Nest Box Bacterial Loads Are Affected by Cavity Use by Secondary Hole Nesters.

Among the environmental factors that affect bird nesting in nest boxes, the influence of microbial communities is relatively poorly understood. In this study, nest boxes used for breeding by secondary cavity nesters were sampled before the start of the breeding season to assess the bacterial loads of the nest box in relation to their previous year status. Different parts of the wooden nest box offer variable conditions for the development of bacteria. During the breeding season, the nest box entrance hole is wiped out by birds, delivering bacteria to their bodies, but during winter, it is exposed to unfavourable external conditions. The interior of the nest box, in turn, is also wiped by birds, but the conditions during winter are more stable there. Therefore, samples from the entrance hole and the interior of the nest box were taken at two different study sites: an urban parkland and a natural forest. We predicted that both the occupancy of the nest boxes during the previous breeding season by birds and the nesting sites would influence the bacterial load of the nest box. To verify this prediction, two categories of nest boxes were sampled at both study sites: nest boxes occupied by any of the two tit species (Great Tit or Blue Tit) in the previous season for breeding and nest boxes that had remained empty that year. The interior bacterial load of the nest box was higher in the nest boxes occupied in the previous breeding season, but only in the forest area. Furthermore, the bacterial load of both the entrance hole of the nest box and the interior was significantly higher in the forest study area in both occupied and unoccupied nest boxes. Our results show that the bacterial load of the nest box is positively related to the presence of nests in the previous breeding season and can vary between different sites.

Microsatellite Instability Assay as a Potential Approach to Evaluate Genotoxicity: Lead Exposure in a Nestling Passerine Bird at the Stage of Intensive Erythropoiesis.

Although many avian studies have investigated the toxic effects of lead on important biochemical and physiological processes, organ and system function, and behavior, studies evaluating the specific genotoxic effects of exposure to lead are scarce. Nowadays, rapid technological advances can provide new molecular techniques in this regard. In this study, as a novel approach in bird studies, we used a panel of ten microsatellite loci to investigate the microsatellite instability (MSI) in response to experimental lead intoxication in a common hole-nesting species, the great tit Parus major. For this purpose, an experiment based on an intentional single supplementation of a lead (II) acetate trihydrate compound was conducted, with the use of two different doses, applied to randomly chosen great tit nestlings from randomly selected broods, being at the stage of intensive erythropoiesis. Although this preliminary study did not find any MSI in the seven microsatellite markers retained for the final comparison, it contributes to the examination of this molecular technique in field conditions as being potentially applicable in ecotoxicological bird studies. We believe that certain issues should be considered in finding an explanation for our result. First, the single doses of lead used in this study may have been too weak to induce genetic instability. Second, the panel of microsatellite markers studied may have been unsusceptible to lead genotoxicity in general. Third, the relatively short time interval (5 days) between the experimental procedure (lead exposure) and the sampling of post-exposure material (blood) for genetic analyses could have limited the effect of lead genotoxicity. Further analyzes are needed to verify these findings and to evaluate the scope of application of the MSI analysis in wild bird population studies.

Phylogeny of Shrew- and Mole-Borne Hantaviruses in Poland and Ukraine.

Earlier, we demonstrated the co-circulation of genetically distinct non-rodent-borne hantaviruses, including Boginia virus (BOGV) in the Eurasian water shrew (Neomys fodiens), Seewis virus (SWSV) in the Eurasian common shrew (Sorex araneus) and Nova virus (NVAV) in the European mole (Talpa europaea), in central Poland. To further investigate the phylogeny of hantaviruses harbored by soricid and talpid reservoir hosts, we analyzed RNAlater®-preserved lung tissues from 320 shrews and 26 moles, both captured during 1990-2017 across Poland, and 10 European moles from Ukraine for hantavirus RNA through RT-PCR and DNA sequencing. SWSV and Altai virus (ALTV) were detected in Sorex araneus and Sorex minutus in Boginia and the Bialowieza Forest, respectively, and NVAV was detected in Talpa europaea in Huta Dlutowska, Poland, and in Lviv, Ukraine. Phylogenetic analyses using maximum-likelihood and Bayesian methods showed geography-specific lineages of SWSV in Poland and elsewhere in Eurasia and of NVAV in Poland and Ukraine. The ATLV strain in Sorex minutus from the Bialowieza Forest on the Polish-Belarusian border was distantly related to the ATLV strain previously reported in Sorex minutus from Chmiel in southeastern Poland. Overall, the gene phylogenies found support long-standing host-specific adaptation.

Ketone body levels in wintering great tits Parus major in sites differing in artificial food availability.

Ketone body levels, among other biochemical blood indices, are important indicators of the physiological condition of birds. Plasma ketone as an indicator of fasting and lipid use is strongly linked to demanding phases in the avian life cycle, such as migration or wintering. The main goal of this study was to check whether ketone body levels differ between the habitats in which individuals stay in winter. To test the above prediction, we used a portable point-of-care device to measure ketone body levels in wintering great tits (Parus major). We assumed that wintering in distinct habitats that differ structurally, particularly with respect to food availability, would affect the metabolic performance of birds and their physiological condition. Individual great tits were trapped in mist nets and blood-sampled in three distinct locations within the city: an urban parkland, the deciduous forest and a city centre. As expected, we showed that the mean ketone level was significantly higher in the area where artificial feeding was irregular than in two areas of regular feeding, indicating the more intense fasting state there. We also checked if the level of ketone bodies differs with respect to the sex of an individual, but we found no such difference.

Effects of ambient temperature during the nestling stage on a stress indicator in nestling pied flycatchers Ficedula hypoleuca.

Long-term and short-term changes in ambient temperature can cause stress in birds, leading to changes in the level of hematological parameters. The H:L ratio (heterophil-to-lymphocyte ratio) is a hematological index that allows for the assessment of the stress induced by environmental changes, including weather conditions. In this paper, we examined the influence of temperatures and the sum of precipitation on the health of nestling pied flycatchers (Ficedula hypoleuca) by using the H:L ratio reflecting the body's response to stress. All examined temperature indicators influenced the H:L ratio, yet the average value of daily minimum temperature during the first 12 days of nestling life had the strongest influence, maximum temperature had the weakest effect, while precipitation had no significant influence. Our research indicates that even a small increase in temperature caused a stress reaction in nestling pied flycatchers, which was reflected by an increase in the H:L ratio. The increase in the stress index (H:L ratio) was probably a result of poor weather conditions (precipitation, low temperature), which prevented the adult birds from actively foraging and properly feeding the nestlings.

Genetic structure of urban and non-urban populations differs between two common parid species.

Landscape conversions induced by human activities can affect dispersal patterns of various bird species and, as a result, affect genetic structure of their populations. Genetic differentiation of bird populations may be enhanced by habitat variation, especially in urban-non-urban systems. The majority of population genetic studies focus on single species, which inflicts limitations for direct comparisons of genetic responses of avian populations to urbanization. Here, we used a set of microsatellite markers to examine genetic diversity, gene flow and population structure in two common parid species, great tits Parus major and blue tits Cyanistes caeruleus occupying three sites in habitats with contrasting urbanization level in central Poland. We found low but significant divergence of urban park population with both suburban and non-urban forest great tit populations, while no differentiation was found between suburban forest and non-urban forest populations. In contrast, no evidence for genetic differentiation was found between blue tit populations from the urban park, suburban forest and non-urban forest sites. We conclude that great tits and blue tits respond to urbanization-related changes in a different way, which may be a result of different rates of migration and/or dispersal, likely higher in blue tits. Some impact may be also induced by interspecific competition. We suggest that changing the focus of urban genetic research from single to multiple species may provide novel insights into how natural populations respond to the processes of urbanization.

Extreme temperature drop alters hatching delay, reproductive success, and physiological condition in great tits.

It has been suggested that extreme weather events may be treated as natural experiments that may unravel the mechanisms by which birds adjust their phenology and breeding parameters to environmental variability. In 2017, a sudden and heavy drop of temperatures for several days affected many European bird populations. This event occurred during the laying-early incubation period in the great tit (Parus major) population in central Poland, causing a large delay in hatching and had sustained reproductive consequences. This cold snap occurring once breeding activity had already started in 2017 was followed by the warm and invariable breeding season of 2018. This natural experiment had an essential influence on great tit reproductive parameters. We found a significant difference in hatching date, number of fledglings, hatching success, and fledging success between 2017 and 2018. In 2017, there were about two fledglings per nest fewer than in 2018. Fledging success was positively associated with hatching delay in 2017, while the relation was negative in 2018. Hatching success differed significantly between both years, being higher in 2018. Mean level of hemoglobin (used as index of body, physiological condition) in the blood of nestling great tits was higher in 2017 than in 2018. We argue that the moment of hatching may be (at least in some years) more tightly related to the moment of maximum food demand of tit nestlings than the traditionally used laying date. Also in extreme years, phenotypic plasticity of hatching delays may be insufficient to adjust the timing of breeding to the upcoming extreme weather events. Further examining its limits may be an important goal for future research.

Effects of experimental lead exposure on physiological indices of nestling great tits Parus major: haematocrit and heterophile-to-lymphocyte ratio.

Lead (Pb) is recognized as one of the most toxic trace elements that can induce a wide range of negative health effects in wildlife. Because the investigation of basic environment-sensitive physiological indicators is easy to perform in wild birds, they have been considered as valuable bioindicators of lead contamination. The harmfulness of lead poisoning can depend on the type of exposure, and in most studies of birds, an effect of chronic lead exposition has been considered. In this study, we investigated whether a single exposure to specific doses of lead affected such physiological indices as haematocrit and the heterophil-to-lymphocyte ratio (H/L ratio). For this purpose, we conducted an experiment based on intentional lead supplementation, with the use of two different doses, applied to randomly chosen great tit (Parus major) nestlings from randomly selected broods. A few days after the exposure (when the nestlings were 15 days old), we determined haematocrit and the H/L ratio as potentially sensitive physiological indicators of lead intoxication. We found that the treatment with lead resulted in a significant decline in haematocrit level. In addition, we found that the age when lead exposure occurred can be considered as an important factor influencing haematocrit variation. A decrease in haematocrit was observed on consecutive days of nestling life. In contrast, the H/L ratio did not show any significant response to lead exposure. These results clearly show that the assessment of haematocrit level for nestling great tits can provide a simple and rapid method of indirect evaluation of physiological effects of lead intoxication caused by a single exposure.

Physiological condition of nestling great tits (Parus major) declines with the date of brood initiation: a long term study of first clutches.

In seasonal environments, a temporal decline in breeding performance (e.g. clutch size, nestling condition, and fledging success) of altricial bird species is a well-known phenomenon. In this study, we present the effect of laying phenology on the physiological condition of nestling great tits (Parus major) in 14 consecutive breeding seasons. We used blood haemoglobin and baseline glucose concentrations as indicators of nestling physiological condition. Nestling blood haemoglobin reflects food base quality and availability during the breeding period. Blood glucose concentration can be used as a supplementary reverse index of condition, since it is negatively related to environmental quality. It might be indicative of the stress caused by unfavourable extrinsic factors, though, due to potential confounding factors such as adverse weather conditions, low food quality, or feeding interruptions, glucose levels should be used in this ecological context with caution. Great tit nestlings from earlier broods were characterised by higher mean haemoglobin concentrations, indicating a seasonal decline in food quality and availability. The blood glucose concentration displayed an opposite pattern, with nestlings from earlier broods being characterised by lower mean concentrations than those from later broods. However, very little of the variation in blood glucose concentration can be explained by the variation in laying date, which suggests that blood glucose concentration is of little importance in the context of breeding phenology. Our results show that the physiological condition of nestlings of this species decreases as the breeding season progresses, most probably due to environmental factors.

Physiological condition of nestling great tits Parus major in response to experimental reduction in nest micro- and macro-parasites.

Most passerines use nests as the exclusive place to lay and incubate eggs and bring nestlings up to fledging. Nests of secondary cavity nesters, like tits, provide a moist, warm and protected habitat for reproduction of blood parasites. Offspring fitness depends on interactions between parental care and environmental constraints. Life-history theory suggests that macro- and micro-parasites may generate selection pressures by affecting host health. In the present study, we replaced natural great tit Parus major nests in two, structurally and floristically contrasting sites (an urban parkland and a rich deciduous forest, located 10 km apart in Lódz, central Poland), with fresh, sterilized, artificial moss-cotton wool nests, twice, on the fifth and tenth day of nestlings life. We then examined haematological condition indicators (haemoglobin and glucose concentrations) of about 14-day-old nestlings. Nestlings that were developing in treated nests improved their health status in comparison with control nestlings. The mean haemoglobin and glucose concentrations (treated and control) also varied between both study areas. Our study confirms that the level of haemoglobin and especially the level of glucose may be treated as reliable indicator of environmental characteristics in great tits.

A consistent long-lasting pattern of spatial variation in egg size and shape in blue tits (Cyanistes caeruleus).

BACKGROUND: Interspecies variation in avian egg shape and size is understandable in terms of adaptation, allometry and phylogeny. Within-species variation in egg properties influences offspring fitness and can be explained by differences in allocation of resources into reproductive components of life history in mulidimensionally variable environments. Egg size is inherently traded-off with clutch size, which may also be true of egg shape in some cases. We investigated long-term variation in egg shape and size between two geographically close populations of blue tits Cyanistes caeruleus in relation to clutch size and habitat differences. RESULTS: The main finding is that there exists a persistent long-lasting pattern of spatial variation of egg size and shape between the two study populations of blue tits, 10 km apart, controlling for clutch size. Eggs in the urban park site were on average larger in volume and less spherical in shape than eggs in the forest site over 12 years of this study. Egg sizes were negatively associated with clutch sizes. Egg shape was not correlated with clutch size. CONCLUSIONS: Our findings suggest that the pattern of variation in egg size and shape results from different trophic richness of the breeding habitats of the study populations, demanding different allocation of resources and, especially, from the contrasting difference in the availability of calcium.

Hatching delays in great tits and blue tits in response to an extreme cold spell: a long-term study.

Variation in ambient temperature affects various life stages of organisms. It has been suggested that climate change not only implies higher global temperatures but also more unpredictable weather and more frequent extreme weather events. Temperature has a major influence on the optimal laying-incubation-hatching dates of insectivorous passerines, because it poses energetic constraints and affects the timing of food abundance. We have been studying breeding characteristics of great tits Parus major and blue tits Cyanistes caeruleus in two areas, an urban parkland and a deciduous forest, around the city of Lódz since 2002. During the egg-laying period in 2017, both tit species at both study areas faced an unusual cold spell as reflected by a sudden decrease in the mean ambient temperature to ca. 2-3 °C for about 5 days, which caused mean hatching delays of up to 6 days. Since flexibility of behavior plays a major role in adjusting to unpredictable weather conditions, examining its limits may be an important goal for future research.

Larvae of Hydropsyche angustipennis (Trichoptera, Hydropsychidae) as indicators of stream contamination by heavy metals in Lódz agglomeration.

A biomonitoring technique was employed to assess metal contamination of several degraded urban streams in the Lódz agglomeration. Presence of heavy metals (Zn, Cu, Fe, Cd, Pb, Cr, was determined in whole bodies of Hydropsyche angustipennis larvae. Larvae of H. angustipennis were the only caddisflies living in most of  investigated streams. These metals as well as other environmental variables were investigated in sediments or free waters of urban streams and in a stretch of a similar size of the Grabia River (reference site), outside the city but still in the vicinity of the Lódz agglomeration. The results of our research demonstrated different levels of contamination of urban streams as well as a correlation between environmental and larval tissue concentrations of heavy metals. Significant differences among sites were observed for metal concentrations in H. angustipennis larval bodies and larval responses to metals were time specific. The highest concentration of heavy metals was observed during the spring months. Differences were evident among various heavy metal levels in the bodies of larvae collected from different streams and at different sampling stations in the same stream.

Effects of extreme thermal conditions on plasticity in breeding phenology and double-broodedness of Great Tits and Blue Tits in central Poland in 2013 and 2014.

Many avian species in Europe breed earlier as a result of higher temperatures caused by global climate changes. Climate change means not only higher temperatures but also more frequent extreme weather events, sometimes contrasting with the long-term trends. It was suggested that we should look closely at every extreme phenomenon and its consequences for the phenology of organisms. Examining the limits of phenotypic plasticity may be an important goal for future research. Extremely low spring temperatures in 2013 (coldest spring in 40 years) resulted in birds laying unusually late, and it was followed in 2014 by the earliest breeding season on record (warmest spring in 40 years). Here, we present results concerning breeding phenology and double-broodedness in the Great Tit (Parus major) and the Blue Tit (Cyanistes caeruleus) in 2013 and 2014 in an urban parkland and a deciduous forest in central Poland. Great Tits started laying eggs 18.2 days later in 2013 than in 2014 in the parkland, whereas the analogous difference was 21.1 days in the forest. Blue Tits started laying eggs in the parkland 18.5 days later in 2013 than in 2014, while the analogous difference was 21.6 days in the forest. The difference in the proportion of second clutches in Great Tits between 2013 (fewer second clutches) and 2014 (more second clutches) was highly significant in the parkland and in the forest. This rather large extent of breeding plasticity has developed in reaction to challenges of irregular inter-annual variability of climatic conditions. Such a buffer of plasticity may be sufficient for Blue Tits and Great Tits to adjust the timing of breeding to the upcoming climate changes.

Isolation and partial characterization of a highly divergent lineage of hantavirus from the European mole (Talpa europaea).

Genetically distinct hantaviruses have been identified in five species of fossorial moles (order Eulipotyphla, family Talpidae) from Eurasia and North America. Here, we report the isolation and partial characterization of a highly divergent hantavirus, named Nova virus (NVAV), from lung tissue of a European mole (Talpa europaea), captured in central Poland in August 2013. Typical hantavirus-like particles, measuring 80-120 nm in diameter, were found in NVAV-infected Vero E6 cells by transmission electron microscopy. Whole-genome sequences of the isolate, designated NVAV strain Te34, were identical to that amplified from the original lung tissue, and phylogenetic analysis of the full-length L, M and S segments, using maximum-likelihood and Bayesian methods, showed that NVAV was most closely related to hantaviruses harbored by insectivorous bats, consistent with an ancient evolutionary origin. Infant Swiss Webster mice, inoculated with NVAV by the intraperitoneal route, developed weight loss and hyperactivity, beginning at 16 days, followed by hind-limb paralysis and death. High NVAV RNA copies were detected in lung, liver, kidney, spleen and brain by quantitative real-time RT-PCR. Neuropathological examination showed astrocytic and microglial activation and neuronal loss. The first mole-borne hantavirus isolate will facilitate long-overdue studies on its infectivity and pathogenic potential in humans.

Whole-Genome Sequence of a Novel Hantavirus Isolated from the European Mole (Talpa europaea).

The complete genome sequence of Nova virus, a novel hantavirus isolated from a European mole (Talpa europaea) captured in central Poland, was determined. The availability of this sequence will facilitate the search for other mole-borne hantaviruses and will accelerate the acquisition of new knowledge about their phylogeography and evolutionary origin.

Assessment of water quality in urban streams based on larvae of Hydropsyche angustipennis (Insecta, Trichoptera).

Hydropsyche angustipennis (Insecta, Trichoptera) larvae were used as indicators of stream contamination in the city of Lódz, Poland. The larvae of H. angustipennis were present at 9 sampling sites established for this study. Significant differences between the sampling sites were noted for environmental parameters as well as concentration of chemicals in water and biodiversity of aquatic invertebrates. Statistical analyses showed significant correlations between quantity and quality of water pollutants and density of H. angustipennis larvae, concentration of metals in larval bodies, and the appearance of morphological anomalies in tracheal gills and anal papillae. In comparison to literature data, the level of contaminants in water, including heavy metals, for each of the studied streams of Lódz was surprisingly low while concentration of these metals in the whole bodies of H. angustipennis larvae was very high. Some of the heavy metals present in the water might be identified only after conducting analyses of their concentration in the larval bodies. Therefore, long life cycle of H. angustipennis and heavy metal tolerance with a possibility of their accumulation in the larval bodies may constitute a support to traditional chemical assessment of water quality or traditional biomonitoring.

Long-term variation in hemoglobin concentration in nestling great tits Parus major.

Several studies have previously proposed that blood hemoglobin concentration in nestling passerines is a reliable index of individual condition and nutritional state. In this paper we present results concerning variation in hemoglobin concentration in the blood of ca. 14-day-old nestling great tits Parus major in central Poland in an 11-year-long period, 2003-2013, in two distinct habitat types: urban park and deciduous forest. The most important findings of the study were: (i) variation in hemoglobin concentration was consistent within broods, (ii) hemoglobin concentration of nestlings varied markedly across years, (iii) hemoglobin concentration was significantly higher in the forest study site which is richer in terms of food abundance during the short period of tits breeding season and (iv) high hemoglobin level was a predictor of nestling survival from hatching to fledging.

Co-circulation of soricid- and talpid-borne hantaviruses in Poland.

Previously, we reported the discovery of a genetically distinct hantavirus, designated Boginia virus (BOGV), in the Eurasian water shrew (Neomys fodiens), as well as the detection of Seewis virus (SWSV) in the Eurasian common shrew (Sorex araneus), in central Poland. In this expanded study of 133 shrews and 69 moles captured during 2010-2013 in central and southeastern Poland, we demonstrate the co-circulation of BOGV in the Eurasian water shrew and SWSV in the Eurasian common shrew, Eurasian pygmy shrew (Sorex minutus) and Mediterranean water shrew (Neomys anomalus). In addition, we found high prevalence of Nova virus (NVAV) infection in the European mole (Talpa europaea), with evidence of NVAV RNA in heart, lung, liver, kidney, spleen and intestine. The nucleotide and amino acid sequence variation of the L segment among the SWSV strains was 0-18.8% and 0-5.4%, respectively. And for the 38 NVAV strains from European moles captured in Huta Dlutowska, the L-segment genetic similarity ranged from 94.1%-100% at the nucleotide level and 96.3%-100% at the amino acid level. Phylogenetic analyses showed geographic-specific lineages of SWSV and NVAV in Poland, not unlike that of rodent-borne hantaviruses, suggesting long-standing host-specific adaptation. The co-circulation and distribution of BOGV, SWSV and NVAV in Poland parallels findings of multiple hantavirus species co-existing in their respective rodent reservoir species elsewhere in Europe. Also, the detection of SWSV in three syntopic shrew species resembles spill over events observed among some rodent-borne hantaviruses.

Spatial and temporal variation of lead, cadmium, and zinc in feathers of great tit and blue tit nestlings in Central Poland.

In this study, we examined heavy-metal concentrations in feathers of nestling great tits Parus major and blue tits Cyanistes caeruleus at two different sites (urban parkland vs. deciduous forest) located in the Lódz agglomeration in relation to interyear variation. We found that tit species did not differ significantly in lead and cadmium concentrations. Zinc concentration was significantly higher in blue tits. We also found that lead and cadmium levels in blue tit nestlings and the level of lead in great tit nestlings were higher in the parkland site than in the woodland site. We explain habitat variation in heavy-metal concentrations in feathers of nestlings by different levels of contamination at study sites. For both tit species, significant variation in heavy-metal amounts accumulated by nestlings was found between years with the lowest value in a year with the lowest value of rainfall. We suggest that the interyear variation may be accounted for by differences in rainfall, thus influencing quantities of trace elements bioavailable in the environment.