A Study of Oribatid Mites as Potential Intermediate Hosts of Anoplocephalid Tapeworms of Tatra chamois and Tatra marmots from the Tatra Mountains, Central Europe, and Report of a New Intermediate Host for Andrya cuniculi, the Parasite of Leporidae.

Tatra chamois (Rupicapra rupicapra tatrica (Blahout 1972)) and Tatra marmot (Marmota marmota latirostris (Kratochvíl 1961)) are significant endemic subspecies of the subalpine and alpine ranges of the Tatra Mountains in Central Europe. In four studied localities in the range of their typical biotopes in Slovakia and Poland, we investigated intestinal parasites of Tatra chamois and Tatra marmots, with an emphasis on anoplocephalid tapeworms. We also studied the occurrence, species diversity, and abundance of oribatid mites as intermediate hosts thereof, and the prevalence of cysticercoid larval stages of anoplocephalid tapeworms in collected oribatids using morphological and molecular methods. Coprological analyses revealed the average positivity of Moniezia spp. in chamois faeces at 23.5% and Ctenotaenia marmotae in marmot samples at 71.1%, with significant differences between the localities under study. Morphological analyses determined the presence of cysticercoids in five oribatid species: Ceratozetes gracilis, Edwardzetes edwardsi, Scheloribates laevigatus, Trichoribates novus, and Tectocepheus velatus sarekensis. This is the first record of T. v. sarekensis as an intermediate host of anoplocephalid tapeworms, as well as the first report of Andrya cuniculi occurrence in the territory of the Tatra Mountains, confirmed also by molecular methods.

Stress and reproductive hormones in hair associated with contaminant metal(loid)s of European brown bear (Ursus arctos).

Environmental contaminants like arsenic (As), cadmium (Cd), mercury (Hg) or lead (Pb) may disrupt hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes due to their endocrine toxicity potential. Resulting long-term physiological stress or adverse effects on wildlife reproduction and ontogeny may cause detrimental effects at the individual and population levels. However, data on environmental metal(loid)s' impact on reproductive and stress hormones in wildlife, especially large terrestrial carnivores, are scarce. Hair cortisol, progesterone and testosterone concentrations were quantified and modelled with hair As, Cd, total Hg, Pb, biological, environmental and sampling factors to test for potential effects in free-ranging brown bears (Ursus arctos) from Croatia (N = 46) and Poland (N = 27). Testosterone in males (N = 48) and females (N = 25) showed positive associations with Hg and an interaction between Cd and Pb, but a negative association with interaction between age and Pb. Higher testosterone was found in hair during its growth phase compared to quiescent phase. Body condition index was negatively associated with hair cortisol and positively associated with hair progesterone. Year and conditions of sampling were important for cortisol variation, while maturity stage for progesterone variation (lower concentrations in cubs and yearlings compared to subadult and adult bears). These findings suggest that environmental levels of Cd, Hg and Pb might influence the HPG axis in brown bears. Hair was shown to be a reliable non-invasive sample for investigating hormonal fluctuations in wildlife while addressing individual and sampling specificities.

90Sr and stable element levels in bones of brown bears: long-term trends in bear populations from Croatia and Poland.

The aim of this study was to investigate the temporal trends and geographical differences in 90Sr and stable element (Ba, Ca, Mn, Sr, Pb, Zn) levels in the bones of Croatian and Polish brown bear (Ursus arctos) populations. Experimental data suggest that in the decades after nuclear weapon tests and the Chernobyl accident, 90Sr bone activity concentrations decreased from 352 to 11 Bq kg-1 in the Croatian bear population (period 1982-2015) and from 831 to 27 Bq kg-1 in Polish bears (period 1962-2020). Calculated effective and ecological half-lives were 9 and 13 years for Croatian bears, and 15 and 31 years for Polish bears, respectively. Different temporal trends were noted in levels of Ba, Mn, Pb and Zn between the two countries with majority of bones having lower Pb, Sr and Zn in Croatian than in Polish bears. Estimated values for the soil-to-bear transfer of 90Sr were the same order of magnitude in the studied populations. Contrary to this, the estimated transfer of stable Sr was an order of magnitude lower for the Croatian bear population compared to Polish bears. The observed differences in soil-to-bear transfer between stable Sr and 90Sr found for Croatian bears might suggest the need for careful consideration on the use of stable Sr data as an analogue for 90Sr. To our knowledge, this is the first study that analysed 90Sr activity in tissue of brown bears. As such, it provides insight into the fate and behaviour of one of the most relevant anthropogenic radionuclides at the top of the food chain.

Pilot research on gastrointestinal parasites of the Tatra chamois (Rupicapra rupicapra tatrica).

INTRODUCTION AND OBJECTIVE: The Tatra chamois (Rupicapra rupicapra tatrica) is a significant representative of the High Tatra Mountains endemic fauna species. In terms of health hazards for these animals, an important role is played by parasitic infections that can lead to a significant depletion of the entire population. OBJECTIVE: The aim of the study was to describe the occurrence of gastrointestinal parasites of Tatra chamois in the current environmental and climatic conditions of the High Tatra Mountains. MATERIAL AND METHODS: During the pilot project in 2014 - 2017, a total of 494 chamois faecal samples were collected from the Slovak High Tatra and 114 samples in the Polish part of the mountain and examined using standard coprological methods. RESULTS: The results revealed that the overall positivity for gastrointestinal parasites in chamois of the Slovak High Tatra reached 74.7%. Most frequent were protozoa - Eimeria spp. (42.7%), helminths were represented by Moniezia spp. tapeworms (23.5%), eggs of GIS-strongylids (7.1%), and sporadically Capillaria spp. (1.4%). The chamois from the Polish Tatra Mts. were infected with Eimeria spp. (43.9%), GIT-strongylids (9.6%), and Moniezia spp. (6.1%). Parasitic infection was determined in 59.6 % of faecal samples from the Polish part of the mountains. Statistical analyses revealed a significant difference in Moniezia spp. occurrence in different Slovak Tatra Mts. Regions, as well as between Slovak and Polish Tatra Mts. CONCLUSIONS: Initial research on the gastrointestinal parasites of the Tatra chamois revealed one indisputable finding - a relatively high prevalence of the genus Moniezia, which is closely linked to the climate and microclimate conditions of the mountains. Further intensive research on parasite composition and distribution in Tatra chamois is needed in broader temporal, ecological, and zoological contexts.

Occurrence of Echinococcus spp. in red foxes and wolves in the protected area of the Tatra National Park in southern Poland - a threat to human health.

INTRODUCTION: Echinococcus multilocularis has been endemic in red foxes in eastern and central parts of Europe, and E. granulosus s. l. identified in wolves in some countries. In recent years, wolves hale emerged as potentially important definitive hosts of E. multilocularis. OBJECTIVE: This aim of the survey was to record indirectly using nested-PCR test with faecal samples the presence of Echinococcus multilocularis and E. granulosus s. l. in the two species of wild canids in the protected area of the Tatra National Park (TNP) in Western Carpathian, southern mountainous part of Poland. MATERIAL AND METHODS: From February to June 2019, experienced staff of TNP randomly collected fox and wolf faeces on and off hiking trails at altitudes from 850 m to 2,000 m above sea level. In total, 91 faecal samples from red foxes and 19 from wolves were collected. Genomic DNA was obtained by direct extraction from faecal samples using a commercial kit, and from taeniid eggs retrieved from the same samples after flotation. RESULTS: A nested PCR screening of 91 red fox faeces indicated the prevalence of E. multilocularis of 4.4%. Positive samples were confirmed by sequencing parts of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Neither genomic DNA of E. multilocularis nor of E. granulosus s.l. was obtained from 19 wolves faeces, nor from taeniid eggs retrieved from these samples by initial flotation. CONCLUSIONS: The current results show that humans might be exposed to a risk of fox tapeworm infection in nature, even at high altitude inan alpine zone, in an environment contaminated by roaming red foxes encouraged by food leftovers on mountain trails.

Hybridization and introgression of native and foreign Sorbus tree species in unique environments of protected mountainous areas.

Hybridization and introgression are important processes influencing the genetic diversity and evolution of species. These processes are of particular importance in protected areas, where they can lead to the formation of hybrids between native and foreign species and may ultimately result in the loss of parental species from their natural range. Despite their importance, the contribution of hybridization and introgression to genetic diversity in Sorbus genus remains not fully recognized. We analysed the genetic and morphological variability of several Sorbus species including native (Sorbus aria), foreign (S. intermedia) and potentially hybrid (S. carpatica) individuals from the Polish Carpathian range. Patterns of variation at 13 nuclear microsatellite loci show hybridization between the tested species and confirm the existence of the hybrid form S. carpatica. Biometric analysis on leaves, based of 10 metric features and three parameters, identified several characters for preliminary taxonomic classification; however, none of them could be used as a fully diagnostic marker for faultless annotation of S. intermedia and S. carpatica. The genetic structure analysis indicated complex patterns of population differentiation and its diverse origin. The results allow assessment of genetic variation and identification of parental species participating in hybridization. This knowledge will advance the management of genetic diversity and development of conservation strategies for efficient maintenance of the unique protected ecosystem.

Losing seasonal patterns in a hibernating omnivore? Diet quality proxies and faecal cortisol metabolites in brown bears in areas with and without artificial feeding.

Bears are omnivores particularly well-adapted to variations in the nutritional composition, quality and availability of food resources. Artificial feeding practices have been shown to strongly influence diet composition and seasonality, as well as to cause alterations in wintering and movement in brown bears (Ursus arctos). In this study, we investigated seasonal differences (hypophagia vs hyperphagia) in food quality of two brown bear subpopulations in the Polish Carpathians using faecal nitrogen (FN) and carbon (FC) estimates. The subpopulations inhabit areas that differ in artificial feeding practices: no artificial feeding occurs in the western subpopulation (Tatra Mountains), while artificial food targeted to ungulates is provided and used year-round in the eastern subpopulation (Bieszczady Mountains). We also compared these results with faecal cortisol metabolites (FCM) to explore how FN and FC correlate with the hypothalamic-pituitary-adrenal axis activity and if the seasonal patterns are apparent. We found that in Tatra Mts bears fed on significantly higher quality diet, as shown by FN and FC values, and had significantly higher FC levels in hyperphagia, when they accumulate fat reserves for wintering. The pattern in FCM levels for Tatra subpopulation followed the changes in energy intake during the seasons of hypo- and hyperphagia, while in Bieszczady Mts, the area with intensive feeding, no seasonal patterns could be observed. Artificial feeding practices may disrupt nutrient phenology and seasonality, relative to subpopulations with natural diets. We showed that the availability of human-provided foods may alter not only the overall dietary quality, but also hormonal patterns linked to seasonal nutritional requirements. Combining FN, FC and FCM proved to be a useful tool for reconstructing diet quality and related physiological patterns.

Metal(loid) exposure assessment and biomarker responses in captive and free-ranging European brown bear (Ursus arctos).

We investigated the level of five non-essential metal(loid)s (As, Cd, Hg, Tl, Pb) and nine essential metals (Mg, Ca, Mn, Fe, Co, Cu, Zn, Se, Mo) in hair and blood components of captive and free-ranging European brown bear populations in Croatia and Poland. Metal(loid) associations with biomarkers of oxidative stress (superoxide dismutase, SOD; glutathione-peroxidase, GSH-Px; malondialdehyde, MDA) and metal exposure (metallothionein, MT) were estimated in this top predatory mammal. Lead was the most abundant non-essential metal(loid) in both blood and hair, with 4 of 35 individuals having blood levels over 100 µg/L. A positive association was found between Pb level and SOD activity in blood. Free-ranging bears had higher blood SOD activity, Mn, Zn and Cd levels, hair Co, Cd, Tl and Pb compared to captive individuals, while the opposite was true for Mg and hair Ca thereby reflecting habitat and diet differences. With increasing age, animals showed lower levels of SOD activity and certain essential metals. Females had higher SOD activity and blood levels of some essential metals than males. Hair showed a higher Fe and Co level when sampled during the growth phase and was not predictive of non-essential metal(loid) blood levels. The established metal(loid) baseline values will enable future risk assessment in both captive and wild European brown bear populations.

Dynamic range expansion leads to establishment of a new, genetically distinct wolf population in Central Europe.

Local extinction and recolonization events can shape genetic structure of subdivided animal populations. The gray wolf (Canis lupus) was extirpated from most of Europe, but recently recolonized big part of its historical range. An exceptionally dynamic expansion of wolf population is observed in the western part of the Great European Plain. Nonetheless, genetic consequences of this process have not yet been fully understood. We aimed to assess genetic diversity of this recently established wolf population in Western Poland (WPL), determine its origin and provide novel data regarding the population genetic structure of the grey wolf in Central Europe. We utilized both spatially explicit and non-explicit Bayesian clustering approaches, as well as a model-independent, multivariate method DAPC, to infer genetic structure in large dataset (881 identified individuals) of wolf microsatellite genotypes. To put the patterns observed in studied population into a broader biogeographic context we also analyzed a mtDNA control region fragment widely used in previous studies. In comparison to a source population, we found slightly reduced allelic richness and heterozygosity in the newly recolonized areas west of the Vistula river. We discovered relatively strong west-east structuring in lowland wolves, probably reflecting founder-flush and allele surfing during range expansion, resulting in clear distinction of WPL, eastern lowland and Carpathian genetic groups. Interestingly, wolves from recently recolonized mountainous areas (Sudetes Mts, SW Poland) clustered together with lowland, but not Carpathian wolf populations. We also identified an area in Central Poland that seems to be a melting pot of western, lowland eastern and Carpathian wolves. We conclude that the process of dynamic recolonization of Central European lowlands lead to the formation of a new, genetically distinct wolf population. Together with the settlement and establishment of packs in mountains by lowland wolves and vice versa, it suggests that demographic dynamics and possibly anthropogenic barriers rather than ecological factors (e.g. natal habitat-biased dispersal patterns) shape the current wolf genetic structure in Central Europe.

Airborne fungi as indicators of ecosystem disturbance: an example from selected Tatra Mountains caves (Poland).

We report on the determination of the spore concentration and the species composition of the airborne fungi in selected caves of the Tatra Mountains, Poland. The following caves were surveyed: Mylna, Oblazkowa, Mrozna, Zimna and Naciekowa. The sampling was carried out in July 2015 and in January 2016. The aeromycological analyses were performed with the impact method, using the Air Ideal 3P apparatus and potato dextrose agar (PDA, Biocorp) culture medium. In the course of the July 2015 analysis, 17 species of fungi were isolated and 11 species were isolated in January 2016. In Mylna and Naciekowa caves, the dominant species were Cladosporium cladosporioides and Stachybotrys cylindrospora. In Oblazkowa cave, Rhizoctonia predominated and in Zimna cave-the colonies of the yeast-like fungi, along with S. cylindrospora. In Mrozna cave, Penicillium notatum was the most abundant taxon. In the winter time, in the majority of the caves Penicillium spp. predominated, with the exception of Mrozna and Naciekowa caves where Aspergillus niger was dominant. We propose that aeromycological monitoring be performed regularly in the following caves: Mrozna, Naciekowa and Zimna.

A modified method for molecular identification of Baylisascaris transfuga in European brown bears (Ursus arctos).

Baylisascaris transfuga is a roundworm that has been reported worldwide in most bear species. In mammals and possibly humans, the larvae of B. transfuga can migrate in the tissues of aberrant hosts with larva migrans syndrome. The current study was performed to identify B. transfuga in faecal samples from free-ranging brown bears in the Tatra Mountains National Park in southern Poland. A commercial kit was used to extract genomic DNA directly from faecal samples. Additionally, a Chelex resin-based technique was successfully implemented to prepare a PCR template from eggs retrieved by flotation. Based on the flotation results of 32 collected faecal samples, the prevalence of B. transfuga was 15.6%. The parasite was confirmed in samples found to be positive during the initial flotation by a molecular assay using DNA isolated directly from faeces. The retrieved eggs were confirmed as B. transfuga after their DNA was extracted using the Chelex protocol. Based on PCR amplification and sequencing of a 413-bp segment of cytochrome c oxidase 1 (COI), the obtained sequence was 100% identical to the COI segment of B. transfuga after a BLAST comparison to the GenBank™ database. The current study includes the first molecular confirmation of B. transfuga in brown bears in the western part of the Carpathians. We show that direct extraction of parasite DNA from bear faeces is efficient for molecular assays. As an alternative, we present the effectiveness of a Chelex-based technique for fast and convenient DNA isolation from the difficult-to-disrupt eggs of B. transfuga for PCR. Molecular tests of parasite DNA extracted directly from faecal material have limits of detection related to the amount of eggs in the samples. Thus, using classical flotation to obtain eggs for PCR may increase the credibility of the results, particularly in cases with a low number of excreted eggs. The Chelex resin protocol has potential for application in studies of intestinal parasites in wildlife for which conventional flotation is routinely used for microscopy.

Histological, chemical and behavioural evidence of pedal communication in brown bears.

Most mammals rely upon scent for intraspecific communication. As most bear species have large home ranges and are non-territorial, scent deposit while walking could be an effective way to communicate with conspecifics. Here, we investigate the existence of pedal glands in brown bears and their role in chemical communication from a histological, biochemical and behavioural perspective. We found eccrine glands in footpads, and prominent apocrine and sebaceous glands in the interdigital, metacarpal and metatarsal skin sections. Pedal scent contained 26 compounds including carboxylic acids, important constituents of mammalian secretions. Six of these compounds were exclusive for males. Finally, we describe a specific marking gait recorded in the field, mostly performed by males. Our study supports the existence of chemical communication through pedal marking in brown bears and suggests sex-coding potential of pedal scent.

First molecular characterization of Sarcocystis tenella in Tatra chamois (Rupicapra rupicapra tatrica) in Poland.

In this study, sarcocysts from three Polish Tatra chamois were isolated and identified using morphological and molecular methods for the first time. Six cysts were found in the latissimus dorsi muscle and another two in the diaphragm. No sarcocysts were detected in the myocardium, tongue, and esophagus. The isolated cysts were long with rounded ends, 0.35-0.61 mm in length, and 0.02-0.06 mm in width. All the sarcocysts were identified as Sarcocystis tenella on the basis of light microscopy and sequencing of cytochrome C oxidase subunit I (cox1) and small-subunit rRNA (ssu rRNA) genes. Comparative analysis showed a 99.23% identity of the cox1 gene sequences from Tatra chamois and sheep sarcocysts, and an even higher degree of sequence identity (99.88%) was documented in the case of the ssu rRNA gene. When compared at a haplotype level, all the sheep sequences of cox1 differed from those isolated from Tatra chamois. In contrast, one out of the two ssu rRNA haplotypes from the sheep isolates was identical with the haplotype from Tatra chamois. In conclusion, we showed that cox1 and ssu rRNA genes can be used as genetic markers for identification of the S. tenella, with cox1 gene providing better resolution during phylogenetic analyses. However, both genetic population analysis and phylogenetic inference with cox1 and ssu rRNA genes demonstrated that they do not constitute good markers for spatial differentiation of S. tenella.

Growth responses to climate in a multi-species tree-ring network in the Western Carpathian Tatra Mountains, Poland and Slovakia.

We analyzed growth responses to climate of 24 tree-ring width and four maximum latewood density chronologies from the greater Tatra region in Poland and Slovakia. This network comprises 1183 ring-width and 153 density measurement series from four conifer species (Picea abies (L.) Karst., Larix decidua Mill., Abies alba (L.) Karst., and Pinus mugo (L.)) between 800 and 1550 m a.s.l. Individual spline detrending was used to retain annual to multi-decadal scale climate information in the data. Twentieth century temperature and precipitation data from 16 grid-boxes covering the 48-50 degrees N and 19-21 degrees E region were used for comparison. The network was analyzed to assess growth responses to climate as a function of species, elevation, parameter, frequency and site ecology. Twenty ring-width chronologies significantly correlated (P<0.05) with June-July temperatures, whereas the latewood density chronologies were correlated with the April-September temperatures. Climatic effects of the previous-year summer generally did not significantly influence ring formation, whereas site elevation and frequency of growth variations (i.e., inter-annual and decadal) were significant variables in explaining growth response to climate. Response to precipitation increased with decreasing elevation. Correlations between summer temperatures and annual growth rates were lower for Larix decidua than for Picea abies. Principal component analysis identified five dominant eigenvectors that express somewhat contrasting climatic signals. The first principal component contained highest loadings from 11 Picea abies ring-width chronologies and one Pinus mugo ring-width chronology and explained 42% of the network's variance. The mean of these 12 high-elevation chronologies was significantly correlated at 0.62 with June-July temperatures, whereas the mean of three latewood density chronologies, which loaded most strongly on the fourth principal component, significantly correlated at 0.69 with April-September temperatures (P<0.001 over the 1901-2002 period in both cases). These groupings allow for a robust estimation of June-July (1661-2004) and April-September (1709-2004) temperatures, respectively. Comparison with reconstructions from the Alps and Central Europe supports the general rule of the dominant influence of growing season temperature on high-elevation forest growth.