Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites.

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.

Detection of Three Sarcocystis Species (Apicomplexa) in Blood Samples of the Bank Vole and Yellow-Necked Mouse from Lithuania.

The genus Sarcocystis is an abundant group of Apicomplexa parasites found in mammals, birds, and reptiles. These parasites are characterised by the formation of sarcocysts in the muscles of intermediate hosts and the development of sporocysts in the intestines of definitive hosts. The identification of Sarcocystis spp. is usually carried out in carcasses of animals, while there is a lack of studies on the detection of Sarcocystis species in blood samples. In the current study, blood samples of 214 yellow-necked mice (Apodemus flavicollis) and 143 bank voles (Clethrionomys glareolus) from Lithuania were examined for Sarcocystis. The molecular identification of Sarcocystis was carried out using nested PCR of cox1 and 28S rRNA and subsequent sequencing. Sarcocystis spp. were statistically (p < 0.01) more frequently detected in the bank vole (6.3%) than in yellow-necked mice (0.9%). The analysed parasites were observed in four different habitats, such as mature deciduous forest, bog, natural meadow, and arable land. Three species, Sarcocystis funereus, Sarcocystis myodes, and Sarcocystis cf. glareoli were confirmed in the bank vole, whereas only Sarcocystis myodes were found in yellow-necked mice. The obtained results are important in the development of molecular identification of Sarcocystis parasites in live animals.

The extinct Sicilian wolf shows a complex history of isolation and admixture with ancient dogs.

The Sicilian wolf remained isolated in Sicily from the end of the Pleistocene until its extermination in the 1930s-1960s. Given its long-term isolation on the island and distinctive morphology, the genetic origin of the Sicilian wolf remains debated. We sequenced four nuclear genomes and five mitogenomes from the seven existing museum specimens to investigate the Sicilian wolf ancestry, relationships with extant and extinct wolves and dogs, and diversity. Our results show that the Sicilian wolf is most closely related to the Italian wolf but carries ancestry from a lineage related to European Eneolithic and Bronze Age dogs. The average nucleotide diversity of the Sicilian wolf was half of the Italian wolf, with 37-50% of its genome contained in runs of homozygosity. Overall, we show that, by the time it went extinct, the Sicilian wolf had high inbreeding and low-genetic diversity, consistent with a population in an insular environment.

Bifidobacterium castoris strains isolated from wild mice show evidence of frequent host switching and diverse carbohydrate metabolism potential.

Members of the gut microbiota genus Bifidobacterium are widely distributed human and animal symbionts believed to exert beneficial effects on their hosts. However, in-depth genomic analyses of animal-associated species and strains are somewhat lacking, particularly in wild animal populations. Here, to examine patterns of host specificity and carbohydrate metabolism capacity, we sequenced whole genomes of Bifidobacterium isolated from wild-caught small mammals from two European countries (UK and Lithuania). Members of Bifidobacterium castoris, Bifidobacterium animalis and Bifodobacterium pseudolongum were detected in wild mice (Apodemus sylvaticus, Apodemus agrarius and Apodemus flavicollis), but not voles or shrews. B. castoris constituted the most commonly recovered Bifidobacterium (78% of all isolates), with the majority of strains only detected in a single population, although populations frequently harboured multiple co-circulating strains. Phylogenetic analysis revealed that the mouse-associated B. castoris clades were not specific to a particular location or host species, and their distribution across the host phylogeny was consistent with regular host shifts rather than host-microbe codiversification. Functional analysis, including in vitro growth assays, suggested that mouse-derived B. castoris strains encoded an extensive arsenal of carbohydrate-active enzymes, including putative novel glycosyl hydrolases such as chitosanases, along with genes encoding putative exopolysaccharides, some of which may have been acquired via horizontal gene transfer. Overall, these results provide a rare genome-level analysis of host specificity and genomic capacity among important gut symbionts of wild animals, and reveal that Bifidobacterium has a labile relationship with its host over evolutionary time scales.

Ecological indices and factors influencing communities of ectoparasitic laelapid mites (Acari, Mesostigmata, Laelapidae) of small mammals in Lithuania.

Family Laelapidae is an ecologically diverse group that includes free-living species and parasites of vertebrates and invertebrates. At least seven genera in this family are associated with small mammals. In this study, ectoparasitic laelapid mites of rodents and shrews were investigated in Lithuania. In total, 2,274 small mammal specimens of 12 species were trapped and 6,089 laelapid mites were collected. The updated list of ectoparasitic mites in Lithuania included 21 mite species. Seven mite species were identified as highly specific for a host species or genus, one species was moderately specific, and four mite species were assigned to generalist parasites. All host species had one or two superdominant mite species. The prevalence and mean intensity varied significantly depending on host species and habitat. We analyzed the influence of the host (species, sex, age) and environmental factors (landscape morphology type, habitat, anthropogenic effect) on the abundance of the mite community and most numerous mite species, as well as the impact of the host community (Shannon's diversity index, species richness, host abundance) on mean abundance of the mite community. Only particular host species (Apodemus flavicollis, Microtus agrestis, and Microtus arvalis) and habitats (pastures, mixed forests) influenced the abundance of mites.

Effects of laboratory domestication on the rodent gut microbiome.

The domestication of the laboratory mouse has influenced the composition of its native gut microbiome, which is now known to differ from that of its wild ancestor. However, limited exploration of the rodent gut microbiome beyond the model species Mus musculus has made it difficult to interpret microbiome variation in a broader phylogenetic context. Here, we analyse 120 de novo and 469 public metagenomically-sequenced faecal and caecal samples from 16 rodent hosts representing wild, laboratory and captive lifestyles. Distinct gut bacterial communities were observed between rodent host genera, with broadly distributed species originating from the as-yet-uncultured bacterial genera UBA9475 and UBA2821 in the families Oscillospiraceae and Lachnospiraceae, respectively. In laboratory mice, Helicobacteraceae were generally depleted relative to wild mice and specific Muribaculaceae populations were enriched in different laboratory facilities, suggesting facility-specific outgrowths of this historically dominant rodent gut family. Several bacterial families of clinical interest, including Akkermansiaceae, Streptococcaceae and Enterobacteriaceae, were inferred to have gained over half of their representative species in mice within the laboratory environment, being undetected in most wild rodents and suggesting an association between laboratory domestication and pathobiont emergence.

Blood parasites (Babesia, Hepatozoon and Trypanosoma) of rodents, Lithuania: part I. Molecular and traditional microscopy approach.

Wild rodents, as natural reservoir hosts carrying various species of pathogens, play an important role in the evolution and emergence of zoonotic diseases. In this study, protist parasites, namely Babesia sp., Trypanosoma sp. and Hepatozoon sp. were studied in rodent populations in Lithuania. Two hundred forty rodent specimens of seven species were analysed by a combined approach using polymerase chain reaction (PCR)-based techniques and traditional microscopic examination. The total prevalence of blood parasites reached 35% in rodent communities. The prevalence of Hepatozoon sp. reached the highest value (32%), followed by Trypanosoma sp. (5%) and Babesia sp. (3%). Myodes glareolus and Microtus agrestis were the most heavily infected rodent species. Comparison of microscopy and PCR-based methods showed that the two approaches might give different results and thus can lead to an underestimation of the actual prevalence and abundance of parasites. In our study, PCR-based assays were more sensitive and robust than traditional microscopy. However, precise molecular results for the estimation of the prevalence of Babesia sp. and Hepatozoon sp. were achieved only by using several sets of primers. To avoid inaccurate results, the improvement and detailed description of molecular and microscopy protocols are required.

The evolution of ecological facilitation within mixed-species biofilms in the mouse gastrointestinal tract.

The eco-evolutionary interactions among members of the vertebrate gut microbiota that ultimately result in host-specific communities are poorly understood. Here we show that Lactobacillus reuteri coexists with  species that belong to the Lactobacillus johnsonii cluster (L. johnsonii, L. gasseri, and L taiwanensis) in a taxonomically wide range of rodents, suggesting cohabitation over evolutionary times. The two dominant Lactobacillus species found in wild mice establish a commensalistic relationship in gastric biofilms when introduced together into germ-free mice in which L. reuteri facilitates colonization of L. taiwanensis. Genomic analysis revealed allopatric diversification in strains of both species that originated from geographically separated locations (Scotland and France). Allopatry of the strains resulted in reduced formation of mixed biofilms in vitro, indicating that interspecies interactions in gastric Lactobacillus-biofilms are the result of an adaptive evolutionary process that occurred in a biogeographical context. In summary, these findings suggest that members within the vertebrate gut microbiota can evolve inter-dependencies through ecological facilitation, which could represent one mechanism by which host-specific bacterial communities assemble across vertebrate species and an explanation for their spatial and biogeographic patterns.

Wolf population genetics in Europe: a systematic review, meta-analysis and suggestions for conservation and management.

The grey wolf (Canis lupus) is an iconic large carnivore that has increasingly been recognized as an apex predator with intrinsic value and a keystone species. However, wolves have also long represented a primary source of human-carnivore conflict, which has led to long-term persecution of wolves, resulting in a significant decrease in their numbers, genetic diversity and gene flow between populations. For more effective protection and management of wolf populations in Europe, robust scientific evidence is crucial. This review serves as an analytical summary of the main findings from wolf population genetic studies in Europe, covering major studies from the 'pre-genomic era' and the first insights of the 'genomics era'. We analyse, summarize and discuss findings derived from analyses of three compartments of the mammalian genome with different inheritance modes: maternal (mitochondrial DNA), paternal (Y chromosome) and biparental [autosomal microsatellites and single nucleotide polymorphisms (SNPs)]. To describe large-scale trends and patterns of genetic variation in European wolf populations, we conducted a meta-analysis based on the results of previous microsatellite studies and also included new data, covering all 19 European countries for which wolf genetic information is available: Norway, Sweden, Finland, Estonia, Latvia, Lithuania, Poland, Czech Republic, Slovakia, Germany, Belarus, Russia, Italy, Croatia, Bulgaria, Bosnia and Herzegovina, Greece, Spain and Portugal. We compared different indices of genetic diversity in wolf populations and found a significant spatial trend in heterozygosity across Europe from south-west (lowest genetic diversity) to north-east (highest). The range of spatial autocorrelation calculated on the basis of three characteristics of genetic diversity was 650-850 km, suggesting that the genetic diversity of a given wolf population can be influenced by populations up to 850 km away. As an important outcome of this synthesis, we discuss the most pressing issues threatening wolf populations in Europe, highlight important gaps in current knowledge, suggest solutions to overcome these limitations, and provide recommendations for science-based wolf conservation and management at regional and Europe-wide scales.

Microtus arvalis and Arvicola scherman: Key Players in the Echinococcus multilocularis Life Cycle.

A broad range of rodent species are described as potential intermediate hosts for Echinococcus multilocularis, a wide-spread zoonotic cestode causing alveolar echinococcosis. However, little is known about the relative contribution of these species for parasite reproduction and the maintenance of its life cycle. In a comparative study in a high endemic region in Zurich, Switzerland, we investigated prevalence rates and fertility of E. multilocularis in the most abundant vole species as well as the predation rate of foxes on these species. To ensure that the fox families had access to different vole species and that these voles were exposed to the same environmental contamination with parasite eggs, we selected eight study plots where at least two rodent species co-occurred. The parasite prevalence in Microtus arvalis [11.0%, confidence intervals (CI) 8.9-13.4] was significantly higher than in Arvicola scherman (5.3%, 3.9-7.1) and Myodes glareolus (3.9%, 2.0-6.7). None of the, only 29 individuals of, Microtus agrestis was infected (0%, 0.0-9.8) and the species was excluded for further analyses. Logistic regression models for the prevalences revealed significant differences between nearby study plots and higher infection rates for females, heavier individuals, and individuals trapped during spring, when the prevalence in M. arvalis peaked up to 65% (CI 50-79) in one plot. Furthermore, we detected significantly higher percentages of fertile infections in M. arvalis and M. glareolus than in A. scherman (OR 11.2 and 6.4, respectively) and a significantly higher protoscolex number in M. glareolus (median 100,000) than in M. arvalis (13,500) and A. scherman (4,290). The most abundant fox prey remains were of the genera Microtus (12.3%, CI 8.4-17.2) and Arvicola (11.5%, 7.7-16.3), whereas Myodes was never recorded as prey (0.0-1.3%). We conclude that M. arvalis and to a lesser extent A. scherman can be regarded as key intermediate hosts in Western and Central European high-endemic regions whereas M. glareolus and M. agrestis play a marginal role. We, therefore, postulate that distribution models of these species could contribute to predict parasite occurrence on a more detailed spatial scale than models of the distribution of foxes which have a very broad and uniform distribution.

Homogenous Population Genetic Structure of the Non-Native Raccoon Dog (Nyctereutes procyonoides) in Europe as a Result of Rapid Population Expansion.

The extent of gene flow during the range expansion of non-native species influences the amount of genetic diversity retained in expanding populations. Here, we analyse the population genetic structure of the raccoon dog (Nyctereutes procyonoides) in north-eastern and central Europe. This invasive species is of management concern because it is highly susceptible to fox rabies and an important secondary host of the virus. We hypothesized that the large number of introduced animals and the species' dispersal capabilities led to high population connectivity and maintenance of genetic diversity throughout the invaded range. We genotyped 332 tissue samples from seven European countries using 16 microsatellite loci. Different algorithms identified three genetic clusters corresponding to Finland, Denmark and a large 'central' population that reached from introduction areas in western Russia to northern Germany. Cluster assignments provided evidence of long-distance dispersal. The results of an Approximate Bayesian Computation analysis supported a scenario of equal effective population sizes among different pre-defined populations in the large central cluster. Our results are in line with strong gene flow and secondary admixture between neighbouring demes leading to reduced genetic structuring, probably a result of its fairly rapid population expansion after introduction. The results presented here are remarkable in the sense that we identified a homogenous genetic cluster inhabiting an area stretching over more than 1500km. They are also relevant for disease management, as in the event of a significant rabies outbreak, there is a great risk of a rapid virus spread among raccoon dog populations.

Migration and isolation during the turbulent Ponto-Caspian Pleistocene create high diversity in the crustacean Paramysis lacustris.

The Ponto-Caspian brackish-water fauna inhabits estuaries and rivers of the Black, Azov and Caspian seas and is fragmented by higher salinity waters and a major interbasin watershed. The fauna is known for the high levels of endemism, complex zoogeographic histories, and as a recent source of successful invasive species. It remains debated whether the Black and Azov Sea brackish-water populations survived unfavourable Pleistocene conditions in multiple separate refugia or whether the two seas were (repeatedly) recolonized from the Caspian. Using microsatellite and mtDNA markers, we demonstrate deep among- and within-basin subdivisions in a widespread Ponto-Caspian mysid crustacean Paramysis lacustris. Five genetic clusters were identified, but their relationships did not reflect the geography of the region. The Azov cluster was the most distinct (4-5% COI divergence), despite its geographic position in the corridor between Black and Caspian seas, and may represent a new species. In the northern Black Sea area, the Dnieper cluster was closer to the Caspian cluster than to the neighbouring Danube-Dniester-Bug populations, suggesting separate colonizations of the Black Sea. Overall, the data implied a predominant gene flow from the east to the Black Sea and highlight the importance of Caspian Sea transgressions in facilitating dispersal. Yet, the presence of distinct lineages in the Black Sea points to the persistence of isolated refugial populations that have gained diagnostic differences under presumably high mutation rates and large population sizes. The unfavourable Pleistocene periods in the Black Sea therefore appear to have promoted diversification of the brackish-water lineages, rather than extirpated them.