Ancient diversity in host-parasite interaction genes in a model parasitic nematode.

Host-parasite interactions exert strong selection pressures on the genomes of both host and parasite. These interactions can lead to negative frequency-dependent selection, a form of balancing selection that is hypothesised to explain the high levels of polymorphism seen in many host immune and parasite antigen loci. Here, we sequence the genomes of several individuals of Heligmosomoides bakeri, a model parasite of house mice, and Heligmosomoides polygyrus, a closely related parasite of wood mice. Although H. bakeri is commonly referred to as H. polygyrus in the literature, their genomes show levels of divergence that are consistent with at least a million years of independent evolution. The genomes of both species contain hyper-divergent haplotypes that are enriched for proteins that interact with the host immune response. Many of these haplotypes originated prior to the divergence between H. bakeri and H. polygyrus, suggesting that they have been maintained by long-term balancing selection. Together, our results suggest that the selection pressures exerted by the host immune response have played a key role in shaping patterns of genetic diversity in the genomes of parasitic nematodes.

Convergent evolution of a parasite-encoded complement control protein-scaffold to mimic binding of mammalian TGF-ß to its receptors, TßRI and TßRII.

The mouse intestinal helminth Heligmosomoides polygyrus modulates host immune responses by secreting a transforming growth factor (TGF)-ß mimic (TGM), to expand the population of Foxp3+ Tregs. TGM comprises five complement control protein (CCP)-like domains, designated D1-D5. Though lacking homology to TGF-ß, TGM binds directly to the TGF-ß receptors TßRI and TßRII and stimulates the differentiation of naïve T-cells into Tregs. However, the molecular determinants of binding are unclear. Here, we used surface plasmon resonance, isothermal calorimetry, NMR spectroscopy, and mutagenesis to investigate how TGM binds the TGF-ß receptors. We demonstrate that binding is modular, with D1-D2 binding to TßRI and D3 binding to TßRII. D1-D2 and D3 were further shown to compete with TGF-ß(TßRII)2 and TGF-ß for binding to TßRI and TßRII, respectively. The solution structure of TGM-D3 revealed that TGM adopts a CCP-like fold but is also modified to allow the C-terminal strand to diverge, leading to an expansion of the domain and opening potential interaction surfaces. TGM-D3 also incorporates a long structurally ordered hypervariable loop, adding further potential interaction sites. Through NMR shift perturbations and binding studies of TGM-D3 and TßRII variants, TGM-D3 was shown to occupy the same site of TßRII as bound by TGF-ß using both a novel interaction surface and the hypervariable loop. These results, together with the identification of other secreted CCP-like proteins with immunomodulatory activity in H. polygyrus, suggest that TGM is part of a larger family of evolutionarily plastic parasite effector molecules that mediate novel interactions with their host.

Role of l-arginine and CD11b+Gr-1+ cells in immunosuppression induced by Heligmosomoides polygyrus bakeri.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous population of monocyte and granulocyte progenitors that are highly suppressive against T cells. In BALB/c mice infected with a nematode Heligmosomoides polygyrus bakeri, we studied the dynamics of MDSCs, identified as CD11b+Gr-1+, induction in different tissues along with the development of parasite infection. We observed that MDSC-like cells are induced both by larvae and adult stages of H polygyrus bakeri. Gr-1+ cells of suppressive phenotype are recruited in the bone marrow, peripheral blood and peritoneal cavity during histotropic phase of infection and are present at that time in the intestine wall, where worms reside. Later, during intestinal phase, suppressive Gr-1+ cells increased in mesenteric lymph nodes and the spleen. l-arginine metabolism was important for the protective immunity, and parasite-induced Gr-1+ cells showed elevated arginase-1 and iNOS expression. Inhibition of arginase-1 and l-arginine administration caused reduced level of infection that coincided with weaker suppressive phenotype of Gr-1+ cells. We identified that l-arginine pathway activation and induction of MDSC-like cells characterize immunosuppressive state during H polygyrus bakeri infection in mice. Our findings confirm the role of MDSCs in parasitic infections and point l-arginine pathway as a potential target for immunomodulation during nematode infections.

Parasitic Nematodes Exert Antimicrobial Activity and Benefit From Microbiota-Driven Support for Host Immune Regulation.

Intestinal parasitic nematodes live in intimate contact with the host microbiota. Changes in the microbiome composition during nematode infection affect immune control of the parasites and shifts in the abundance of bacterial groups have been linked to the immunoregulatory potential of nematodes. Here we asked if the small intestinal parasite Heligmosomoides polygyrus produces factors with antimicrobial activity, senses its microbial environment and if the anti-nematode immune and regulatory responses are altered in mice devoid of gut microbes. We found that H. polygyrus excretory/secretory products exhibited antimicrobial activity against gram+/- bacteria. Parasites from germ-free mice displayed alterations in gene expression, comprising factors with putative antimicrobial functions such as chitinase and lysozyme. Infected germ-free mice developed increased small intestinal Th2 responses coinciding with a reduction in local Foxp3+RORγt+ regulatory T cells and decreased parasite fecundity. Our data suggest that nematodes sense their microbial surrounding and have evolved factors that limit the outgrowth of certain microbes. Moreover, the parasites benefit from microbiota-driven immune regulatory circuits, as an increased ratio of intestinal Th2 effector to regulatory T cells coincides with reduced parasite fitness in germ-free mice.

A structurally distinct TGF-ß mimic from an intestinal helminth parasite potently induces regulatory T cells.

Helminth parasites defy immune exclusion through sophisticated evasion mechanisms, including activation of host immunosuppressive regulatory T (Treg) cells. The mouse parasite Heligmosomoides polygyrus can expand the host Treg population by secreting products that activate TGF-ß signalling, but the identity of the active molecule is unknown. Here we identify an H. polygyrus TGF-ß mimic (Hp-TGM) that replicates the biological and functional properties of TGF-ß, including binding to mammalian TGF-ß receptors and inducing mouse and human Foxp3+ Treg cells. Hp-TGM has no homology with mammalian TGF-ß or other members of the TGF-ß family, but is a member of the complement control protein superfamily. Thus, our data indicate that through convergent evolution, the parasite has acquired a protein with cytokine-like function that is able to exploit an endogenous pathway of immunoregulation in the host.

HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33.

Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy.

TPL-2 restricts Ccl24-dependent immunity to Heligmosomoides polygyrus.

TPL-2 (COT, MAP3K8) kinase activates the MEK1/2-ERK1/2 MAPK signaling pathway in innate immune responses following TLR, TNFR1 and IL-1R stimulation. TPL-2 contributes to type-1/Th17-mediated autoimmunity and control of intracellular pathogens. We recently demonstrated TPL-2 reduces severe airway allergy to house dust mite by negatively regulating type-2 responses. In the present study, we found that TPL-2 deficiency resulted in resistance to Heligmosomoides polygyrus infection, with accelerated worm expulsion, reduced fecal egg burden and reduced worm fitness. Using co-housing experiments, we found resistance to infection in TPL-2 deficient mice (Map3k8-/-) was independent of microbiota alterations in H. polygyrus infected WT and Map3k8-/-mice. Additionally, our data demonstrated immunity to H. polygyrus infection in TPL-2 deficient mice was not due to dysregulated type-2 immune responses. Genome-wide analysis of intestinal tissue from infected TPL-2-deficient mice identified elevated expression of genes involved in chemotaxis and homing of leukocytes and cells, including Ccl24 and alternatively activated genes. Indeed, Map3k8-/-mice had a significant influx of eosinophils, neutrophils, monocytes and Il4GFP+ T cells. Conditional knockout experiments demonstrated that specific deletion of TPL-2 in CD11c+ cells, but not Villin+ epithelial cells, LysM+ myeloid cells or CD4+ T cells, led to accelerated resistance to H. polygyrus. In line with a central role of CD11c+ cells, CD11c+ CD11b+ cells isolated from TPL-2-deficient mice had elevated Ccl24. Finally, Ccl24 neutralization in TPL-2 deficient mice significantly decreased the expression of Arg1, Retnla, Chil3 and Ear11 correlating with a loss of resistance to H. polygyrus. These observations suggest that TPL-2-regulated Ccl24 in CD11c+CD11b+ cells prevents accelerated type-2 mediated immunity to H. polygyrus. Collectively, this study identifies a previously unappreciated role for TPL-2 controlling immune responses to H. polygyrus infection by restricting Ccl24 production.

Plastic and micro-evolutionary responses of a nematode to the host immune environment.

Parasitic organisms have to cope with the defences deployed by their hosts and this can be achieved adopting immune evasion strategies or optimal life history traits according to the prevailing pattern of immune-mediated mortality. Parasites often encounter variable immune environments both within and between hosts, promoting the evolution of plastic strategies instead of fixed responses. Here, we explored the plasticity and micro-evolutionary responses of immunomodulatory mechanisms and life history traits to the immune environment provided by the host, using the parasitic nematode Heligmosomoides polygyrus. To test if the parasite responds plastically to the immune environment, we stimulated the systemic inflammatory response of mice and we assessed i) the expression of two genes with candidate immunomodulatory functions (Hp-Tgh2 and Hp-CPI); ii) changes in the number of eggs shed in the faeces. To test if the immune environment induces a micro-evolutionary response in the parasite, we maintained the nematode in mice whose inflammatory response was up- or down-regulated during four generations. We found that H. polygyrus plastically responded to a sudden rise of pro-inflammatory cytokines, up-regulating the expression of two candidate genes involved in the process of immune modulation, and enhancing egg output. At the micro-evolutionary level, parasites maintained in hosts experiencing different levels of inflammation did not have differential expression of Hp-Tgh2 and Hp-CPI genes when infecting unmanipulated, control, mice. However, parasites maintained in mice with an up-regulated inflammation shed more eggs compared to the control line. Overall, our study shows that H. polygyrus can plastically adjust the expression of immunomodulatory genes and life history traits, and responds to selection exerted by the host immune system.

Microevolutionary response of a gut nematode to intestinal inflammation.

Parasitic helminths interfere with the immune response of their hosts to establish long-lasting, chronic infections. While favorable to the parasite, the capacity to dampen the immune response can also provide a benefit to the host in terms of reduced risk of immune disorders and immunopathology. The immunomodulatory role of nematodes has been exploited in clinical trials to treat a number of inflammatory and immune diseases. However, how parasites adapt to an inflammatory environment remains a poorly explored question. Here, we conducted a serial passage experiment where the gut nematode Heligmosomoides polygyrus was maintained for nine generations in mice with a drug-induced intestinal inflammation or in control hosts. The life history traits of parasites from the selected lines were assessed in hosts that were either exposed to the inflammatory environment or kept as controls. In addition to the nematode life history traits, we assessed the severity of the intestinal inflammation. We found that H. polygyrus adapted to the inflammatory environment through both plastic and microevolutionary responses. In particular, per capita fecundity was globally enhanced in worms that experienced intestinal inflammation and that were selected in the inflammatory environment. Interestingly, we also found that worms selected in the inflammatory environment were better able, after nine generations of selection, to alleviate the inflammatory symptoms. This latter result further highlights the potential therapeutic role of gut nematodes in the treatment of inflammatory diseases.

Novel O-linked methylated glycan antigens decorate secreted immunodominant glycoproteins from the intestinal nematode Heligmosomoides polygyrus.

Glycan molecules from helminth parasites have been associated with diverse biological functions ranging from interactions with neighbouring host cell populations to down-modulation of specific host immunity. Glycoproteins secreted by the intestinal nematode Heligmosomoides polygyrus are of particular interest as the excretory-secretory products (termed HES) of this parasite contain both heat-labile and heat-stable components with immunomodulatory effects. We used MALDI-TOF-MS and LC-MS/MS to analyse the repertoire of N- and O-linked glycans released from Heligmosomoides polygyrus excretory-secretory products by PNGase A and F, ß-elimination and hydrazinolysis revealing a broad range of structures including novel methylhexose- and methylfucose-containing glycans. Monoclonal antibodies to two immunodominant glycans of H. polygyrus, previously designated Glycans A and B, were found to react by glycan array analysis to a methyl-hexose-rich fraction and to a sulphated LacDiNAc (LDN; GalNAcß1-4GlcNAc) structure, respectively. We also analysed the glycan repertoire of a major glycoprotein in Heligmosomoides polygyrus excretory-secretory products, VAL-2, which contains many glycan structures present in Heligmosomoides polygyrus excretory-secretory products including Glycan A. However, it was found that this set of glycans is not responsible for the heat-stable immunomodulatory properties of Heligmosomoides polygyrus excretory-secretory products, as revealed by the inability of VAL-2 to inhibit allergic lung inflammation. Taken together, these studies reveal that H. polygyrus secretes a diverse range of antigenic glycoconjugates, and provides a framework to explore the biological and immunomodulatory roles they may play within the mammalian host.

Contrasting patterns of structural host specificity of two species of Heligmosomoides nematodes in sympatric rodents.

Host specificity is a fundamental property of parasites. Whereas most studies focus on measures of specificity on host range, only few studies have considered quantitative aspects such as infection intensity or prevalence. The relative importance of these quantitative aspects is still unclear, mainly because of methodological constraints, yet central to a precise assessment of host specificity. Here, we assessed simultaneously two quantitative measures of host specificity of Heligmosomoides glareoli and Heligmosomoides polygyrus polygyrus infections in sympatric rodent hosts. We used standard morphological techniques as well as real-time quantitative PCR and sequencing of the rDNA ITS2 fragment to analyse parasite infection via faecal sample remains. Although both parasite species are thought to be strictly species-specific, we found morphologically and molecularly validated co- and cross-infections. We also detected contrasting patterns within and between host species with regard to specificity for prevalence and intensity of infection. H. glareoli intensities were twofold higher in bank voles than in yellow-necked mice, but prevalence did not differ significantly between species (33 vs. 18%). We found the opposite pattern in H. polygyrus infections with similar intensity levels between host species but significantly higher prevalence in mouse hosts (56 vs. 10%). Detection rates were higher with molecular tools than morphological methods. Our results emphasize the necessity to consider quantitative aspects of specificity for a full view of a parasites' capacity to replicate and transmit in hosts and present a worked example of how modern molecular tools help to advance our understanding of selective forces in host-parasite ecology and evolution.

Proteomic analysis of excretory-secretory products of Heligmosomoides polygyrus assessed with next-generation sequencing transcriptomic information.

The murine parasite Heligmosomoides polygyrus is a convenient experimental model to study immune responses and pathology associated with gastrointestinal nematode infections. The excretory-secretory products (ESP) produced by this parasite have potent immunomodulatory activity, but the protein(s) responsible has not been defined. Identification of the protein composition of ESP derived from H. polygyrus and other relevant nematode species has been hampered by the lack of genomic sequence information required for proteomic analysis based on database searches. To overcome this, a transcriptome next generation sequencing (RNA-seq) de novo assembly containing 33,641 transcripts was generated, annotated, and used to interrogate mass spectrometry (MS) data derived from 1D-SDS PAGE and LC-MS/MS analysis of ESP. Using the database generated from the 6 open reading frames deduced from the RNA-seq assembly and conventional identification programs, 209 proteins were identified in ESP including homologues of vitellogenins, retinol- and fatty acid-binding proteins, globins, and the allergen V5/Tpx-1-related family of proteins. Several potential immunomodulators, such as macrophage migration inhibitory factor, cysteine protease inhibitors, galectins, C-type lectins, peroxiredoxin, and glutathione S-transferase, were also identified. Comparative analysis of protein annotations based on the RNA-seq assembly and proteomics revealed processes and proteins that may contribute to the functional specialization of ESP, including proteins involved in signalling pathways and in nutrient transport and/or uptake. Together, these findings provide important information that will help to illuminate molecular, biochemical, and in particular immunomodulatory aspects of host-H. polygyrus biology. In addition, the methods and analyses presented here are applicable to study biochemical and molecular aspects of the host-parasite relationship in species for which sequence information is not available.

Heligmosomoides bakeri: a new name for an old worm?

A popular model system for exploring the host-parasite relationship of gastrointestinal nematodes is commonly known as Heligmosomoides polygyrus bakeri. Recently, this parasite was raised to full species level as H. bakeri, to distinguish it from a close relative, H. polygyrus sensu stricto, the dominant intestinal nematode of wood mice in Western Europe, which is unable to infect laboratory mice (Mus sp.) without the aid of powerful immunosuppressants. Herein, the argument is presented that it is necessary to rename this parasite, and that H. bakeri is the correct name for the species used widely throughout the world as a laboratory research model. Supporting this claim, key evidence is presented demonstrating that H. bakeri and H. polygyrus are two quite distinct species.

C-type lectins from the nematode parasites Heligmosomoides polygyrus and Nippostrongylus brasiliensis.

The C-type lectin superfamily is highly represented in all metazoan phyla so far studied. Many members of this superfamily are important in innate immune defences against infection, while others serve key developmental and structural roles. Within the superfamily, many proteins contain multiple canonical carbohydrate-recognition domains (CRDs), together with additional non-lectin domains. In this report, we have studied two gastrointestinal nematode parasites which are widely used in experimental rodent systems, Heligmosomoides polygyrus and Nippostrongylus brasiliensis. From cDNA libraries, we have isolated 3 new C-type lectins from these species; all are single-CRD proteins with short additional N-terminal domains. The predicted Hp-CTL-1 protein contains 156 aa, Nb-CTL-1 191 aa and Nb-CTL-2 183 aa; all encode predicted signal peptides, as well as key conserved sequence motifs characteristic of the CTL superfamily. These lectins are most similar to C. elegans CLEC-48, 49 and 50, as well as to the lectin domains of mammalian immune system proteins CD23 and CD206. RT-PCR showed that these H. polygyrus and N. brasiliensis genes are primarily expressed in the gut-dwelling adult stages, although Nb-CTL-2 transcripts are also prominent in the free-living infective larval (L3) stage. Polyclonal antibodies raised to Hp-CTL-1 and Nb-CTL-1 reacted to both proteins by ELISA, and in Western blot analysis recognised a 15-kDa band in secreted proteins of adult N. brasiliensis (NES) and a 19-kDa band in H. polygyrus ES (HES). Anti-CTL-1 antibody also bound strongly to the cuticle of adult H. polygyrus. Hence, live parasites release C-type lectins homologous to some key receptors of the mammalian host immune system, raising the possibility that these products interfere in some manner with immunological recognition or effector function.

Attempts to establish RNA interference in the parasitic nematode Heligmosomoides polygyrus.

To analyze the potential of RNA interference (RNAi) in the intestinal nematode Heligmosomoides polygyrus, we delivered double-stranded RNA (dsRNA) of tropomyosin to various life stages of the parasite. Three different methods were examined for their potential use. First, feeding of recombinant bacteria that expressed dsRNA did neither result in phenotypical changes of H. polygyrus nor in a significant reduction of tropomyosin mRNA levels. In contrast, feeding of such bacteria to Caenorhabditis elegans elicited the expected phenotypes. Quantification of bacteria ingested by C. elegans and H. polygyrus larvae (L1) revealed that the parasitic worms took up only a fraction of the bacteria ingested by C. elegans. Second, electroporation of L1 failed to transport siRNA through the cuticle and was lethal to the larvae. However, the cuticle of adult worms was penetrated by dye-labeled RNA, but no systemic spreading was observed. Third, soaking of adult H. polygyrus in tropomyosin dsRNA led to a higher proportion of worms showing symptoms of ageing, such as a disintegrated gut and ovaries, but did not induce reduction of tropomyosin mRNA levels. Database analysis revealed that orthologous proteins involved in dsRNA-uptake and -systemic spread in C. elegans are missing in the parasitic nematodes Brugia malayi and Haemonchus contortus, whereas proteins responsible for dsRNA-processing, -amplification and mRNA-regulation are present. Thus, our data indicate that the study of gene function by RNAi in H. polygyrus is limited, possibly due to deficiencies of genes involved in RNA-uptake and spread.

Molecular evidence that Heligmosomoides polygyrus from laboratory mice and wood mice are separate species.

The gastro-intestinal (GI) nematode Heligmosomoides polygyrus is an important experimental model in laboratory mice and a well-studied parasite of wood mice in the field. Despite an extensive literature, the taxonomy of this parasite in different hosts is confused, and it is unclear whether laboratory and field systems represent the same or different Operational Taxonomic Units (OTUs). Molecular analyses reveal high sequence divergence between H. p. bakeri (laboratory) and H. p. polygyrus (field); 3% difference in the ribosomal DNA Internal Transcribed Spacers (ITS) and 8.6% variation in the more rapidly evolving mitochondrial cytochrome c oxidase I (COI) gene. The COI sequence of U.K. H. p. polygyrus is more similar to H. glareoli from voles than to H. p. bakeri, while a single isolate of H. p. polygyrus from Guernsey confirms the extent of genetic variation between H. p. polygyrus populations. Analysis of molecular variance demonstrated that mtCOI sequence variation is associated primarily with groups with distinct ITS2 sequences, and with host identity, but is not partitioned significantly with a single combined taxon H. polygyrus incorporating European and North American isolates. We conclude therefore that the laboratory OTU should be raised to the level of a distinct species, as H. bakeri from the laboratory mouse Mus musculus, and we reject the hypothesis that H. bakeri has diverged from H. polygyrus in the recent past following introduction into America. However, we are unable to reject the hypothesis that H. polygyrus and H. bakeri are sister taxa, and it may be that H. polygyrus is polyphyletic or paraphyletic.

Mapping of chromosomal regions influencing immunological responses to gastrointestinal nematode infections in mice.

This paper reports the results of a genome-wide search for quantitative trait loci (QTL) influencing immunological responses to infection with the gastro-intestinal nematode parasite Heligmosomoides polygyrus in an F2 population created by crossing the resistant SWR and the susceptible CBA inbred mouse strains. Following infections, intestinal granuloma score at post mortem, mucosal mast cell protease 1, and IgE and IgG1 titres were recorded. The susceptible CBA mice had significantly higher IgG1, but significantly lower IgE, mucosal mast cell protease 1 and granuloma scores than SWR mice. Significant QTL were mapped to chromosomes 4, 11, 13 and 17 for granuloma score; chromosomes 12 and 17 for IgE; chromosome 10, 17 and 18 for IgG1 and chromosomes 1, 9, 10, 11, 17 and 18 for mucosal mast cell protease 1. Chromosomes 10, 11, 17 and 18 had QTL affecting more than one trait, and these are most likely to represent single QTL with multiple effects rather than multiple QTL. Some of these QTL map to regions known to harbour genes responsible for the induction of immunological responses to intestinal worms.

Activity of the cyclic depsipeptide emodepside (BAY 44-4400) against larval and adult stages of nematodes in rodents and the influence on worm survival.

The present investigations deal with the activity of the cyclic depsipeptide emodepside (BAY 44-4400) against larval and adult stages of three rodent nematodes. While emodepside acts strongly against the adult stages of the rat nematodes Nippostrongylus brasiliensis and Strongyloides ratti, as well as against the mouse nematode Heligmosomoides polygyrus, its actions against the larval stages of these nematodes vary according to the species. Thus, emodepside is highly effective against the lung and intestine larval stages of N. brasiliensis and S. ratti. By contrast. the larval stages of H. polygyrus in the intestine are only partly affected by higher emodepside dosages.

Genomic variability within laboratory and wild isolates of the trichostrongyle mouse nematode Heligmosomoides polygyrus.

PCR-RFLP techniques have been used to characterize wild and laboratory isolates of the trichostrongyle nematode Heligmosomoides polygyrus from the wood mouse Apodemus sylvaticus and the laboratory mouse Mus musculus respectively. Both isolates can be distinguished by eight endonuclease digestions of the ITS region of the rDNA repeat namely, Alu I, Dde I, Hpa II, Hae III, Hinf I, Hha I, Pvu II and Sal I. In two of the digests, Hinf I and Rsa I, a minor polymorphism was observed in the wild isolate of H. polygyrus which has been cultured in laboratory-bred A. sylvaticus for several generations when compared with H. p. polygyrus from wild A. sylvaticus. A minor polymorphism was also identified in further wild isolates of H. polygyrus collected from A. sylvaticus in a field site in Egham, Surrey. However no evidence of polymorphism was observed in the laboratory isolate of H. polygyrus from the CD1 strain of M. musculus and the laboratory-bred A. sylvaticus. Reasons for this are discussed and further studies on the population genetics of H. polygyrus are suggested.