Detection and quantification of coccidia in studies of wildlife can be challenging. Therefore, prevalence of coccidia is often not assessed at the parasite species level in non-livestock animals. Parasite species – specific prevalences are especially important when studying evolutionary questions in wild populations. We tested whether increased host population density increases prevalence of individual Eimeria species at the farm level, as predicted by epidemiological theory. We studied free-living commensal populations of the house mouse ( Mus musculus ) in Germany, and established a strategy to detect and quantify Eimeria infections. We show that a novel diagnostic primer targeting the apicoplast genome (Ap5) and coprological assessment after flotation provide complementary detection results increasing sensitivity. Genotyping PCRs confirm detection in a subset of samples and cross-validation of different PCR markers does not indicate bias towards a particular parasite species in genotyping. We were able to detect double infections and to determine the preferred niche of each parasite species along the distal-proximal axis of the intestine. Parasite genotyping from tissue samples provides additional indication for the absence of species bias in genotyping amplifications. Three Eimeria species were found infecting house mice at different prevalences: Eimeria ferrisi (16.7%; 95% CI 13.2–20.7), E. falciformis (4.2%; 95% CI 2.6–6.8) and E. vermiformis (1.9%; 95% CI 0.9–3.8). We also find that mice in dense populations are more likely to be infected with E. falciformis and E. ferrisi . We provide methods for the assessment of prevalences of coccidia at the species level in rodent systems. We show and discuss how such data can help to test hypotheses in ecology, evolution and epidemiology on a species level.

Genetic diversity in animal immune systems is usually beneficial. In hybrid recombinants, this is less clear, as the immune system could also be impacted by genetic conflicts. In the European house mouse hybrid zone, the long‐standing impression that hybrid mice are more highly parasitized and less fit than parentals persists despite the findings of recent studies. Working across a novel transect, we assessed infections by intracellular protozoans, Eimeria spp., and infections by extracellular macroparasites, pinworms. For Eimeria, we found lower intensities in hybrid hosts than in parental mice but no evidence of lowered probability of infection or increased mortality in the centre of the hybrid zone. This means ecological factors are very unlikely to be responsible for the reduced load of infected hybrids. Focusing on parasite intensity (load in infected hosts), we also corroborated reduced pinworm loads reported for hybrid mice in previous studies. We conclude that intensity of diverse parasites, including the previously unstudied Eimeria, is reduced in hybrid mice compared to parental subspecies. We suggest caution in extrapolating this to differences in hybrid host fitness in the absence of, for example, evidence for a link between parasitemia and health.

Host defense mechanisms evolve to alleviate the detrimental effect of parasites. They can be categorized into two components: resistance and tolerance (Råberg et al., 2009). Resistance is the ability of a host to reduce parasite burden, resulting from defense against parasite infection or proliferation early after infection (Schmid-Hempel, 2013). The negative effect of resistance on parasite fitness can lead to antagonistic coevolution. According to theoretical models, fluctuating host and parasite genotypes arise, and

Detection and quantification of coccidia in studies of wildlife can be challenging. Therefore, prevalence of coccidia is often not assessed at the parasite species level in non-livestock animals.Parasite species -specific prevalences are especially important when studying evolutionary questions in wild populations. We might expect, for example, highly prevalent parasite species to have a lower virulence than lowly prevalent species.We studied free-living commensal populations of the house mouse (Mus musculus) in Germany, and established a strategy to detect and quantify Eimeria infections. We show that a novel diagnostic primer targeting the apicoplast genome (Ap5) and coprological assessment after flotation provide complementary detection results increasing sensitivity. Genotyping PCRs confirm detection in a subset of samples and cross-validation of different PCR markers does not indicate bias towards a particular parasite species in genotyping. We were able to detect double infections and to determine the preferred parasite occurrence along the distal-proximal axis of the intestine.Parasite genotyping from tissue samples provides additional indication for the absence of species bias in genotyping amplifications. Three Eimeria species were found infecting house mice at different prevalences: Eimeria ferrisi (16.1%; 95% CI 12.7 -20.2), E. falciformis (4.2%; 95% CI 2.6 -6.8) and E. vermiformis (1.1%; 95% CI 0.4 -2.7).We provide methods for the assessment of prevalences of coccidia at the species level in rodent systems. We discuss the need for broader species level assessment in Coccidia. Prevalence negatively correlates with virulence for Eimeria species of house mice, as the more prevalent E. ferrisi has been shown to be less virulent than E. falciformis. It is an open question whether house mouse Eimeria are host specialist and whether prevalence in our system correlates with host range.

Intracellular parasites of the genus Eimeria are described as tissue/host‐specific. Phylogenetic classification of rodent Eimeria suggested that some species have a broader host range than previously assumed. We explore whether Eimeria spp. infecting house mice are misclassified by the most widely used molecular markers due to a lack of resolution, or whether, instead, these parasite species are indeed infecting multiple host species. With the commonly used markers (18S/COI), we recovered monophyletic clades of E. falciformis and E. vermiformis from Mus that included E. apionodes identified in other rodent host species (Apodemus spp., Myodes glareolus, and Microtus arvalis). A lack of internal resolution in these clades could suggest the existence of a species complex with a wide host range infecting murid and cricetid rodents. We question, however, the power of COI and 18S markers to provide adequate resolution for assessing host specificity. In addition to the rarely used marker ORF470 from the apicoplast genome, we present multilocus genotyping as an alternative approach. Phylogenetic analysis of 35 nuclear markers differentiated E. falciformis from house mice from isolates from Apodemus hosts. Isolates of E. vermiformis from Mus are still found in clusters interspersed with non‐Mus isolates, even with this high‐resolution data. In conclusion, we show that species‐level resolution should not be assumed for COI and 18S markers in coccidia. Host–parasite cospeciation at shallow phylogenetic nodes, as well as contemporary coccidian host ranges more generally, is still open questions that need to be addressed using novel genetic markers with higher resolution.

Currently, methods for monitoring changes of gut barrier integrity and the associated immune response via non-invasive means are limited. Therefore, we aimed to develop a novel non-invasive technique to investigate immunological host responses representing gut barrier changes in response to infection. We identified the mucous layer on feces from mice to be mainly composed of exfoliated intestinal epithelial cells. Expression of RELM-β, a gene prominently expressed in intestinal nematode infections, was used as an indicator of intestinal cellular barrier changes to infection. RELM-β was detected as early as 6 days post-infection (dpi) in exfoliated epithelial cells. Interestingly, RELM-β expression also mirrored the quality of the immune response, with higher amounts being detectable in a secondary infection and in high dose nematode infection in laboratory mice. This technique was also applicable to captured worm-infected wild house mice. We have therefore developed a novel non-invasive method reflecting gut barrier changes associated with alterations in cellular responses to a gastrointestinal nematode infection.

Ethics statement:Trapping and handling of mice was approved by local authorities ("Landesamt fuer Gesundheit, Umwelt und Verbrauchersschutz Brandenburg") under permit number 35 -101 2014-2347. Data Accessibility Statement:there is no data to be archived. Intensity of infection with intracellular Eimeria spp. and pinworms is reduced in hybrid mice compared to parental subspecies AbstractThe longstanding impression that hybrid mice are more highly parasitized and therefore less fit than parentals persists despite the findings of recent studies. Working across a novel transect of the European House Mouse hybrid zone we assessed intracellular infections by Eimeria, a parasite of high pathogenicity, and infections by pinworms, assumed to be less pathogenic. For Eimeria we found lower intensities in hybrid hosts than in parental mice but no evidence of lowered probability of infection in the centre of the hybrid zone. This means ecological and epidemiological factors are very unlikely to be responsible for the reduced load of infected hybrids. Focussing on parasite intensity (load in infected hosts) we also corroborated reduced pinworm loads reported for hybrid mice in previous studies. In addition we questioned whether differences in body condition during infection would indicate different impacts on hybrid vs. parental hosts' health.We couldn't show such an effect. We conclude that intensity of diverse parasites, including the previously unstudied Eimeria, is reduced in hybrid mice compared to parental subspecies. We suggest caution in extrapolating this to differences in hybrid host fitness in the absence of, for example, evidence for a link between parasitemia and health.

Resistance (the host's capacity to reduce parasite burden) and tolerance (the host's capacity to reduce impact on host health of a given parasite burden) manifest two different lines of immune defenses. In some host-parasite systems these two defenses are balanced against each other, while in others they are uncoupled. In hybrid hosts, resistance has sometimes been interpreted as having an effect on fitness without considering the modulatory effect of tolerance. Here, we used two closely related parasite species of genus Eimeria and measured proxies for resistance and tolerance in four wild-derived strains of inbred mice from two subspecies during controlled infection.We found a negative correlation between resistance and tolerance against E. falciformis, while the two are uncoupled against E. ferrisi. This might be explained by trade-offs, as resistance limits infection load and thereby the scope of possible tolerance, and both resistance and tolerance can be costly in terms of resource allocation. Resistance can be assumed to be limited by immunopathogenicity, tolerance by carrying capacity of the host or energy drained by the parasite.Findings of resistance in natural populations of hybrid mice have to be interpreted carefully in this context. Resistance and tolerance have to be studied in conjunction. 2019) which were also used for serial passaging of all the isolates. Parasite infective forms (oocysts) were recovered by flotation in saturated NaCl solution followed by washing and observation under light microscope (following the protocole described in Clerc, Fenton, Babayan, & Pedersen, 2019) and stored at room temperature in 1mL of 2% potassium dichromate for a maximum of 2 months before infection of the wild-derived mouse strains.Oocysts were allowed to sporulate 10 days before infection in a water bath at 30°C.