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.

Interspecies transmission of antimicrobial-resistant bacteria between wild birds and mammals in urban environment.

The transmission of antibiotic-resistant bacteria among wild animal species may hold significant epidemiological implications. However, this issue is seldom explored due to the perceived complexity of these systems, which discourages experimental investigation. To address this knowledge gap, we chose a configuration of birds and mammals coexisting in an urban green area as a research model: the rook Corvus frugilegus and the striped field mouse Apodemus agrarius. The indirect transmission of antimicrobial-resistant bacteria between these species is possible because rodents inhabiting rook colonies frequently come into contact with the birds' faeces and pellets. The study was conducted in two cities in eastern Poland (Central Europe) - Lublin and Chelm. Among 71 Escherichia (E.) coli isolates studied, 19.7% showed resistance to from one to six of the antibiotics tested, with much higher prevalence of antibiotic-resistant bacteria in the birds (32%) than in the rodents (7%). Whole genome sequencing was performed on 10 selected E. coli isolates representing similar resistance phenotypes. The following antimicrobial resistance genes were detected: blaTEM-1b, tet(A), tet(B), aph(6)-Id, aph(3'')-Ib, aadA1, aadA2, catA1, floR, cmlA, sul2, sul3, dfrA14, and dfrA2. Birds from the same city and also from both neighbouring cities shared E. coli bacteria with the same sequence types, whereas isolates detected in birds were not found to have been transferred to the mammalian population, despite close contact. This demonstrates that even intensive exposure to sources of these pathogens does not necessarily lead to effective transmission of antibiotic-resistant E. coli strains between birds and mammals. Further efforts should be dedicated to investigating actual transmission of antimicrobial-resistant bacteria in various ecological systems, including those that are crucial for public health, such as urban environments. This will facilitate the development of more accurate models for epidemiological threats and the formulation of well-balanced decisions regarding the coexistence of humans and urban wildlife.

Concentration of metals and metalloids in livers of birds of various foraging guilds collected during the autumn migration period in Poland.

During migration, birds explore various habitats at stopover sites that differ in food resources and contamination levels. In this study, hepatic concentrations of 21 elements (metals and metalloids) in 11 species of birds, representing various foraging habitats (such as aquatic, aquatic/terrestrial, and terrestrial) and migration modes (migratory and sedentary) representing various foraging guilds (omnivores, piscivores, and molluscivores), were analyzed. The samples (N = 84) were collected during the autumn migration period in Poland. The concentrations of elements determined in this study exhibited high inter-species variability, reflecting the diversity in contamination levels depending on food resources used by specific bird groups. Many of the investigated individuals from different species showed exceeded levels of subclinical toxicity and moderate clinical poisoning due to Cd and Hg. Higher concentrations of As, Hg, and Ba and lower V concentrations were found in migratory birds as compared to sedentary birds. Species foraging in terrestrial habitat had different concentrations of some elements compared to aquatic and aquatic/terrestrial species. Some specific inter-species differences in hepatic elemental concentrations were found. Differences in elemental concentrations among various groups can primarily be attributed to their foraging guilds, with certain elements, particularly As, V, and Hg, playing a significant role in the dissimilarity of elemental concentrations between foraging habitat groups and migratory mode groups. The data collected confirmed the limited ability of As to enter ecosystem pathways. The results of this study contribute to understanding the year-round exposure of migratory birds to environmental contamination, which can have carry-over effects on their performance in wintering and breeding grounds.

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.

Honey analysis in terms of nicotine, patulin and other mycotoxins contamination by UHPLC-ESI-MS/MS - method development and validation.

Fifty-seven samples of honey of different types and origins were screened for nicotine and nine mycotoxins (aflatoxin B1, aflatoxin B2, fusarenon X, ochratoxin A, penicillic acid, zearalenone, sterigmatocystin, gliotoxin, and patulin). The sample set consisted of monofloral, multifloral, nectar, honeydrew, cream, and artificial honey originating mainly from Poland. The physicochemical characterization of honey was performed by determining colour (by Pfund method), water content (by refractometry), total phenolics and flavonoids content (by spectrophotometry). For nicotine and mycotoxins determination a QuEChERS-based UHPLC-ESI-MS/MS method was developed and validated. Analyses were carried out in alkaline conditions to ensure patulin-methanol adduct formation and facilitate this mycotoxin detection. About 33% of tested honey samples were contaminated by nicotine or/and mycotoxins. However, the presence of mycotoxins was not related to herein evaluated physicochemical parameters of honey samples.

Comorbidities and concentration of trace elements in livers of European bison from Bieszczady Mountains (Poland).

European bison is a species for which health monitoring is essential in conservation activities. So far, little research has been carried out on the concentration of elements in this species. Most previous studies did not associate the concentration of elements with susceptibility to diseases. In this study we investigate the relationship between comorbidities in European bison and concentrations of a wide spectrum of elements in the liver. Samples were collected during the monitoring of the European bison population in Bieszczady (southeast Poland) over the 2020-2022 period. Each individual was also visually inspected by a veterinarian in the field for the presence of lesions as a part of a post-mortem examination. The animals were divided into 3 groups: group A-one type of clinical sign; group B-two types of clinical signs; group C-three or more types of clinical signs. The ICP-OES method was applied to assess the concentration of 40 elements in livers. Discriminant analysis showed clear differences between the mineral status of individuals in the groups with one, two, and at least three types of clinical signs. Detailed analysis of selected elements showed that, in the case of eight elements, there was a relationship with age, sex, or comorbidities. Cu, Se, and Zn showed significant differences in relation to comorbidities, but only Cu concentration was lower when the frequency of lesions was higher. We concluded that in research on the mineral status of the population, apart from the availability of trace elements in the environment, the health condition of the studied individuals should also be considered. However, inferring the mineral status of the population on the basis of randomly obtained samples from dead individuals may give an incomplete view of the population, especially in the case of species susceptible to diseases, such as European bison.

Detection of spatial avoidance between sousliks and moles by combining field observations, remote sensing and deep learning techniques.

Nowadays, remote sensing is being increasingly applied in ecology and conservation, and even underground animals can successfully be studied if they leave clear signs of their presence in the environment. In this work, by combining a field study, analysis of high-resolution aerial images, and machine learning techniques, we investigated the interspecies relationships of two small burrowing mammals: the spotted souslik Spermophilus suslicus and the European mole Talpa europaea. The study was conducted for 3 years (2018-2020) at a 105-ha grass airfield where both species coexist (Poland). Both field studies and the analysis of aerial imagery showed that, in the period of low population numbers, the souslik avoided coexistence with the European mole, and the presence of the mole was found to reduce the area of the habitat suitable for the souslik. The presence of other burrowing species may be an important element in the habitat selectivity of the souslik, but this has not yet been included in the conservation guidelines for this species. We discuss the contribution of our results to the knowledge of the ecology of burrowing mammals and their interspecies relationships. We also assess the possibility of using remote sensing and deep learning methods in ecology and conservation of small burrowing mammals.

Genetic Structure of the Root Vole Microtus oeconomus: Resistance of the Habitat Specialist to the Natural Fragmentation of Preferred Moist Habitats.

Climate-related changes have a severe impact on wetland ecosystems and pose a serious challenge for wetland-dependent animals as their preferred habitats decline, lose spatial continuity, and appear as isolated islands in the landscape. In this paper, we studied the effects of long-term habitat changes (drying out and fragmentation of wet non-forest habitats) on the genetic structure of the population of the root vole Microtus oeconomus, a species preferring moist habitats. We intended to check what barriers and what distances affected its genetic isolation on a local scale. The study was conducted in the area of Kampinoski National Park in central Poland (Europe). DNA variability of 218 root vole individuals was assessed by genotyping nine microsatellite loci. Despite its spatial fragmentation, the studied population did not seem to be highly structured, and isolation through distance was the main differentiating factor. Even a distance of several kilometres of unfavourable natural habitats and unfavourable terrain did not exclude the exchange of genes between subpopulations. Our results suggest that the genetic effects of the fragmentation of wetlands have been considerably compensated (delayed) due to the migratory abilities of this species. Our study does not provide clear results on the impact of anthropogenic barriers but suggests that such barriers may have a much stronger effect than natural barriers.

Description of the Nymphal Stages of Hoplopleura affinis (Anoplura: Hoplopleuridae) from the Striped Field Mouse Apodemus agrarius with a Global Checklist of Hoplopleura Species from the Genus Apodemus.

The genus Hoplopleura is the most speciose genus of sucking lice observed parasitizing rodents and lagomorphs (pikas). Despite the fact that the majority of Anoplura are believed to be monoxenic, many species within Hoplopleura may be oligoxenic. In addition, the occurrence of more than one parasite species per host species on individuals has been confirmed. As such, a precise species identification of the parasite, especially of the nymphs, is of high significance. The study is based on the material of 245 sucking louse specimens taken from 179 individuals of the striped field mouse Apodemus agrarius collected between 2008 and 2017. The study employs scanning microscopy to provide superior quality resolution of the studied traits. The study presents the first record of the characters of the nymphal stages of H. affinis, one of the common Eurasian species of the genus. Additional aspects of the biology and the host-parasite relationship of H. affinis are presented, e.g., female, male and nymphs of lice, showing different preferences in the choice of location (topography) on the host body. In addition, a global checklist has been made of all the species of Hoplopleura found parasitizing rodents of the genus Apodemus. Generally, the ranges of the occurrence of lice of this genus coincide with the geographic distribution of typical hosts, although this has not always been confirmed by local studies.

Stress hormone level and the welfare of captive European bison (Bison bonasus): the effects of visitor pressure and the social structure of herds.

BACKGROUND: Captive European bison (Bison bonasus) play an active role in conservation measures for this species; this includes education, which may conflict with these animals' welfare. The effect of the presence of visitors on the welfare of captive animals can be negative, positive or neutral. However, the response of a given species to visitors is difficult to predict, since even closely related species display varying levels of tolerance to captivity. The aim of the study was to compare immunoreactive fecal cortisol levels (regarded as an indicator of the level of physiological stress) in groups of captive European bison that differed in terms of their social structure and the level of visitor pressure. The second aim was to determine if there was a correlation between intestinal parasitic burden and immunoreactive fecal cortisol levels. RESULTS: Immunoreactive fecal cortisol levels were not influenced by sex or age. However, study site and the interaction between study site and visitor pressure were statistically significant. European bison in one enclosure presented higher levels of immunoreactive fecal cortisol on weekdays than at weekends. In the other two study sites, the levels did not differ between weekdays and weekends. No correlation was found between parasitological infestation and immunoreactive fecal cortisol levels. CONCLUSIONS: Measurement of fecal cortisol metabolites could be a valuable method for further research into the welfare of European bison in captivity. More subtle factors such as individual animal characteristics, feeding systems, and the arrangement of enclosures can be of great importance in terms of the effect of visitors on animals. The results of this study can be used in guidelines for the management of European bison populations.

How Is Wildlife Affected by the COVID-19 Pandemic? Lockdown Effect on the Road Mortality of Hedgehogs.

Globally, wildlife is affected by unprecedented changes related to the COVID-19 pandemic. In this paper, the lockdown effect on the traffic-related mortality in hedgehogs in an urban area was studied. Comparing the pre-pandemic (2018 and 2019) and pandemic (2020) years, we showed that hedgehog roadkill levels during the lockdown period were over 50% lower (which means a decrease greater than the decrease in road traffic in the same period measured by the number of accidents or the average number of vehicles per day). Based on literature data, we showed that this may mean at least tens of thousands of hedgehogs have survived on a national scale. We report the need to start intensive research on the possible demographic and genetic effects of this unique phenomenon. We also ask how stable the effect of the COVID-19 pandemic will be on wildlife and whether the lockdown (which is an anthropause) may reverse the negative trends in the decline in the number of wild species, including hedgehogs.

Human-Wildlife Conflict: The Human Dimension of European Bison Conservation in the Bieszczady Mountains (Poland).

An important limitation for the population growth of European bison in the Bieszczady Mountains may be the level of social acceptance. The study aimed to compare attitudes to European bison of local village inhabitants in Bieszczady and city dwellers in Rzeszów. We also investigated whether damage caused by European bison or other wild species changes peoples' perceptions of this animal. Our study showed that not only does the growing European bison population cause an increase in negative attitudes among local village communities, but this species also causes more conflict than any other herbivore in the Bieszczady Mountains. Village residents believed that the main threats that arise from European bison were the damage they cause and forest use limitations. The current compensation system for the damage caused by this species does not solve the problem because over 60% of damage is not effectively reported to the state administration. The city dwellers of Rzeszów displayed a different attitude towards the European bison. We concluded that while educational workshops for local villagers may alleviate conflict in the short-term, ultimately it is only by restricting the growth of the European bison population that a long-term effect will be achieved.

Essential differences in the mineral status of free-ranging European bison Bison bonasus populations in Poland: The effect of the anthroposphere and lithosphere.

Tracking the concentration of elements in the organs of endangered animal species can provide significant knowledge about environmental hazards and allows more targeted protective measures to be taken. In this paper the differences in concentrations of 27 elements in the livers of free-ranging European bison were studied. The ICP-OES method was applied to study the four main Polish populations, which live in lowland (Bialowieska, Knyszynska and Borecka forests) and mountain (Bieszczady) habitats. The study was based on a unique and relatively large amount of comparative material. It was found that European bison populations that live in different locations differ from each other in terms of the hepatic concentrations of 17 of the elements studied in this work. The mountain population in Bieszczady was most distinctive due to the higher concentrations of Ba, Ca, Cd, Se, and the lower concentrations of Mo and V (compared to the other populations). Additionally, even geographically close lowland populations sometimes have different hepatic concentrations of particular elements. The sex and age of individuals have a weak effect on mineral status. Although the lithosphere is the primary factor that affects the availability of elements for the European bison, the soil parameters and biosphere can significantly affect the availability of elements, e.g. by accumulation. It was also shown that the anthroposphere plays a role, but this is mainly through agriculture as feeding on arable fields significantly changes the mineral status of European bison. The need for further research, especially on Cd, Se and Cu, was indicated. It was concluded that toxic elements or deficiencies in essential elements in the populations of the studied species should be considered locally as this would allow better understanding of the health condition of individual populations.

Serine Protease Inhibitors-New Molecules for Modification of Polymeric Biomaterials.

Three serine protease inhibitors (AEBSF, soy inhibitor, α1-antitrypsin) were covalently immobilized on the surface of three polymer prostheses with the optimized method. The immobilization efficiency ranged from 11 to 51%, depending on the chosen inhibitor and biomaterial. The highest activity for all inhibitors was observed in the case of immobilization on the surface of the polyester Uni-Graft prosthesis, and the preparations obtained showed high stability in the environment with different pH and temperature values. Modification of the Uni-Graft prosthesis surface with the synthetic AEBSF inhibitor and human α1-antitrypsin inhibited the adhesion and multiplication of Staphylococcus aureus subs. aureus ATCC® 25923TM and Candida albicans from the collection of the Department of Genetics and Microbiology, UMCS. Optical profilometry analysis indicated that, after the immobilization process on the surface of AEBSF-modified Uni-Graft prostheses, there were more structures with a high number of protrusions, while the introduction of modifications with a protein inhibitor led to the smoothing of their surface.

Pesticides and conservation of large ungulates: Health risk to European bison from plant protection products as a result of crop depredation.

The coexistence of large mammals and humans in the contemporary landscape is a big challenge for conservationists. Wild ungulates that forage on arable fields are exposed to the negative effects of pesticides, and this problem also applies to protected species for which intoxication by pesticides may pose a health risk and directly affect the effectiveness of conservation efforts. In this paper we assessed the threat posed by pesticides to the European bison Bison bonasus, a species successfully restituted after being extinct in the wild. We studied samples of B. bonasus liver from three free-living populations in Poland (Bialowieska, Knyszynska, and Borecka forests) and captive individuals from breeding centres. LC-QTOF-MS/MS two-step analysis for the detection, identification and confirmation of pesticide residues in liver samples, which included MS and targeted MS/MS scans, was conducted. It was found that European bison are exposed to pesticides as a result of crop depredation: the presence of tetraconazole, fluopyram and diazinon residues in 12 liver samples was confirmed. The concentration levels of the detected substances were quite low, but in the liver samples more than one substance was usually found, and the potential health risk to European bison may result from the synergistic interaction of these substances. The place of occurrence of the population, abundance, and the management regime affect the exposure of European bison to pesticides. Due to the high conservation status of the European bison, the monitoring of intoxication by pesticides should be included in the conservation plans of this species. This issue should also be more widely included in the study of other wild ungulates because knowledge about the impact of pesticides on wildlife is still insufficient.

Properties of Artificial Phospholipid Membranes Containing Lauryl Gallate or Cholesterol.

Lauryl gallate (LG) is an antioxidant agent. However, it exhibits poor solubility in water. Its interactions with the membrane result in structure evolution thus affecting the membrane functionality. In this paper the Brewster angle microscope coupled with the Langmuir trough was applied to determine the morphology, phase behaviour, thickness and miscibility of ternary Langmuir monolayers with equal mole fractions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and an increasing mole fraction of LG. The results were discussed as regards analogous systems where cholesterol (Chol) was the third component. Moreover, the phosphatidylcholine-lauryl gallate (PC-LG) interactions were monitored by the attenuated total reflectance Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry. Besides lipid composition, the addition of LG was found to be a significant factor to modulate the model membrane properties. The LG molecules adjust themselves to the PC monolayer structure. The hydrophobic fragment is dipped into the membrane interior while the hydroxyl groups of phenolic gallate moiety associate with the polar groups of PC mainly through hydrogen bonding inducing the compacting effect. LG is found to be deeply submerged within DOPC, closer to the double bonds, and its insertion practically does not affect the DPPC/DOPC membrane fluidity. This is crucial for getting more profound insight into the role of LG in stabilizing the non-raft domains, mostly exposed to oxidation in which LG can co-localize and serve its antioxidant function.

Do terrestrial animals avoid areas close to turbines in functioning wind farms in agricultural landscapes?

Most studies on the effects of wind energy on animals have focused on avian and bat activity, habitat use, and mortality, whereas very few have been published on terrestrial, non-volant wildlife. In this paper, we studied the utilization of functioning wind farm areas by four terrestrial animals common to agricultural landscapes: European roe deer, European hare, red fox, and the common pheasant. Firstly, we expected that the studied animals do not avoid areas close to turbines and utilize the whole area of functioning wind farms with a frequency similar to the control areas. Secondly, we expected that there is no relation between the turbine proximity and the number of tracks of these animals. The study was conducted over two winter seasons using the snow-tracking method along 100 m linear transects. In total, 583 transects were recorded. Wind farm operations may affect terrestrial animals both in wind farm interiors and in a 700-m buffer zone around the edge of turbines. The reactions of animals were species specific. Herbivorous mammals (roe deer and European hare) avoided wind farm interiors and proximity to turbines. The common pheasant showed a positive reaction to wind turbine proximity. The red fox had the most neutral response to wind turbines. Although this species visited wind farm interiors less often than the control area, there was no relation between fox track density and turbine proximity. Greater weight should be given to the effects of wind farms on non-flying wildlife than at present. Investors and regulatory authorities should always consider the likely impacts of wind farms during environmental impact assessments and try to reduce these negative effects.

An assessment of non-volant terrestrial vertebrates response to wind farms--a study of small mammals.

The majority of studies on the effects of wind energy development on wildlife have been focused on birds and bats, whereas knowledge of the response of terrestrial, non-flying vertebrates is very scarce. In this paper, the impact of three functioning wind farms on terrestrial small mammal communities (rodents and shrews) and the population parameters of the most abundant species were studied. The study was carried out in southeastern Poland within the foothills of the Outer Western Carpathians. Small mammals were captured at 12 sites around wind turbines and at 12 control sites. In total, from 1200 trap-days, 885 individuals of 14 studied mammal species were captured. There was no difference in the characteristics of communities of small mammals near wind turbines and within control sites; i.e. these types of sites were inhabited by a similar number of species of similar abundance, similar species composition, species diversity (H' index) and species evenness (J') (Pielou's index). For the two species with the highest proportion in the communities (Apodemus agrarius and Microtus arvalis), the parameters of their populations (mean body mass, sex ratio, the proportion of adult individuals and the proportion of reproductive female) were analysed. In both species, none of the analysed parameters differed significantly between sites in the vicinity of turbines and control sites. For future studies on the impact of wind turbines on small terrestrial mammals in different geographical areas and different species communities, we recommend the method of paired 'turbine-control sites' as appropriate for animal species with pronounced fluctuations in population numbers.