Virulence factors of Salmonella spp. isolated from free-living grass snakes Natrix natrix.

Salmonellosis associated with reptiles is a well-researched topic, particularly in China and the United States, but it occurs less frequently in Europe. The growth of the human population and changes in the environment could potentially increase the interaction between humans and free-living reptiles, which are an unidentified source of Salmonella species. In this study, we sought to explore this issue by comparing the microbiota of free-living European grass snakes, scientifically known as Natrix natrix, with that of captive banded water snakes, or Nerodia fasciata. We were able to isolate 27 strains of Salmonella species from cloacal swabs of 59 N. natrix and 3 strains from 10 N. fasciata. Our findings revealed that free-living snakes can carry strains of Salmonella species that are resistant to normal human serum (NHS). In contrast, all the Salmonella species strains isolated from N. fasciata were sensitive to the action of the NHS, further supporting our findings. We identified two serovars from N. natrix: Salmonella enterica subspecies diarizonae and S. enterica subspecies houtenae. Additionally, we identified three different virulotypes (VT) with invA, sipB, prgH, orgA, tolC, iroN, sitC, sifA, sopB, spiA, cdtB and msgA genes, and ß-galactosidase synthesised by 23 serovars. The identification of Salmonella species in terms of their VT is a relatively unknown aspect of their pathology. This can be specific to the serovar and pathovar and could be a result of adaptation to a new host or environment.

The great tit HapMap project: A continental-scale analysis of genomic variation in a songbird.

A major aim of evolutionary biology is to understand why patterns of genomic diversity vary within taxa and space. Large-scale genomic studies of widespread species are useful for studying how environment and demography shape patterns of genomic divergence. Here, we describe one of the most geographically comprehensive surveys of genomic variation in a wild vertebrate to date; the great tit (Parus major) HapMap project. We screened ca 500,000 SNP markers across 647 individuals from 29 populations, spanning ~30 degrees of latitude and 40 degrees of longitude - almost the entire geographical range of the European subspecies. Genome-wide variation was consistent with a recent colonisation across Europe from a South-East European refugium, with bottlenecks and reduced genetic diversity in island populations. Differentiation across the genome was highly heterogeneous, with clear 'islands of differentiation', even among populations with very low levels of genome-wide differentiation. Low local recombination rates were a strong predictor of high local genomic differentiation (FST), especially in island and peripheral mainland populations, suggesting that the interplay between genetic drift and recombination causes highly heterogeneous differentiation landscapes. We also detected genomic outlier regions that were confined to one or more peripheral great tit populations, probably as a result of recent directional selection at the species' range edges. Haplotype-based measures of selection were related to recombination rate, albeit less strongly, and highlighted population-specific sweeps that likely resulted from positive selection. Our study highlights how comprehensive screens of genomic variation in wild organisms can provide unique insights into spatio-temporal evolutionary dynamics.

Measurements of body temperature and oxidative stress reveal differential costs associated with humoral immune function in a passerine bird.

Eco-immunology considers resistance to antigens a costly trait for an organism, but actual quantification of such costs is not straightforward. Costs of the immune response are visible in impaired coloration and reduced growth or reproductive success. Activation of the humoral immune response is a slow, complex and long-lasting process, which makes the quantification of its energetic cost a potential losing game. We implemented near-continuous measurements of body temperature in zebra finches (Taeniopygia guttata) as a proxy for the energetic cost, with a particular focus during activation of the humoral immune response until the peak of antibody release several days later. At the peak of the antibody release we additionally measured oxygen consumption (open-flow respirometry) and markers of oxidative stress (dROMs, OXY). Birds with an activated immune response maintained a higher night-time body temperature during the first 4 nights after an immune challenge in comparison to controls, implying increased night-time energy use. At peak antibody production, we did not find differences in night-time body temperature and oxygen consumption but observed differentiated results for oxygen consumption during the day. Immune-challenged females had significantly higher oxygen consumption compared with other groups. Moreover, we found that activation of the humoral immune response increases oxidative damage, a potential cost of maintaining the higher night-time body temperature that is crucial at the early stage of the immune response. The costs generated by the immune system appear to consist of two components - energetic and non-energetic - and these appear to be separated in time.

The interactive effect of ambient temperature and brood size manipulation on nestling body mass in blue tits: an exploratory analysis of a long-term study.

BACKGROUND: Relatively few studies have examined the interactive effects of ecological factors on physiological responses in wild animals. Nearly all of them have been short-term investigations that did not include experimental manipulations, limiting our ability to understand how climate change will affect natural populations. Using a 10-year brood size manipulation experiment in wild blue tits (Cyanistes caeruleus), we quantified the impact of weather conditions and brood competition on the body mass and structural size (tarsus length) of nestlings just prior to leaving the nest. RESULTS: We found that variation in nestling body mass on day 14 after hatching was explained by an interactive effect between average ambient temperature experienced during nestling period and brood size treatment. Specifically, in control broods nestling body mass was correlated with temperature in a non-linear manner (concave) with the vertex point (maximum body mass) at ca. 13 °C. In contrast, in enlarged broods nestling body mass permanently increased (also non-linearly) as temperature advanced. CONCLUSIONS: Our results highlight the importance of considering the effects of brood rearing conditions alongside other environmental factors experienced during growth while investigating early-life environmental effects on body condition.

Differential effects of steroid hormones on levels of broad-sense heritability in a wild bird: possible mechanism of environment × genetic variance interaction?

Genetic variation is one of the key concepts in evolutionary biology and an important prerequisite of evolutionary change. However, we know very little about processes that modulate its levels in wild populations. In particular, we still are to understand why genetic variances often depend on environmental conditions. One of possible environment-sensitive modulators of observed levels of genetic variance are maternal effects. In this study we attempt to experimentally test the hypothesis that maternally transmitted agents (e.g. hormones) may influence the expression of genetic variance in quantitative traits in the offspring. We manipulated the levels of steroid hormones (testosterone and corticosterone) in eggs laid by blue tits in a wild population. Our experimental setup allowed for full crossing of genetic and rearing effects with the experimental manipulation. We observed that birds treated with corticosterone exhibited a significant decrease in broad-sense genetic variance of tarsus length, and an increase in this component in body mass on the 2nd day post-hatching. Our study indicates, that maternally transmitted substances such as hormones may have measurable impact on the levels of genetic variance and hence, on the evolutionary potential of quantitative traits.

Sexual dichromatism, size dimorphism, and microscale anatomy of white wing stripe in blue tits.

Achromatic patches are a common element of plumage patterns in many bird species and there is growing body of evidence that in many avian taxa they can play a signaling role in mate choice. Although the blue tit Cyanistes caeruleus is a well-established model species in the studies on coloration, its white wing patch has never been examined in the context of sex-specific trait expression. In this exploratory study, we examined sexual size dimorphism and dichromatism of greater covert's dots creating white wing patch and analyzed its correlations with current body condition and crown coloration-a trait with established role in sexual selection. Further, we qualitatively analyzed microstructural barb morphology underlying covert's coloration. We found significant sexual dimorphism in the dot size independent of covert size and sexual dichromatism in both white dot and blue outer covert's vane spectral characteristics. Internal structure of covert barbs within the white dot was similar to the one found in barbs from the blue part that is, with a medullary area consisting of dead keratinocytes containing channel-type ß-keratin spongy nanostructure and centrally located air cavities. However, it lacked melanosomes which was the main observed difference. Importantly, UV chroma of covert's blue vane was positively correlated with crown UV chroma and current condition (the latter only in males), which should be a premise for further research on the signal function of the wing stripe.

Connecting the data landscape of long-term ecological studies: The SPI-Birds data hub.

The integration and synthesis of the data in different areas of science is drastically slowed and hindered by a lack of standards and networking programmes. Long-term studies of individually marked animals are not an exception. These studies are especially important as instrumental for understanding evolutionary and ecological processes in the wild. Furthermore, their number and global distribution provides a unique opportunity to assess the generality of patterns and to address broad-scale global issues (e.g. climate change). To solve data integration issues and enable a new scale of ecological and evolutionary research based on long-term studies of birds, we have created the SPI-Birds Network and Database (www.spibirds.org)-a large-scale initiative that connects data from, and researchers working on, studies of wild populations of individually recognizable (usually ringed) birds. Within year and a half since the establishment, SPI-Birds has recruited over 120 members, and currently hosts data on almost 1.5 million individual birds collected in 80 populations over 2,000 cumulative years, and counting. SPI-Birds acts as a data hub and a catalogue of studied populations. It prevents data loss, secures easy data finding, use and integration and thus facilitates collaboration and synthesis. We provide community-derived data and meta-data standards and improve data integrity guided by the principles of Findable, Accessible, Interoperable and Reusable (FAIR), and aligned with the existing metadata languages (e.g. ecological meta-data language). The encouraging community involvement stems from SPI-Bird's decentralized approach: research groups retain full control over data use and their way of data management, while SPI-Birds creates tailored pipelines to convert each unique data format into a standard format. We outline the lessons learned, so that other communities (e.g. those working on other taxa) can adapt our successful model. Creating community-specific hubs (such as ours, COMADRE for animal demography, etc.) will aid much-needed large-scale ecological data integration.

Differential effects of early growth conditions on colour-producing nanostructures revealed through small angle X-ray scattering and electron microscopy.

The costs associated with the production and maintenance of colour patches is thought to maintain their honesty. Although considerable research on sexual selection has focused on structurally coloured plumage ornaments, the proximate mechanisms of their potential condition dependence, and thus their honesty, is rarely addressed, particularly in an experimental context. Blue tit (Cyanistes caeruleus) nestlings have ultraviolet (UV)-blue structurally coloured tail feathers, providing a unique opportunity for investigation of the causes of variation in their colour. Here, we examined the influence of early growing conditions on the reflectance and structural properties of UV-blue-coloured tail feathers of blue tit nestlings. We applied a two-stage brood size manipulation to determine which stage of development more strongly impacts the quality of tail feather colouration and microstructure. We used small-angle X-ray scattering (SAXS) and electron microscopy to characterise the nanoscale and microscale structure of tail feather barbs. Nestlings from the broods enlarged at a later stage of growth showed a sex-specific rectrix development delay, with males being more sensitive to this manipulation. Contrary to predictions, treatment affected neither the quality of the barbs' nanostructures nor the brightness and UV chroma of feathers. However, at the microscale, barbs' keratin characteristics were impaired in late-enlarged broods. Our results suggest that nanostructure quality, which determines the UV-blue colour in tail feathers, is not sensitive to early rearing conditions. Furthermore, availability of resources during feather growth seems to impact the quality of feather microstructure more than body condition, which is likely to be determined at an earlier stage of nestling growth.

Cloacal Gram-Negative Microbiota in Free-Living Grass Snake Natrix natrix from Poland.

Reptiles appear to be an important vector for Gram-negative pathogens, therefore, they are epidemiologically relevant. However, the composition of reptilian microbiota has been poorly recognized so far. The majority of studies concern exotic reptiles as asymptomatic carriers of Salmonella serovars. Studies of other intestinal bacteria of reptiles are rare. Only recently, the microbiota of free-living European reptiles have been investigated, however, on the basis of small samples, mainly in protected areas. Here, we aim to investigate cloacal Gram-negative microbiota of free-living Natrix natrix. Snakes (N = 45) used in the study were collected in Kraków (Poland) and its vicinity. Nineteen species of Gram-negative bacteria were isolated. The most common species were: Aeromonas hydrophila, Morganella morganii, Proteus vulgaris, Salmonella spp. The bacteria prevalent in N. natrix cloacal swabs are likely to represent the natural intestinal Gram-negative microbiota of the examined snakes. Importantly, the identified bacteria are pathogenic to humans, which clearly highlights the epidemiological potential of free-living N. natrix. The risk of infection is high for immunocompromised humans, children (under 5 years old), elderly persons, and pregnant women. Our study provides the largest dataset on intestinal Gram-negative microbiota of wild snakes. The presence of multiple human pathogens determined by us calls for the necessity of further studies on reptile-transmitted bacteria in anthropogenic environments.

Cellular Turnover: A Potential Metabolic Rate-Driven Mechanism to Mitigate Accumulation of DNA Damage.

Oxidative stress, the imbalance of reactive oxygen species and antioxidant capacity, may cause damage to biomolecules pivotal for cellular processes (e.g., DNA). This may impair physiological performance and, therefore, drive life-history variation and aging rate. Because aerobic metabolism is supposed to be the main source of such oxidative risk, the rate of oxygen consumption should be positively associated with the level of damage and/or antioxidants. Empirical support for such relationships remains unclear, and recent considerations suggest even a negative relationship between metabolic rate and oxidative stress. We investigated the relationship between standard metabolic rate (SMR), antioxidants, and damage in blood plasma and erythrocytes for 35 grass snakes (Natrix natrix). Reactive oxygen metabolites (dROMs) and nonenzymatic antioxidants were assessed in plasma, while two measures of DNA damage and the capacity to neutralize H2O2 were measured in erythrocytes. Plasma antioxidants showed no correlation to SMR, and the level of dROMs was positively related to SMR. A negative relationship between antioxidant capacity and SMR was found in erythrocytes, but no association of SMR with either measure of DNA damage was detected. No increase in DNA damage, despite lower antioxidant capacity at high SMR, indicates an upregulation in other defense mechanisms (e.g., damage repair and/or removal). Indeed, we observed a higher frequency of immature red blood cells in individuals with higher SMR, which indicates that highly metabolic individuals had increased erythrocyte turnover, a mechanism of damage removal. Such DNA protection through upregulated cellular turnover might explain the negligible senescence observed in some ectotherm taxa.

Parental genetic similarity and offspring performance in blue tits in relation to brood size manipulation.

In birds, as in many other taxa, higher genetic similarity of mates has long been known to reduce offspring fitness. To date, the majority of avian studies have focused on examination whether the genetic similarity of social mates predicts hatching success. Yet, increased genetic similarity of mates may also reduce offspring fitness during later life stages, including the nestling period and beyond. Here, we investigated whether parental genetic similarity influences offspring performance using data from free-living blue tits (Cyanistes caeruleus) collected across three breeding seasons. Additionally, we tested whether brood size manipulation affects the magnitude and direction of the relationship between genetic similarity of mates and offspring performance. Sixteen microsatellite markers were used to measure genetic similarity between biological parents. We found that the genetic similarity of parents negatively affects offspring immune response and this effect was independent of the experimental brood size manipulation.

More than just the numbers-contrasting response of snake erythrocytes to thermal acclimation.

Acclimation to lower temperatures decreases energy expenditure in ectotherms but increases oxygen consumption in most endotherms, when dropped below thermoneutrality. Such differences should be met by adjustments in oxygen transport through blood. Changes in hematological variables in correspondence to that in metabolic rates are, however, not fully understood, particularly in non-avian reptiles. We investigated the effect of thermal acclimation on a snake model, the grass snakes (Natrix natrix). After 6 months of acclimation to either 18 °C or 32 °C hematocrit, hemoglobin concentration, erythrocyte number, and size were assessed. All variables revealed significantly lower values under warm compared to cold ambient temperature. Our data suggest that non-avian reptiles, similarly as birds, reduce erythrocyte fraction under energy-demanding temperatures. Due to low deformability of nucleated erythrocytes in sauropsids, such reduced fraction may be important in decreasing blood viscosity to optimize blood flow. Novel findings on flexible erythrocyte size provide an important contribution to this optimization process.

Birds with high lifetime reproductive success experience increased telomere loss.

Lifetime reproductive success (LRS) is what counts in terms of evolution, but investments in reproduction entail costs for an organism. The idea that telomere dynamics may be shaped in response to such costs is already established; however, we still lack information on whether this relation translates to overall fitness. Here, we quantified LRS (number of fledged young) and longitudinal telomere dynamics of small passerine birds-the blue tits ( Cyanistes caeruleus). We found that individual telomere erosion rate was positively associated with lifetime fledgling number. Birds with more fledged young experienced increased telomere attrition. We show that telomere attrition rate, but not telomere length, is related to individual fitness and suggest that telomere dynamics may underlie reproductive costs experienced by animals as a consequence of prioritizing their lifetime fitness. This is the first study, to our knowledge, to provide evidence that more pronounced telomere erosion is associated with higher fitness gain.

Sex-specific effects of parasites on telomere dynamics in a short-lived passerine-the blue tit.

Parasitic infections potentially drive host's life-histories since they can have detrimental effects on host's fitness. Telomere dynamics is a candidate mechanism to underlie life-history trade-offs and as such may correlate with observed fitness reduction in infected animals. We examined the relationship of chronic infection with two genera of haemosporidians causing avian malaria and malaria-like disease with host's telomere length (TL) in a longitudinal study of free-ranging blue tits. The observed overall infection prevalence was 80% and increased with age, constituting a potentially serious selective pressure in our population. We found longer telomeres in individuals infected with a parasite causing lesser blood pathologies i.e. Haemoproteus compared to Plasmodium genus, but this only held true among males. Female TL was independent of the infection type. Our results indicate that parasitic infections could bring about other types of costs to females than to males with respect to TL. Additionally, we detected linear telomere loss with age, however a random regression analysis did not confirm significant heterogeneity in TL of first breeders and telomere shortening rates in further life.

Contrasting response of haematological variables between long-term training and short exercise bouts in zebra finches (Taeniopygia guttata).

Physical aerobic activity is oxygen demanding, but - particularly for birds - there is still little understanding of how blood contributes to oxygen supply under various activity levels. In a two-factorial experimental design, we investigated the long-term effect of daily flight training and the immediate effect of a short exercise bout on a set of haematological variables: haemoglobin (Hb) content, haematocrit (Hct), and red blood cell number (RBCcount) and size (RBCarea) in zebra finches (Taeniopygia guttata). For a period of 6 weeks, birds were either trained daily for 3 h in a flight arena or remained untrained. Subsequently, half of each group was blood sampled either in the resting condition or after a 5 min exercise bout in a flight-hover wheel. We found significantly lower Hb content, Hct and RBCcount compared with that in untrained controls in response to training, while RBCarea did not differ between treatments. Response to an exercise bout revealed the opposite pattern, with significantly higher Hb content and Hct compared with that in non-exercised birds. Additionally, RBCarea was significantly smaller immediately after exercise compared with that in non-exercised birds, and such short-term flexibility represents a novel finding for birds. This contrasting response in erythrocyte characteristics with respect to long-term training and short exercise bouts appears as a clear pattern, presumably underlain by changes in water balance. We infer alterations of blood flow to be involved in adequate oxygen supply. During an exercise bout, RBCarea flexibility may not only enhance oxygen delivery through improved erythrocyte surface area to volume ratio but also improve blood flow through a compensatory effect on blood viscosity.

A biotrophic fungal infection of the great burnet Sanguisorba officinalis indirectly affects caterpillar performance of the endangered scarce large blue butterfly Phengaris teleius.

Interactions between ecological communities of herbivores and microbes are commonly mediated by a shared plant. A tripartite interaction between a pathogenic fungus-host plant-herbivorous insect is an example of such mutual influences. In such a system a fungal pathogen commonly has a negative influence on the morphology and biochemistry of the host plant, with consequences for insect herbivore performance. Here we studied whether the biotrophic fungus Podosphaera ferruginea, attacking the great burnet Sanguisorba officinalis, affects caterpillar performance of the endangered scarce large blue butterfly Phengaris teleius. Our results showed that the pathogenic fungus affected the number and size of inflorescences produced by food-plants and, more importantly, had indirect, plant-mediated effects on the abundance, body mass and immune response of caterpillars. Specifically, we found the relationship between caterpillar abundance and variability in inflorescence size on a plant to be positive among healthy food-plants, and negative among infected food-plants. Caterpillars that fed on healthy food-plants were smaller than those that fed on infected food-plants in one studied season, while there was no such difference in the other season. We observed the relationship between caterpillar immune response and the proportion of infected great burnets within a habitat patch to be positive when caterpillars fed on healthy food-plants, and negative when caterpillars fed on infected food-plants. Our results suggest that this biotrophic fungal infection of the great burnet may impose a significant indirect influence on P. teleius caterpillar performance with potential consequences for the population dynamics and structure of this endangered butterfly.

High oxidative stress despite low energy metabolism and vice versa: Insights through temperature acclimation in an ectotherm.

Reactive oxygen species (ROS) are inescapable byproducts of energy metabolism and may cause costly damage to biomolecules. Organisms have evolved different means to counter oxidative stress, such as modulation of ROS production, neutralization of produced ROS through free radical scavenging and the repair or removal of the damaged structures. A positive relationship between metabolic rate and ROS production is commonly expected, but the oxidative burden of aerobic metabolism remains poorly understood. We investigated whether acclimation to ambient temperatures imposing variation in standard metabolic rate (SMR) is mirrored in the oxidative status of an ectotherm. Grass snakes (Natrix natrix) acclimated for six months to warm (32 °C) conditions revealed seven times higher SMR compared to cold-acclimated (18 °C) individuals. In contrast to SMR, the amount of damage measured as reactive oxygen metabolites test (dROMs) and abundance of micronucleated erythrocytes was significantly lower in warm-acclimated individuals, while non-enzymatic antioxidant capacity of plasma was unaltered by thermal acclimation. Our results support that high SMR may allow snakes to better cope with oxidative stress, possibly through tissue repair or removal of damaged tissues that also requires energy costs. The reversed association between self-maintenance metabolism and oxidative damage to biomolecules provides novel rational for temperature dependent life histories of ectotherms. How oxidative stress may contribute to the known reduced rates of ectotherm growth or reproduction under cold temperatures or if oxidative stress may even drive such life history trait declines are now important challenges to be addressed.

Effects of interspecific coexistence on laying date and clutch size in two closely related species of hole-nesting birds.

Coexistence between great tits Parus major and blue tits Cyanistes caeruleus, but also other hole-nesting taxa, constitutes a classic example of species co-occurrence resulting in potential interference and exploitation competition for food and for breeding and roosting sites. However, the spatial and temporal variations in coexistence and its consequences for competition remain poorly understood. We used an extensive database on reproduction in nest boxes by great and blue tits based on 87 study plots across Europe and Northern Africa during 1957-2012 for a total of 19,075 great tit and 16,729 blue tit clutches to assess correlative evidence for a relationship between laying date and clutch size, respectively, and density consistent with effects of intraspecific and interspecific competition. In an initial set of analyses, we statistically controlled for a suite of site-specific variables. We found evidence for an effect of intraspecific competition on blue tit laying date (later laying at higher density) and clutch size (smaller clutch size at higher density), but no evidence of significant effects of intraspecific competition in great tits, nor effects of interspecific competition for either species. To further control for site-specific variation caused by a range of potentially confounding variables, we compared means and variances in laying date and clutch size of great and blue tits among three categories of difference in density between the two species. We exploited the fact that means and variances are generally positively correlated. If interspecific competition occurs, we predicted a reduction in mean and an increase in variance in clutch size in great tit and blue tit when density of heterospecifics is higher than the density of conspecifics, and for intraspecific competition, this reduction would occur when density of conspecifics is higher than the density of heterospecifics. Such comparisons of temporal patterns of means and variances revealed evidence, for both species, consistent with intraspecific competition and to a smaller extent with interspecific competition. These findings suggest that competition associated with reproductive behaviour between blue and great tits is widespread, but also varies across large spatial and temporal scales.