Pathogens detected in ticks (Ixodes ricinus) feeding on red squirrels (Sciurus vulgaris) from city parks in Warsaw.

The European red squirrel (Sciurus vulgaris) is a common host for Ixodes ricinus ticks in urban and rural habitats, however, studies on ticks and tick-borne pathogens (TBPs) of squirrels have not been conducted in Poland yet. Thus, the aims of the current study were to assess and compare the prevalence and abundance of ticks on red squirrels trapped at two sites in the Warsaw area (in an urban forest reserve and an urban park) and using molecular tools, to assess the genetic diversity of three pathogens (Borrelia burgdorferi sensu lato, Rickettsia and Babesia spp.) in I. ricinus ticks collected from squirrels. For the detection of Rickettsia spp. a 750 bp long fragment of the citrate synthase gltA gene was amplified; for B. burgdorferi s.l. 132f/905r and 220f/824r primers were used to amplify the bacterial flaB gene fragments (774 and 605 bp, respectively) and for Babesia spp., a 550 bpfragment of 18S rRNA gene was amplified. In total, 91 red squirrels were examined for ticks. There were differences in tick prevalence and mean abundance of infestation in squirrels from the urban forest reserve and urban park. Three species of B. burgdorferi s.l., Rickettsia spp., and Babesia microti were detected in ticks removed from the squirrels. Our results broaden knowledge of S. vulgaris as an important host for immature I. ricinus stages and support the hypothesis that red squirrels act as a reservoir of B. burgdorferi. Moreover, we conclude that red squirrels may also play a role in facilitating the circulation of other pathogens causing serious risk of tick-borne diseases in natural and urban areas.

Long-term, medium-term and acute stress response of urban populations of Eurasian red squirrels affected by different levels of human disturbance.

Animals in urban areas often encounter novel and potentially stressful conditions. It is important to understand how wildlife cope with anthropogenic disturbance. To investigate this specific adaptation we live-trapped squirrels in two study sites in Warsaw: a forest reserve and an urban park and we estimated stress responses at three levels: long-term and medium-term stress (the level of stress hormones, i.e. cortisol and cortisone concentrations, in hair and feces) and acute reaction to human-induced stress (measured during handling with the aid of the three indices: breath rate, struggle rate, and vocalization). According to GLMM models no difference in the stress hormones level was found between the two populations. The only differences in cortisol concentrations clearly depended on the season, i.e. being higher in autumn and winter comparying to other seasons. There was no influence of sex, or reproductive status on stress hormones. Forest squirrels had significantly higher breath rates, suggesting they were more stressed by handling. There was no difference in the struggle rate between study areas, this index was mostly affected by season (i.e. being highest in winter). First-trapped squirrels vocalized less than during the subsequent trappings. Assumingly, during the first, and more stressful trapping, squirrels used 'freezing' and/or little vocalization, while during next captures they used alarm calls to warn conspecifics. Overall, we showed that the two squirrel populations differed only in terms of their breath rate. This suggests that they did not differ in medium-term and long-term stress in general, but they can differ in acute response to handling. This also suggests that both populations were similarly affected by environmental factors. The lack of clear effects may also be due to population heterogeneity. Thus, in order to assess the effects of anthropogenic stressors a broader range of indicators and diverse analytical methods, including behavioral analyses, should be employed.

A validated LC-MS/MS method for simultaneous determination of key glucocorticoids in animal hair for applications in conservation biology.

A new method for the determination of main glucocorticoids (cortisol, cortisone, and corticosterone) in hair by liquid chromatography-tandem mass spectrometry was developed. Glucocorticoids were extracted from hair shafts using methanol followed by solid-phase extraction. A validation test was performed using hair from three species of wild mammals with different body size (0.2-800 kg), lifestyle (terrestrial, burrowing and arboreal species), social organization (living in herds or solitary), and different predicted type of hair glucocorticoids: European bison (Bison bonasus), European hamster (Cricetus cricetus), and Eurasian red squirrel (Sciurus vulgaris). Regardless of the species evaluated, the method shows good linearity for all analytes accompanied by satisfactory accuracy (91-114%) and precision (RSD < 13%). Depending on the analyte and hair origin, the calculated limits of quantification were between 0.05 and 1.19 ng/mL, which corresponds to 1.28-31.51 pg/mg. Using cortisol and cortisone as examples, we have demonstrated that measuring multiple glucocorticoids simultaneously provides more comprehensive information than solely concentrating on one, thereby contributing to a more balanced and reliable interpretation of the acquired results. However, the utility of cortisol metabolites as markers of stress response in keratinized tissues should be substantiated by additional experimental studies on targeted animals. We posit that this paper could serve as a crucial catalyst to prompt such experiments.

Body Condition and Breeding of Urban Red Squirrels: Comparison of Two Populations Affected by Different Levels of Urbanization.

The red squirrel is among the mammals that have adjusted well to urban habitats. Here, we focused on the two populations inhabiting Warsaw: in a park (with year-round supplemental feeding) and in an urban forest. We hypothesised that park squirrels would have higher body mass (and better body condition), being more stable over the year, and would have a higher breeding rate (i.e., the share of breeding females). Contrary to our hypothesis, forest squirrels were heavier and had better body condition than park squirrels. The body masses of squirrels from both areas were quite stable (with the highest values obtained in spring). Females in better body conditions were more likely to breed. More breeding females and sub-adults were trapped in the park. Regardless of the study site, the highest share of breeding females was in spring, but they also bred in winter and in the remaining seasons. The lower body mass/condition of park squirrels may be possibly explained by high intraspecific competition, or by stable food (and thermal as typical for the city) conditions, in which accumulating fat was not crucial. Mild winter conditions may have also enabled squirrels to breed early. This study showed the high plasticity of red squirrels living in human-transformed habitats.

Alien or Native? How to Distinguish Feces of Fallow and Roe Deer Using Central Poland as a Case Study.

The method of pellet group count is commonly used for estimating population trends of ungulates; however, in the case of species of similar body size, the misidentification rate can be high. Our aim was to find a metrical threshold between pellet groups of roe deer (native species) and fallow deer (alien species) to be applied during fieldwork. The study was conducted in spring 2020 and 2021 in central Poland (lowlands) in areas were only roe or fallow deer occurred. We measured the number of feces in the group, the length and width of five randomly selected feces from each pellet group and the length/width ratio. Roe deer pellets were shorter, narrower and less elongated than those of fallow deer; yet, length was found to be the best discriminant. The most accurate threshold was 1.2 cm, i.e., 12-15% of pellets were over/below this value. The mean number of pellets in a group was lower for roe deer (39.6, SE = 1.6) than for fallow deer (64.5, SE = 1.5). A value of 50-52 pellets best differentiated between the two species. To conclude, combining these two measurements could be an objective method to distinguish between pellet groups of the two species.

Colonization of Warsaw by the red fox Vulpes vulpes in the years 1976-2019.

The red fox is one of the most adaptable carnivores inhabiting cities. The aim of our study was to describe the process of Warsaw colonization by the red fox. We focused on: (1) the fox distribution in Warsaw on the basis of presence-absence data (2005-2012) over a grid of 1 × 1 km2, (2) the process of settlement in 29 green areas (study periods 1976-1978, 2004-2012, and 2016-2019) in relation to habitat type, and (3) temporal and spatial patterns of the red fox incidents (1998-2015) reported by Warsaw citizens. We found out that: (1) the red fox penetrated the whole city (i.e. its presence was confirmed in all squares of the grid), (2) 21% of the green areas were colonized in 1976-1978 but 93% in 2016-2019. Forests and riparian habitats were occupied more frequently than parks and cemeteries in 1976-1978 with no difference in the further years; (3) the probability of the fox incidents increased over years, was higher in June-October, on working days, and around noon, and with the share of discontinuous urban fabric in the buffers around incident locations. Nevertheless, the incidents only partially reflect population abundance trends and activity patterns of the species, so should be treated cautiously.

Food Niche Overlap of Avian Predators (Falconiformes, Strigiformes) in a Field and Forest Mosaic in Central Poland.

Food niche overlap statistics are a common way to show competition for food resources in a group of animals. Niche breadths of various species are very variable and their diet composition changes reflecting prey availability. The aim of this study was to evidence the food niche overlap of the whole assemblage of avian predators (eight raptor and owl species, some of them reaching very high densities) in a field and forest mosaic of central Poland. The diet composition was assessed on the basis of pellet analyses and the identification of prey remains found under the nests in the breeding period. The extent of the niche overlap was calculated using a Pianka formula. The food niche overlap indices ranged from 0.02 to 0.93 (mostly below 0.5). The most separate food niche was that of the white-tailed eagle, who regularly preyed on fish. The highest niche overlap was recorded for the common buzzard and common kestrel, two species preying on field rodents, switching to soricomorphs when the former were scarce. Our results confirmed that the food niches of species coexisting in the same area were considerably separate, which is a result of preying on various prey species or searching for them in different habitats.

Eurasian Beaver (Castor fiber) Winter Foraging Preferences in Northern Poland-The Role of Woody Vegetation Composition and Anthropopression Level.

We studied beavers' dietary preferences and the role of several factors (such as plant species, size and anthropopression level) that affect the beavers' foraging in northern Poland. Woody plants along the river were measured and classified according to species in six 100 m-long transects that were characterized by a diversified human disturbance level. Ivlev's electivity index was used to present the beavers' preferences for various plant species and sizes, and the generalized linear model was used to assess the significance of studied factors in beavers' browsing choices. Most popular in the beavers' diets were willows (Salix), maples (Acer) and alder (Alnus), but only willows and maples were preferred. We noted a decrease in the beavers' foraging preference in parallel to an increase in the shoot diameter; plants with a diameter below 10 cm were preferred. All factors included in the generalized linear model (GLM) were significant in shaping the beavers' foraging choices. A negative correlation between the shoot diameter and the human disturbance level was found, but the species composition of the browsed woody plants was the same in each transect. Beavers' foraging preferences, as observed in our study, were similar to those described in the literature and confirmed the role of woody species and their diameters in shaping the beavers' diet. We also suggested the potential role of anthropopression in the shaping of the beavers' foraging behaviors.

The choice and handling of supplemental food by red squirrels in an urban park.

One of the main factors that allow the red squirrel (Sciurus vulgaris) to live in cities may be the availability of supplemental food provided by people. In this article, preferences of red squirrels in relation to supplemental food, how it is handled, and whether change seasonally, is investigated. A cafeteria test was performed, in which squirrels could choose one from six kind of nuts. Three types of nuts were used: walnuts, hazelnuts and peanuts, with unshelled and shelled nuts of each offered. In most cases squirrels took an offered nut. If a nut was not taken, it was mostly due to lack of interest, without signs of fear. Regardless of season, whole, unshelled walnuts were selected most frequently - the most high-energy and least perishable from offered food. The least frequently selected overall were peanuts. Caching behaviour changed seasonally - nuts were mostly eaten in spring and hoarded in autumn and it took less time to cache nuts in autumn and winter. It may reflect optimal caching strategy at the time when food is abundant and there is a desire to secure as big a store of food as possible. Results from the study suggest that supplemental food is an important component of squirrels' diet and have an impact on their caching strategies.

Pigeon and Poultry Breeders, Friends or Enemies of the Northern Goshawk Accipiter gentilis? A Long-Term Study of a Population in Central Poland.

In this study, we focused on a goshawk population in central Poland (study area 105 km²,forests 24 km², seven small forest complexes) which was monitored long-term (with high densitiesrecorded in the 1980s of 16.3 pairs/100 km² despite persecution by farmers) to analyse howenvironmental factors (prey availability and changes in the forest structure) influenced populationabundance, breeding parameters, and diet composition. The study was undertaken from 2011-2018,and the results were compared with published data from two previous study periods (1982-1992and 2001-2003). The number of breeding pairs dropped from 17.1 to 8.0; the breeding success wasaround 75% in all study periods. The selection of nesting trees followed the changes in stand speciesand age structure. More nesting attempts per one nest were recorded in the current time period (1.7 vs.1.1), which probably reflected lower anthropopressure (i.e., no cases of persecution were recordedin this study). Diet composition seemed to follow changes in the prey availability: The share ofdomestic pigeons and poultry (the main prey in the 1980s) as well as small game dropped, whilethe share of Eurasian jay and wood pigeon increased. Our studies suggested that anthropogenicfood (poultry and domestic pigeons) played a key role for the goshawk population in thetransformed habitats of the field and forest mosaic.

Indirect Influence of African Swine Fever Outbreak on the Raven (Corvus corax) Population.

Carrion plays a crucial role in the raven's diet. In the past, domestic pig carrion was widely available in Poland. This changed with an African swine fever (ASF) outbreak and the introduction of strict procedures aimed at stopping the virus from spreading. We compared data from Central Poland (field and forest mosaic, study area of 105 km²) for two periods, i.e., before (2011⁻2014) and after the ASF outbreak (2015⁻2018). In breeding seasons, nests of ravens were found, juveniles were counted, and the time when juveniles left their nests was recorded. Diet composition data were based on pellet analysis and direct observations of feeding birds. The number of breeding pairs dropped from 12.3 to 7.5 in the second period. Breeding parameters were similar. However, birds in the second period had fewer fledglings per successful pair. Domestic pig carrion was found to be an important food item, and with its limited supply, ravens changed their diet, i.e., they fed on the carrion of dogs and cats or preyed on small vertebrates more often. Overall, our study points to a crucial role of the availability of the carrion of big farm animals (i.e., domestic pig) in maintaining the high density of breeding raven populations.