High genetic structuring of Tula hantavirus.

Tula virus (TULV) is a vole-associated hantavirus with low or no pathogenicity to humans. In the present study, 686 common voles (Microtus arvalis), 249 field voles (Microtus agrestis) and 30 water voles (Arvicola spec.) were collected at 79 sites in Germany, Luxembourg and France and screened by RT-PCR and TULV-IgG ELISA. TULV-specific RNA and/or antibodies were detected at 43 of the sites, demonstrating a geographically widespread distribution of the virus in the studied area. The TULV prevalence in common voles (16.7 %) was higher than that in field voles (9.2 %) and water voles (10.0 %). Time series data at ten trapping sites showed evidence of a lasting presence of TULV RNA within common vole populations for up to 34 months, although usually at low prevalence. Phylogenetic analysis demonstrated a strong genetic structuring of TULV sequences according to geography and independent of the rodent species, confirming the common vole as the preferential host, with spillover infections to co-occurring field and water voles. TULV phylogenetic clades showed a general association with evolutionary lineages in the common vole as assessed by mitochondrial DNA sequences on a large geographical scale, but with local-scale discrepancies in the contact areas.

White-Toothed Shrews (Genus Crocidura): Potential Reservoirs for Zoonotic Leptospira spp. and Arthropod-Borne Pathogens?

Three species of white-toothed shrews of the order Eulipotyphla are present in central Europe: the bicolored (Crocidura leucodon), greater (Crocidura russula) and lesser (Crocidura suaveolens) white-toothed shrews. Their precise distribution in Germany is ill-defined and little is known about them as reservoirs for zoonotic pathogens (Leptospira spp., Coxiella burnetii, Brucella spp., Anaplasma phagocytophilum, Babesia spp., Neoehrlichia mikurensis and Bartonella spp.). We investigated 372 Crocidura spp. from Germany (n = 341), Austria (n = 18), Luxembourg (n = 2) and Slovakia (n = 11). West European hedgehogs (Erinaceus europaeus) were added to compare the presence of pathogens in co-occurring insectivores. Crocidura russula were distributed mainly in western and C. suaveolens mainly in north-eastern Germany. Crocidura leucodon occurred in overlapping ranges with the other shrews. Leptospira spp. DNA was detected in 28/227 C. russula and 2/78 C. leucodon samples. Further characterization revealed that Leptospira kirschneri had a sequence type (ST) 100. Neoehrlichia mikurensis DNA was detected in spleen tissue from 2/213 C. russula samples. Hedgehogs carried DNA from L. kirschneri (ST 100), L. interrogans (ST 24), A. phagocytophilum and two Bartonella species. This study improves the knowledge of the current distribution of Crocidura shrews and identifies C. russula as carrier of Leptospira kirschneri. However, shrews seem to play little-to-no role in the circulation of the arthropod-borne pathogens investigated.

To the canopy and beyond: Air dispersal as a mechanism of ubiquitous protistan pathogen assembly in tree canopies.

Cercozoa and Oomycota contain a huge biodiversity and important pathogens of forest trees and other vegetation. We analyzed air dispersal of these protistan phyla with an air sampler near-ground (~2 m) and in tree crowns (~25 m) of three tree species (oak, linden and ash) in a temperate floodplain forest in March (before leafing) and May (after leaf unfolding) 2019 with a cultivation-independent high-throughput metabarcoding approach. We found a high diversity of Cercozoa and Oomycota in air samples with 122 and 81 OTUs, respectively. Especially oomycetes showed a significant difference in community composition between both sampling dates. Differences in community composition between air samples in tree canopies and close to the ground were however negligible, and also tree species identity did not affect communities in air samples, indicating that the distribution of protistan propagules through the air was not spatially restricted in the forest ecosystem. OTUs of plant pathogens, whose host species did not occur in the forest, demonstrate dispersal of propagules from outside the forest biome. Overall, our results lead to a better understanding of the stochastic processes of air dispersal of protists and protistan pathogens, a prerequisite to understand the mechanisms of their community assembly in forest ecosystems.

On the phenology of protists: recurrent patterns reveal seasonal variation of protistan (Rhizaria: Cercozoa and Endomyxa) communities in tree canopies.

Tree canopies are colonized by billions of highly specialized microorganisms that are well adapted to the highly variable microclimatic conditions, caused by diurnal fluctuations and seasonal changes. In this study, we investigated seasonality patterns of protists in the tree canopies of a temperate floodplain forest via high-throughput sequencing with group-specific primers for the phyla Cercozoa and Endomyxa. We observed consistent seasonality, and identified divergent spring and autumn taxa. Tree crowns were characterized by a dominance of bacterivores and omnivores, while eukaryvores gained a distinctly larger share in litter and soil communities on the ground. In the canopy seasonality was largest among communities detected on the foliar surface: In spring, higher variance within alpha diversity of foliar samples indicated greater heterogeneity during initial colonization. However, communities underwent compositional changes during the aging of leaves in autumn, highly reflecting recurring phenological changes during protistan colonization. Surprisingly, endomyxan root pathogens appeared to be exceptionally abundant across tree canopies during autumn, demonstrating a potential role of the canopy surface as a physical filter for air-dispersed propagules. Overall, about 80% of detected OTUs could not be assigned to known species-representing dozens of microeukaryotic taxa whose canopy inhabitants are waiting to be discovered.

From Forest Soil to the Canopy: Increased Habitat Diversity Does Not Increase Species Richness of Cercozoa and Oomycota in Tree Canopies.

Tree canopies provide habitats for diverse and until now, still poorly characterized communities of microbial eukaryotes. One of the most general patterns in community ecology is the increase in species richness with increasing habitat diversity. Thus, environmental heterogeneity of tree canopies should be an important factor governing community structure and diversity in this subsystem of forest ecosystems. Nevertheless, it is unknown if similar patterns are reflected at the microbial scale within unicellular eukaryotes (protists). In this study, high-throughput sequencing of two prominent protistan taxa, Cercozoa (Rhizaria) and Oomycota (Stramenopiles), was performed. Group specific primers were used to comprehensively analyze their diversity in various microhabitats of a floodplain forest from the forest floor to the canopy region. Beta diversity indicated highly dissimilar protistan communities in the investigated microhabitats. However, the majority of operational taxonomic units (OTUs) was present in all samples, and therefore differences in beta diversity were mainly related to species performance (i.e., relative abundance). Accordingly, habitat diversity strongly favored distinct protistan taxa in terms of abundance, but due to their almost ubiquitous distribution the effect of species richness on community composition was negligible.

Green lacewings (Neuroptera: Chrysopidae) are commonly associated with a diversity of rickettsial endosymbionts.

BACKGROUND: Bacterial symbionts transmitted from mothers to offspring are found in the majority of arthropods. Numerous studies have illustrated their wide impact on host biology, such as reproduction, behavior, and physiology One of the most common inherited symbionts is Rickettsia spp. (Alphaproteobacteria, Rickettsiales), which are found in about one-quarter of terrestrial arthropods, as well as in other invertebrates. In insect populations, Rickettsia spp. have been reported to cause reproductive modifications and fecundity-enhancing effects. Here, we investigated the incidence and genetic diversity of Rickettsia symbionts in green lacewings (Neuroptera, Chrysopidae), which are best known for their use as biological control agents against crop pests. RESULTS: We screened 18 species of green lacewings and allies for Rickettsia and found the symbiont in 10 species, infecting 20-100% of sampled individuals. Strain characterization based on multiple bacterial loci revealed an unprecedented diversity of Rickettsia associated with lacewings, suggesting multiple independent acquisitions. Further, the detected Rickettsia lineages are restricted to a specific lineage (i.e., species or genus) of investigated lacewings, and these associations are stable across multiple sampled locations and points in time. CONCLUSIONS: We conclude that Rickettsia-lacewing symbioses are common and evolutionarily stable. The role of these symbionts remains to be identified, but is potentially important to optimizing their use in biological pest control.

Tula virus infections in the Eurasian water vole in Central Europe.

Recent reports of novel hantaviruses in shrews and moles and the detection of rodent-borne hantaviruses in different rodent species raise important questions about their host range and specificity, evolution, and host adaptation. Tula virus (TULV), a European hantavirus, is believed to be slightly or non-pathogenic in humans and was initially detected in the common vole Microtus arvalis, the East European vole M. levis (formerly rossiaemeridionalis), and subsequently in other Microtus species. Here we report the first multiple RT-PCR detection and sequence analyses of TULV in the Eurasian water vole Arvicola amphibius from different regions in Germany and Switzerland. Additional novel TULV S-, M-, and L-segment sequences were obtained from M. arvalis and M. agrestis trapped in Germany at sites close to trapping sites of TULV-RT-PCR-positive water voles. Serological investigations using a recombinant TULV nucleocapsid protein revealed the presence of TULV-reactive antibodies in RT-PCR-positive and a few RT-PCR-negative water voles. Phylogenetic analyses revealed a geographical clustering of the novel S-, M-, and L-segment sequences from A. amphibius with those of M. arvalis- and M. agrestis-derived TULV lineages, and may suggest multiple TULV spillover or a potential host switch to A. amphibius. Future longitudinal studies of sympatric Microtus and Arvicola populations and experimental infection studies have to prove the potential of A. amphibius as an additional TULV reservoir host.

Mitochondrial genomes reveal slow rates of molecular evolution and the timing of speciation in beavers (Castor), one of the largest rodent species.

BACKGROUND: Beavers are one of the largest and ecologically most distinct rodent species. Little is known about their evolution and even their closest phylogenetic relatives have not yet been identified with certainty. Similarly, little is known about the timing of divergence events within the genus Castor. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced complete mitochondrial genomes from both extant beaver species and used these sequences to place beavers in the phylogenetic tree of rodents and date their divergence from other rodents as well as the divergence events within the genus Castor. Our analyses support the phylogenetic position of beavers as a sister lineage to the scaly tailed squirrel Anomalurus within the mouse related clade. Molecular dating places the divergence time of the lineages leading to beavers and Anomalurus as early as around 54 million years ago (mya). The living beaver species, Castor canadensis from North America and Castor fiber from Eurasia, although similar in appearance, appear to have diverged from a common ancestor more than seven mya. This result is consistent with the hypothesis that a migration of Castor from Eurasia to North America as early as 7.5 mya could have initiated their speciation. We date the common ancestor of the extant Eurasian beaver relict populations to around 210,000 years ago, much earlier than previously thought. Finally, the substitution rate of Castor mitochondrial DNA is considerably lower than that of other rodents. We found evidence that this is correlated with the longer life span of beavers compared to other rodents. CONCLUSIONS/SIGNIFICANCE: A phylogenetic analysis of mitochondrial genome sequences suggests a sister-group relationship between Castor and Anomalurus, and allows molecular dating of species divergence in congruence with paleontological data. The implementation of a relaxed molecular clock enabled us to estimate mitochondrial substitution rates and to evaluate the effect of life history traits on it.