humoral, adaptive cellular, and innate immune state. Firstly, we show how immune variation is structured among mouse populations, finding that there can be extensive immune discordance among neighbouring populations. Secondly, we identify the principal factors that underlie the immunological differences among mice, showing that body condition promotes and age constrains individuals' immune state, while factors such as microparasite infection and season are comparatively unimportant. By applying a multifactorial analysis to an immune system-wide analysis, our results bring a new and unified understanding of the immunobiology of a wild mammal.

Predicting the effects of parasite co-infection across species boundaries.

It is normal for hosts to be co-infected by parasites. Interactions among co-infecting species can have profound consequences, including changing parasite transmission dynamics, altering disease severity and confounding attempts at parasite control. Despite the importance of co-infection, there is currently no way to predict how different parasite species may interact with one another, nor the consequences of those interactions. Here, we demonstrate a method that enables such prediction by identifying two nematode parasite groups based on taxonomy and characteristics of the parasitological niche. From an understanding of the interactions between the two defined groups in one host system (wild rabbits), we predict how two different nematode species, from the same defined groups, will interact in co-infections in a different host system (sheep), and then we test this experimentally. We show that, as predicted, in co-infections, the blood-feeding nematode Haemonchus contortus suppresses aspects of the sheep immune response, thereby facilitating the establishment and/or survival of the nematode Trichostrongylus colubriformis; and that the T. colubriformis-induced immune response negatively affects H. contortus This work is, to our knowledge, the first to use empirical data from one host system to successfully predict the specific outcome of a different co-infection in a second host species. The study therefore takes the first step in defining a practical framework for predicting interspecific parasite interactions in other animal systems.

The genomic basis of nematode parasitism.

Nematodes are highly abundant animals, and many species have a parasitic lifestyle. Nematode parasites are important pathogens of humans and other animals, and there is considerable interest in understanding their molecular and genomic adaptations to nematode parasitism. This has been approached in three main ways: comparing the genomes of closely related parasitic and free-living taxa, comparing the gene expression of parasitic and free-living life cycle stages of parasitic nematode species, and analysing the molecules that parasitic nematodes excrete and secrete. To date, these studies show that many species of parasitic nematodes have genomes that have large gene families coding for proteases/peptidases, protease inhibitors, SCP/TAPS proteins and acetylcholinesterases, and in many cases there is evidence that these appear to be used by parasitic stages inside hosts, and are often secreted. Many parasitic nematodes have taxa-restricted gene families that also appear to be involved in parasitism, emphasizing that there is still much to be discovered about what it takes to be a parasitic nematode.

The Immunology of Wild Rodents: Current Status and Future Prospects.

Wild animals' immune responses contribute to their evolutionary fitness. These responses are moulded by selection to be appropriate to the actual antigenic environment in which the animals live, but without imposing an excessive energetic demand which compromises other component of fitness. But, exactly what these responses are, and how they compare with those of laboratory animals, has been little studied. Here, we review the very small number of published studies of immune responses of wild rodents, finding general agreement that their humoral (antibody) responses are highly elevated when compared with those of laboratory animals, and that wild rodents' cellular immune system reveals extensive antigenic exposure. In contrast, proliferative and cytokine responses of ex vivo-stimulated immune cells of wild rodents are typically depressed compared with those of laboratory animals. Collectively, these responses are appropriate to wild animals' lives, because the elevated responses reflect the cumulative exposure to infection, while the depressed proliferative and cytokine responses are indicative of effective immune homeostasis that minimizes immunopathology. A more comprehensive understanding of the immune ecology of wild animals requires (i) understanding the antigenic load to which wild animals are exposed, and identification of any key antigens that mould the immune repertoire, (ii) identifying immunoregulatory processes of wild animals and the events that induce them, and (iii) understanding the actual resource state of wild animals, and the immunological consequences that flow from this. Together, by extending studies of wild rodents, particularly addressing these questions (while drawing on our immunological understanding of laboratory animals), we will be better able to understand how rodents' immune responses contribute to their fitness in the wild.