Puumala Virus in Bank Voles, Lithuania.

Little is known about the presence of human pathogenic Puumala virus (PUUV) in Lithuania. We detected this virus in bank voles (Myodes glareolus) in a region of this country in which previously PUUV-seropositive humans were identified. Our results are consistent with heterogeneous distributions of PUUV in other countries in Europe.

High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species.

Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent-the Harvest mouse Micromys minutus-as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.

Insight into Body Condition Variability in Small Mammals.

The body condition index (BCI) is an indicator of both reproductive success and health in small mammals and might help to understand ecological roles of species. We analyzed BCI data from 28,567 individuals trapped in Lithuania between 1980 and 2023. We compared BCIs between species and examined differences in age groups, gender, and reproductive statuses within each species. Seven out of eighteen species had sample sizes with N < 100. In terms of species, we found that seven of the eight species with the highest average BCIs are granivores or omnivores, which can consume animal-based food at least seasonally. The two contrasting (decreasing or increasing) BCI patterns observed during ontogeny can be related to diet differences among juveniles, subadults, and adult animals. Our results demonstrate that reproductive stress has a negative impact on the BCI of adult females in all analyzed species and nearly all adult males. Although the animals with extremely low BCI consisted mostly of shrews, for the first time we found 23 common and pygmy shrews exhibiting the Chitty effect, i.e., a very high body mass resulting in a BCI > 5.0. This is the first multi-species approach of body condition at middle latitudes. The results increase our understanding of how changing environmental conditions are affecting small mammals.

Trends and Characteristics of Human Casualties in Wildlife-Vehicle Accidents in Lithuania, 2002-2022.

We analyzed 474 human casualties in wildlife-vehicle accidents (WVAs) that occurred between 2002 and 2022 in Lithuania, which is a small northern European country. The study revealed the escalating trend of WVAs, since 2018 surpassing other transport accidents, although the number of casualties per WVA was ca. 100 times lower compared to other transport accidents. Moose was the primary contributor, responsible for 66.7% of fatalities and 47.2% of injuries, despite much lower species abundance compared to roe deer, which is the main species involved in WVAs without human casualties. Temporal patterns highlighted seasonal, daily, and hourly variations, with the majority of casualties occurring during dusk or dawn in May and September, on weekends, and between 20:00 and 22:00. Spatially, main roads with high traffic density exhibited the highest casualties per unit length. Most casualties occurred after hitting an animal directly with cars and motorcycles being most vulnerable vehicles. The effectiveness of WVA prevention measures was inconclusive: 9.5% of fatalities and 1.4% of injuries were registered in the area of the warning sign, and 10.4% of all casualties occurred on fenced road segments. These findings suggest the need for a critical evaluation of the current prevention strategies in reducing human casualties associated with WVAs.

Low Genetic Variability of the Tundra Vole in Lithuania.

The distribution and spread of the tundra vole (Alexandromys oeconomus) in Lithuania have been documented over the last 70 years, but the genetic diversity of the species has not been studied. In this study, we examined A. oeconomus trapped in three sites in northern and western Lithuania using mtDNA sequence analysis of the cytb and control region. The western and northern sites are separated by anthropogenic landscape barriers. The western site is subject to regular spring flooding. Phylogenetic analyses of the studied individuals placed them in the Central European phylogroup, suggesting that Lithuanian A. oeconomus originated from northeastern Poland. In Lithuania, the genetic diversity of A. oeconomus at both mtDNA loci was relatively low (Hd < 0.6, π < 0.002) compared to that found in other European samples (Hd = 0.833-0.958; π = 0.00402-0.01552). Individuals analyzed in Lithuania were genetically different from samples collected in Poland and Northern Europe (ΦST > 0.15, p < 0.05). The genetic divergence between the western and northern samples of A. oeconomus in Lithuania, together with the low genetic variability among the voles studied, provides new insights into the phylogeography of the species and the influence of barriers on the colonization of the country.

Abundance and Population Structure of Small Rodents in Fruit and Berry Farms.

Fruit and berry farms are anthropogenic habitats still inhabited by small mammals, though their presence is constantly affected by agricultural activities. Based on trapping data from 2018-2022, we analyzed the abundance and population structure of the dominant rodent species to assess changes in gender and age ratios by year and habitat, the annual and seasonal dynamics of relative abundance, and the relationship between breeding parameters and abundance. The relative abundance of the dominant species, common vole, yellow-necked mouse, striped field mouse, and bank vole, and their proportion in the investigated community varied according to year, season, and habitat. No outbreaks were recorded during the study period. The abundance of the striped field mouse exhibited a downward trend independently of habitat, while the abundance and proportions of the other three species were habitat-dependent. There was no consistent pattern between litter size and relative abundance in the same or following years. Given the ongoing conflict between biodiversity conservation in Europe and agriculture, the results contribute to a better understanding of the functioning and viability of rodent populations in fruit farms and may be used in agroecology and sustainable farming.

Mammal Roadkills in Lithuanian Urban Areas: A 15-Year Study.

We investigated roadkills in urban areas in Lithuania from 2007 to 2022, including two periods with COVID-19 restrictions on people's movement. We analyzed the proportions of wild and domestic animals in roadkill, annual trends, the predominant species involved, and monthly changes during the restrictions. Urban roads were characterized by a low species diversity of roadkilled mammals, with roe deer (Capreolus capreolus) dominating. Total numbers increased exponentially during the study period. The proportion of domestic animals, 12.2%, significantly exceeded that on non-urban roads in the country. The proportion of domestic animals decreased from over 40% in 2007-2009 to 3.7-5.4% in 2020-2022, while the proportion of wild mammals increased from 36.1-39.6% to 89.9-90.6%, respectively. During the periods of COVID-19 restrictions, the number of roadkills in urban areas was significantly higher than expected based on long-term trends. Compared to 2019, the number of roadkilled roe deer in 2020-2021 almost doubled from 700 to 1281-1325 individuals. These anthropause effects were, however, temporary. The imbalance between the roadkill number and transport intensity might require new mitigation strategies to sustain mammal populations in urban areas, at least through improving driver awareness on the issue.

Shrews Under-Represented in Fruit Farms and Homesteads.

Shrews are a less studied group of small mammals than rodents. Between 2018 and 2022, we surveyed 23 sites in Lithuania, including natural and anthropogenic habitats, with the aim to assess the proportion of Soricidae in small mammal communities and their diet based on stable isotope analysis. The average representation of Soricidae was 3.1%, about half the long-term average in other habitats in the country. The highest proportions were in meadows and farmsteads, at 4.9% and 5.0% respectively. Shrews were not trapped on farms or in young orchards, and their relative abundance was very low in intensively managed orchards (0.006 individuals per 100 trap days). Neomys fodiens and N. anomalus were unexpectedly found in homesteads, including in outbuildings. Sorex araneus and S. minutus had similar diets. The trophic carbon/nitrogen discrimination factor between invertebrates and shrew hair was 2.74‱/3.98‱ for S. araneus, 1.90‱/3.78‱ for S. minutus in the orchards. The diet of N. fodiens and N. anomalus at the homesteads requires further investigation. We propose that the under-abundance of shrews may be due to contamination by plant protection products and a lack of invertebrates under intensive agricultural practices.

Lockdown's Silver Lining? Different Levels of Roadkill during the COVID-19 Times in Lithuania.

The impact of COVID-19 restrictions on human and vehicular mobility on the number of roadkills of wild mammals, and roe deer in particular, was assessed in Lithuania. We statistically analyzed the distribution of all mammals and roe deer (Capreolus capreolus) as the most abundant species annually from 2002 to 2022 and monthly from 2020 to 2021, including during the two restriction periods. Notably, there was a marked decrease in roadkill incidents on main, national and regional roads, particularly in April-May 2020 (the beginning of lockdown period I) and November-December 2020 (the beginning of lockdown period II), 5.1-20.8% and 33.6-54.4%, respectively. During these months, collisions with mammals on the A14 main road were reduced by 75-90%. However, there was a significant increase in the number of roadkills in urban areas, resulting in the total number of mammal-vehicle and roe deer-vehicle collisions in 2020 and 2021 being higher than expected based on long-term trends. However, after restrictions were eased, collision numbers with wildlife on the main, national and regional roads increased again and became higher than expected.

Small Mammal Diversity Changes in a Baltic Country, 1975-2021: A Review.

The structure and diversity of small mammal (SM) communities over the long term may show the influences of climate change, landscape changes and local disturbances. We review published data regarding SM trapping and owl pellet analysis from Lithuania (the most southerly of the three Baltic States, Northern Europe), covering the period 1975-2021. Over decades, we analysed trends in the diversity of SM communities and the proportions of species and proportions of trophic groups. The large increase in granivores, from 6.9% in 1975-1980 to 45.4% in 2011-2020 and 54.7% in 2021, coincided with a decrease in omnivores and insectivores. The proportion of herbivores increased less notably. At the species level, significant decreases in the proportions of M. arvalis, C. glareolus and S. araneus were accompanied by notable increases in the proportions of A. flavicollis and A. agrarius, the latter from 1.0% in 1975-1980 to 25.3% in 2021. Concluding, two periods were identified, specifically before the 1990s and subsequently. In the second period, in the aftermath of land-use changes within the country in 1990, diversity increased, and dominance decreased, a situation that has not subsequently changed. Not excluding the concomitant effects of climate change, we relate these patterns to the alterations in habitat and anthropogenic impact.

Long-term changes in a small mammal community in a temperate zone meadow subject to seasonal floods and habitat transformation.

We tested small mammal (SM) community response to the influence of seasonal floods and habitat transformation in a temporal zone meadow (west Lithuania). The habitat transitioned from a natural flooded meadow in 2008-2012 to a mowed and pastured area in 2020. SM were trapped in late summer to autumn using lines of 25 traps (145 in total), the number of trapping sessions being one or several per year. We tested the hypotheses that: (1) any reaction to the multiple disturbances is species-specific, that is, the relative abundance of SM species and their proportion in the community differs with respect to the flood magnitude and habitat transformation, and (2) at the guild level, disturbances favor habitat generalists, thereby changing the characteristics of SM community. The average relative density of SM was 20.25 individuals per 100 trap nights (range 7.56-40.67), with 4-year-long cyclical changes of density observed. In separate years, we recorded from 4 to 9 species; Shannon's diversity index varied from 0.46 to 2.19, with expressed change of the dominant species. Habitat generalist species were favored, while habitat specialists were disadvantaged. As the meadow transformation progressed, unfavored states in the SM community prevailed, with excessive numbers of granivore and herbivore species present. Our study suggests that multiple disturbances may lead to an increase in relative abundance, species richness, and diversity within the SM assemblages.

Factors Affecting Roadkills of Semi-Aquatic Mammals.

We previously showed that registration efforts and traffic intensity explain 90% of variation in mammal roadkill numbers, 70% of variation in the numbers of recorded species and 40% of diversity variation. Here we analyze semiaquatic mammal roadkill in Lithuania in 2002-2021, relating these to the monitoring effort. From 39,936 analyzed roadkill, the European beaver (Castor fiber) was registered 60 times, American mink (Neovison vison) 26 times, otter (Lutra lutra) 22 times and muskrat (Ondatra zibethica) 3 times. The average roadkill indexes were 0.000065, 0.00076, 0.00061 and 0.00010 ind./km/day, and the extrapolated annual roadkill for the country was 44-357, 36-456, 49-464 and 89-144 individuals, respectively. Beaver roadkill numbers correlated with the registration effort and traffic intensity, otter roadkill with registration effort only and mink with hunting bag (number of hunted individuals per year). Roadkill was not always related to proximity to water, with 38-54% of roadkill occurring over 200 m from the nearest water source. With American mink and muskrat being invasive species in the EU and otter protected in many countries, it is valuable to enhance the registrations of their roadkill (using targeted efforts by drivers, hunters or other citizen scientists) to obtain the extrapolated amount of roadkill and to use this knowledge in species management.

The elemental composition of small mammals in a commercial orchard-meadow system.

We analyze concentrations of various elements in small mammals from commercial orchards and berry plantations and test differences between them depending on species, individual body mass, age, gender, type and age of crop and intensity of applied agricultural measures. Skinned front legs (muscle and bones) were used to register the presence and concentration of Ca, Cd, Mo, Ni, Se, Ag, As, Be, Bi, Co, Cr, Cu, Fe, Ga, Mg, Mn, Pb, Sr, U, V and Zn. The most abundant species were common vole (Microtus arvalis), yellow-necked (Apodemus flavicollis) and striped field (A. agrarius) mice. The maximum recorded concentrations exceeded the minimums by 1.7-7.7 times in Mg, Zn, Cu, Ga, Ni and Ca, and 11.0-23.2 times in Mn, Be, Mo, Co, Sr, V, Pb and As. The hypothesis that the use of fertilization and pesticides in commercial orchards should induce differences in the elemental concentrations between crop areas and control habitats was confirmed by the higher concentrations of Cu, Mn, Bi, Co, Cr, Fe, Ni, Sr and Pb in rodents from the crop areas. Spatially controlled generalized linear mixed model confirmed the cumulative influence of species and crop, explaining 30-80% of the distribution of Ca, Ni, Co, Cu, Ga, Mn, Pb and U. The effect of species and the age of the individual was significant for Ni, Co, Cu, Fe, Ga, Mn and Pb, while effect of gender was not expressed. Depletion of Cu in older individuals was found in all three species. With species as a grouping factor, the effect of crop type and the intensity of agricultural practices were significant factors in the accumulation of Ca, Ni, Co, Cu, Ga, Mn and Pb, while effect of crop age was not expressed. The obtained elemental concentrations in rodents indicated orchards to be cleaner than heavy polluted areas.

Cocirculation of Leptospira spp. and multiple orthohantaviruses in rodents, Lithuania, Northern Europe.

In Europe, zoonotic Leptospira spp. and orthohantaviruses are mainly associated with specific rodent hosts. These pathogens cause febrile human diseases with similar symptoms and disease progression. In Lithuania, the presence of Dobrava-Belgrade orthohantavirus (DOBV), Tula orthohantavirus (TULV) and Leptospira spp. in rodent reservoirs is still unknown, and Puumala orthohantavirus (PUUV) was detected in bank voles (Clethrionomys glareolus) at only one site. Therefore, we collected and screened 1617 rodents and insectivores from Lithuania for zoonotic (re-)emerging Leptospira and orthohantaviruses. We detected Leptospira DNA in six rodent species, namely striped field mouse (Apodemus agrarius), yellow-necked mouse (Apodemus flavicollis), bank vole, common vole (Microtus arvalis), field vole (Microtus agrestis) and root vole (Microtus oeconomus). Leptospira DNA was detected with an overall mean prevalence of 4.4% (range 3.7%-7.9% per rodent species). We detected DOBV RNA in 5.6% of the striped field mice, PUUV RNA in 1% of bank voles and TULV RNA in 4.6% of common voles, but no Leptospira DNA in shrews and no hantavirus-Leptospira coinfections in rodents. Based on the complete coding sequences of the three genome segments, two distant DOBV phylogenetic lineages in striped field mice, one PUUV strain in bank voles and two TULV strains in common voles were identified. The Leptospira prevalence for striped field mice and yellow-necked mice indicated a significant negative effect of the distance to water points. The detection of (re-)emerging human pathogenic Leptospira and three orthohantaviruses in rodent reservoirs in Lithuania calls for increased awareness of public health institutions and allows the improvement of molecular diagnostics for pathogen identification.

Roadkills as a Method to Monitor Raccoon Dog Populations.

The raccoon dog (Nyctereutes procyonoides) is one of the most frequently killed species on Lithuanian roads. As an invasive species, up-to-date knowledge of population size, trends and spatial distribution is critically important both for species assessment and for the planning of control measures. In Lithuania, however, raccoon dog surveys have not been carried out since 1997. We investigated, therefore, whether roadkill counts on predefined routes could be used as a proxy for a survey. Our dataset includes survey numbers for the period 1956-1997, hunting bag sizes for 1965-2020 (including the spatial distribution of the hunting bag in 2018-2020) and roadkill data relating to 1551 individuals between 2002-2020. At the most local scale, that of the hunting areas of hunting clubs, correlations between the numbers of hunted and roadkilled individuals were negative and insignificant or absent. At the country scale, however, we found significant correlation both between the numbers surveyed and hunted in 1965-1997 (r = 0.88), and between those hunted and the number of roadkills in 2002-2020 (r = 0.56-0.69). Therefore, we consider that roadkill counts on predefined and stable routes may be used as a proxy for a survey at the country scale. Practical implementation of the method is proposed.

Stable Isotopes Reveal the Dominant Species to Have the Widest Trophic Niche of Three Syntopic Microtus Voles.

Diets and trophic positions of co-occurring animals are fundamental issues in their ecology, and these issues in syntopic rodents have been studied insufficiently. Using carbon (δ13C) and nitrogen (δ15N) stable isotope ratios from hair samples, we analysed the trophic niches of common (Microtus arvalis), field (M. agrestis), and root (M. oeconomus) voles co-occurring in orchards, berry plantations, and nearby meadows (as control habitat to orchards and plantations). We tested if the niche of the dominant common vole was the widest, whether its width depended on the presence of other vole species, and whether there were intraspecific differences. Results suggest stability in the trophic niches of all three Microtus species, as season explained only 2% of the variance. The widest trophic niche was a characteristic of the dominant common vole, the range of δ13C values exceeding the other two species by 1.6, the range of δ15N values exceeding the other two species by 1.9, and the total area of niche exceeding that of the other voles by 2.3-3 times. In the meadows and apple orchards, co-occurring vole species were separated according to δ13C (highest values in the dominant common vole), but they maintained similar δ15N values. Results give new insights into the trophic ecology small herbivores, showing the impact of species co-occurrence.

Isotopic Niche of Syntopic Granivores in Commercial Orchards and Meadows.

In agricultural habitats, diets and trophic positions of syntopic granivorous small mammals are not known sufficiently. Agroecosystems may be quite complex isotopically and the most complex situation concerns the nitrogen-15 isotope as δ15N values are influenced by many internal and external fluxes. We analysed the isotopic niches of striped field (Apodemus agrarius), yellow-necked (Apodemus flavicollis), and harvest (Micromys minutus) mice living sympatrically and syntopically in apple and plum orchards, raspberry and currant plantations, and nearby meadows that were used as control habitats. Carbon (δ13C) and nitrogen (δ15N) stable isotope ratios from hair samples were used as a proxy for their diet. As the main hypothesis, we expected differences in the isotopic niches of these three species, minimising interspecific competition. All species were fully separated according to δ15N values, presuming different amounts of food of animal origin in their diet. The separation of species according to δ13C was not expressed in all habitats. The core dietary niches of these species were fully separated in the apple orchards and raspberry plantations. Intraspecific differences of the isotopic niche were not present in any of the three species: that is, resources were equally used by males and females, adults, subadults, and juveniles.

Identification of a novel hantavirus strain in the root vole (Microtus oeconomus) in Lithuania, Eastern Europe.

Hantaviruses are zoonotic pathogens that can cause subclinical to lethal infections in humans. In Europe, five orthohantaviruses are present in rodents: Myodes-associated Puumala orthohantavirus (PUUV), Microtus-associated Tula orthohantavirus, Traemmersee hantavirus (TRAV)/ Tatenale hantavirus (TATV)/ Kielder hantavirus, rat-borne Seoul orthohantavirus, and Apodemus-associated Dobrava-Belgrade orthohantavirus (DOBV). Human PUUV and DOBV infections were detected previously in Lithuania, but the presence of Microtus-associated hantaviruses is not known. For this study we screened 234 Microtus voles, including root voles (Microtus oeconomus), field voles (Microtus agrestis) and common voles (Microtus arvalis) from Lithuania for hantavirus infections. This initial screening was based on reverse transcription-polymerase chain reaction (RT-PCR) targeting the S segment and serological analysis. A novel hantavirus was detected in eight of 79 root voles tentatively named "Rusne virus" according to the capture location and complete genome sequences were determined. In the coding regions of all three genome segments, Rusne virus showed high sequence similarity to TRAV and TATV and clustered with Kielder hantavirus in phylogenetic analyses of partial S and L segment sequences. Pairwise evolutionary distance analysis confirmed Rusne virus as a strain of the species TRAV/TATV. Moreover, we synthesized the entire nucleocapsid (N) protein of Rusne virus in Saccharomyces cerevisiae. We observed cross-reactivity of antibodies raised against other hantaviruses, including PUUV, with this new N protein. ELISA investigation of all 234 voles detected Rusne virus-reactive antibodies exclusively in four of 79 root voles, all being also RNA positive, but not in any other vole species. In conclusion, the detection of Rusne virus RNA in multiple root voles at the same trapping site during three years and its absence in sympatric field voles suggests root voles as the reservoir host of this novel virus. Future investigations should evaluate host association of TRAV, TATV, Kielder virus and the novel Rusne virus and their evolutionary relationships.

On the Doorstep, Rodents in Homesteads and Kitchen Gardens.

Scarcely investigated in terms of small mammals, kitchen gardens and homesteads form a subset of environments. Using results of snap trapping, we present the first data on small mammal species diversity, gender and age structure, relative abundance, body fitness, and reproduction parameters in these commensal habitats (kitchen gardens, homestead gardens, houses, and outbuildings). We expected that (i) house mice should be the dominant species in buildings, while striped field mice should be dominant in gardens, (ii) body condition should be the highest in buildings, (iii) body condition should increase in the autumn, irrespective of the habitat, and (iv) breeding failures in the form of disrupted pregnancies should be observed. Not all of the predictions were confirmed. From the seven recorded species, gardens and outbuildings were dominated by yellow-necked mice, while bank voles dominated in buildings where food was available. The number of recorded species and diversity index increased during the autumn months. The body condition was highest in rodents that were trapped in gardens. It decreased towards winter, with the exception of the striped field mouse. Breeding disturbances were recorded in all of the most numerous species, comprising 16.7-100% of all observed pregnancies.

Habitat Suitability Based Models for Ungulate Roadkill Prognosis.

Roads do not only have a detrimental effect on nature (fragmenting habitats, isolating populations and threatening biodiversity), but the increasing numbers of wildlife-vehicle collisions are also a direct threat to humans and property. Therefore, mitigation measures should be placed with respect to animal distribution and movements across the roads. We simulated red deer, roe deer and wild boar movements in Lithuania, focusing on the two main highways A1 and A2. Using regional habitat suitability and linkage models, we calculated movement pathways and the most probable crossing zones in 2009. The prognostic value of these models was tested by comparing the pathway predictions to the real roadkill and roadkill cluster locations in 2002-2009 and 2010-2017. Across both periods and on both highways, the roe deer roadkill locations were significantly closer to the model-predicted pathways than to randomly selected points. The prediction of roadkill locations was also good for wild boar. The roe deer roadkill clusters and multi-species clusters were significantly better represented by the model than by random distribution. On both highways, the biggest differences in distance from the predicted locations were near big cities. We recommended wildlife movement models as an additional tool for planning wildlife-vehicle collision mitigation measures and we advise measures for increasing their predicting power.