Host-parasite incongruences in rodent Eimeria suggest significant role of adaptation rather than cophylogeny in maintenance of host specificity.

The degree of host specificity, its phylogenetic conservativeness and origin are virtually unknown in Eimeria. This situation is largely due to the inadequate sample of eimerian molecular data available for reliable phylogenetic analyses. In this study, we extend the data set by adding 71 new sequences of coccidia infecting 16 small-mammal genera, mostly rodents. According to the respective feasibility of PCR gene amplification, the new samples are represented by one or more of the following genes: nuclear 18S rRNA, plastid ORF 470, and mitochondrial COI. Phylogenetic analyses of these sequences confirm the previous hypothesis that Eimeria, in its current morphology-based delimitation, is not a monophyletic group. Several samples of coccidia corresponding morphologically to other genera are scattered among the Eimeria lineages. More importantly, the distribution of eimerians from different hosts indicates that the clustering of eimerian species is influenced by their host specificity, but does not arise from a cophylogenetic/cospeciation process; while several clusters are specific to a particular host group, inner topologies within these clusters do not reflect host phylogeny. This observation suggests that the host specificity of Eimeria is caused by adaptive rather than cophylogenetic processes

Hemolivia species infecting Central American wood turtles (Rhinoclemmys pulcherrima manni) and problems with differential diagnosis within the genus Hemolivia

Blood parasites of the genus Hemolivia Petit, Landau, Baccam and Lainson, 1990 (Adeleorina: Karyolysidae) are hemogregarines of ectothermic vertebrates, such as lizards, chelonians, and toads. Only five species of Hemolivia from vertebrate hosts and one from their tick vector have been described so far. In the present study, Central American wood turtles (Rhinoclemmys pulcherrima manni) originating from Southern Nicaragua were screened for the presence of hemogregarines. Ten out of 30 specimens (33.3%) were positive for Hemolivia using both approaches – microscopy and PCR-based analyses. Phylogenetic analyses based on the 18S rRNA gene revealed the presence of two haplotypes, both placed as sister taxa in the Hemolivia clade. Their phylogenetic position was supported by high bootstrap values and high posterior probabilities, suggesting that there are at least two new distinct haplotypes corresponding to two distinct species. However, the specimens of each haplotype were microscopically indistinguishable from each other based on the gamont morphology, therefore, only a single species could be described and named, as Hemolivia pulcherrima n. sp. We consider that the uniform morphology of the most common blood stages of species of the genus Hemolivia complicates their differential diagnosis. Sequence divergence and different host spectra, therefore, remain the only differentiating tools

Morphological features and origin of the newly obtained samples within this study.

<p>CZ – Czech Republic, SK – Slovakia, UK – England; OW – oocyst wall, MP – micropyle, OR – oocyst residuum.</p

A <i>Skeleton</i> tree.

<p><i>Skeleton</i> tree (ML and BI) of the taxa for which all 3 genes (<i>18S rDNA</i>, <i>ORF 470</i> and <i>COI</i>) are available.</p

<i>Concatenated</i> ML tree.

<p> Letters A–D indicate clusters delimited according to the <i>Skeleton</i> tree (taxa present in the <i>Skeleton</i> tree are labeled with asterisks). Clades A and B are supported by both BI and ML analyses of the <i>Concatenated</i> and <i>Skeleton</i> matrices. The red node indicates a cluster with weak host specificity. Numbers 1–4 indicate lineages that are also supported by BI analyses of the following matrices: 1, <i>Concatenated</i>; 2, <i>ORF 470</i>; 3, <i>COI</i>; 4, <i>18S rDNA</i>. The newly added samples are printed in bold; coccidia from rodents are printed in blue. To decrease the size of the tree for the printed presentation, we removed several of the most basal outgroups.</p

Taxa and sequences included in the phylogenetic analyses.

*<p>: sequences included in the <i>Skeleton</i> matrix.</p><p>•: taxa used as outgroups for the phylogenetic analyses.</p><p>– : the sequence is not available.</p><p>Taxa for which new sequences were obtained in this study and Accession numbers of these sequences are printed in bold.</p

Detection and quantification of house mouse Eimeria at the species level

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.Peer Reviewe

Generalist Eimeria species in rodents

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.Peer Reviewe