Intensity of infection with intracellular Eimeria spp. and pinworms is reduced in hybrid mice compared to parental subspecies.

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.

Concurrent Proteomic Fingerprinting and Molecular Analysis of Cyathostomins.

Rapid, cost-effective, efficient, and reliable helminth species identification is of considerable importance to understand host-parasite interactions, clinical disease, and drug resistance. Cyathostomins (Nematoda: Strongylidae) are considered to be the most important equine parasites, yet research on this group is hampered by the large number of 50 morphologically differentiated species, their occurrence in mixed infections with often more than 10 species and the difficulties associated with conventional identification methods. Here, MALDI-TOF MS, previously successfully applied to identify numerous organisms, is evaluated and compared with conventional and molecular genetic approaches. A simple and robust protocol for protein extraction and subsequent DNA isolation allowing molecular confirmation of proteomic findings is developed, showing that MALDI-TOF MS can discriminate adult stages of the two closely related cyathostomin species Cylicostephanus longibursatus and Cylicostephanus minutus. Intraspecific variability of proteomic profiles within morphospecies demonstrated an identification of morphospecies with an accuracy of close to 100%. In contrast, three genospecies within C. minutus and sex-specific profiles within both morphospecies could not be reliably discriminated using MALDI-TOF MS. In conclusion, MALDI-TOF MS complemented by the molecular protocol is a reliable and efficient approach for cyathostomin species identification.

An epigenetic toolbox for conservation biologists.

Ongoing climatic shifts and increasing anthropogenic pressures demand an efficient delineation of conservation units and accurate predictions of populations' resilience and adaptive potential. Molecular tools involving DNA sequencing are nowadays routinely used for these purposes. Yet, most of the existing tools focusing on sequence-level information have shortcomings in detecting signals of short-term ecological relevance. Epigenetic modifications carry valuable information to better link individuals, populations, and species to their environment. Here, we discuss a series of epigenetic monitoring tools that can be directly applied to various conservation contexts, complementing already existing molecular monitoring frameworks. Focusing on DNA sequence-based methods (e.g. DNA methylation, for which the applications are readily available), we demonstrate how (a) the identification of epi-biomarkers associated with age or infection can facilitate the determination of an individual's health status in wild populations; (b) whole epigenome analyses can identify signatures of selection linked to environmental conditions and facilitate estimating the adaptive potential of populations; and (c) epi-eDNA (epigenetic environmental DNA), an epigenetic-based conservation tool, presents a non-invasive sampling method to monitor biological information beyond the mere presence of individuals. Overall, our framework refines conservation strategies, ensuring a comprehensive understanding of species' adaptive potential and persistence on ecologically relevant timescales.

Aberrant microbiomes are associated with increased antibiotic resistance gene load in hybrid mice.

Antibiotic resistance is a priority public health problem resulting from eco-evolutionary dynamics within microbial communities and their interaction at a mammalian host interface or geographical scale. The links between mammalian host genetics, bacterial gut community, and antimicrobial resistance gene (ARG) content must be better understood in natural populations inhabiting heterogeneous environments. Hybridization, the interbreeding of genetically divergent populations, influences different components of the gut microbial communities. However, its impact on bacterial traits such as antibiotic resistance is unknown. Here, we present that hybridization might shape bacterial communities and ARG occurrence. We used amplicon sequencing to study the gut microbiome and to predict ARG composition in natural populations of house mice (Mus musculus). We compared gastrointestinal bacterial and ARG diversity, composition, and abundance across a gradient of pure and hybrid genotypes in the European House Mouse Hybrid Zone. We observed an increased overall predicted richness of ARG in hybrid mice. We found bacteria-ARG interactions by their co-abundance and detected phenotypes of extreme abundances in hybrid mice at the level of specific bacterial taxa and ARGs, mainly multidrug resistance genes. Our work suggests that mammalian host genetic variation impacts the gut microbiome and chromosomal ARGs. However, it raises further questions on how the mammalian host genetics impact ARGs via microbiome dynamics or environmental covariates.

Shifting focus from resistance to disease tolerance: A review on hybrid house mice.

Parasites have been proposed to modulate the fitness of hybridizing hosts in part based on observations in the European house mouse hybrid zone (HMHZ), a tension zone in which hybrids show reduced fitness. We here review evidence (1) for parasite load differences in hybrid versus parental mice and (2) for health and fitness effects of parasites promoting or preventing introgression and hybridization. The question of relative resistance or susceptibility of hybrids to parasites in the HMHZ has long been controversial. Recent field studies found hybrids to be more resistant than mice from parental subspecies against infections with pinworms and protozoans (Eimeria spp.). We argue that the field studies underlying the contradictory impression of hybrid susceptibility have limitations in sample size, statistical analysis and scope, focusing only on macroparasites. We suggest that weighted evidence from field studies indicate hybrid resistance. Health is a fitness component through which resistance can modulate overall fitness. Resistance, however, should not be extrapolated directly to a fitness effect, as the relationship between resistance and health can be modulated by tolerance. In our own recent work, we found that the relationship between health and resistance (tolerance) differs between infections with the related species E. falciformis and E. ferrisi. Health and tolerance need to be assessed directly and the choice of parasite has made this difficult in previous experimental studies of house mice. We discuss how experimental Eimeria spp. infections in hybrid house mice can address resistance, health and tolerance in conjunction.

DNA-based quantification and counting of transmission stages provides different but complementary parasite load estimates: an example from rodent coccidia (Eimeria).

BACKGROUND: Counting parasite transmission stages in faeces is the classical measurement to quantify "parasite load". DNA-based quantifications of parasite intensities from faecal samples are relatively novel and often validated against such counts. When microscopic and molecular quantifications do not correlate, it is unclear whether oocyst counts or DNA-based intensity better reflects biologically meaningful concepts. Here, we investigate this issue using the example of Eimeria ferrisi (Coccidia), an intracellular parasite of house mice (Mus musculus). METHODS: We performed an infection experiment of house mice with E. ferrisi, in which the intensity of infection correlates with increased health impact on the host, measured as temporary weight loss during infection. We recorded the number of parasite transmissive stages (oocysts) per gram of faeces (OPG) and, as a DNA-based measurement, the number of Eimeria genome copies per gram of faeces for 10 days post-infection (dpi). We assessed weight loss relative to the day of experimental infection as a proxy of host health and evaluated whether DNA or oocyst counts are better predictors of host health. RESULTS: Absolute quantification of Eimeria DNA and oocyst counts showed similar but slightly diverging temporal patterns during 10 dpi. We detected Eimeria DNA earlier than the first appearance of oocysts in faeces. Additionally, Eimeria OPGs within each dpi did not explain parasite DNA intensity. Early dpi were characterized by high DNA intensity with low oocyst counts, while late infections showed the opposite pattern. The intensity of Eimeria DNA was consistently a stronger predictor of either maximal weight loss (1 value per animal during the infection course) or weight loss on each day during the experiment when controlling for between-dpi and between-individual variance. CONCLUSIONS: Eimeria ferrisi oocyst counts correlate weakly with parasite intensity assessed through DNA quantification. DNA is likely partially derived from life-cycle stages other than transmissive oocysts. DNA-based intensities predict health outcomes of infection for the host more robustly than counts of transmissive stages. We conclude that DNA-based quantifications should not necessarily require validation against counts of transmissive stages. Instead, DNA-based load estimates should be evaluated as complementary sources of information with potential specific biological relevance for each host-parasite system.

Coupling between tolerance and resistance for two related Eimeria parasite species.

Resistance (host capacity to reduce parasite burden) and tolerance (host capacity to reduce impact on its health for a given parasite burden) manifest two different lines of defense. Tolerance can be independent from resistance, traded off against it, or the two can be positively correlated because of redundancy in underlying (immune) processes. We here tested whether this coupling between tolerance and resistance could differ upon infection with closely related parasite species. We tested this in experimental infections with two parasite species of the genus Eimeria. We measured proxies for resistance (the (inverse of) number of parasite transmission stages (oocysts) per gram of feces at the day of maximal shedding) and tolerance (the slope of maximum relative weight loss compared to day of infection on number of oocysts per gram of feces at the day of maximal shedding for each host strain) in four inbred mouse strains and four groups of F1 hybrids belonging to two mouse subspecies, Mus musculus domesticus and Mus musculus musculus. We found a negative correlation between resistance and tolerance against Eimeria falciformis, while the two are uncoupled against Eimeria ferrisi. We conclude that resistance and tolerance against the first parasite species might be traded off, but evolve more independently in different mouse genotypes against the latter. We argue that evolution of the host immune defenses can be studied largely irrespective of parasite isolates if resistance-tolerance coupling is absent or weak (E. ferrisi) but host-parasite coevolution is more likely observable and best studied in a system with negatively correlated tolerance and resistance (E. falciformis).

Generalist Eimeria species in rodents: Multilocus analyses indicate inadequate resolution of established markers.

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.

Detection and quantification of house mouse Eimeria at the species level - Challenges and solutions for the assessment of coccidia in wildlife.

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.