Telomere length (i.e., the length of the repeated sequences of DNA at the end of chromosomes) is a promising indicator of overall stress. Our study aimed to compare the effects of a stress-inducing separation process between dams and their calves, with either a gradual or a nose-flap separation method after a three-months dam-calf contact since calving, on relative telomere length (RTL). Due to their nature, the nose-flap and gradual separation method have different effects on behaviour, stress hormone levels and physical development during and after dam-calf separation, which requires an overall measure of the weaning and separation stress during both procedures. We also investigated correlations between behavioural and other physiological stress indicators on RTL. We found no significant effect of the weaning and separation method on RTL in dairy calves after weaning and separation from their dams, but a tendency for shorter RTL in gradually separated dams compared to nose-flap separated dams. No correlations between behavioural and other physiological stress indicators and RTL were found, which may be due to a short interval between the two RTL measurement points. Future studies should aim to analyse the effect of various separation methods over a longer period and preferably include a non-separation group as reference.

Telomeres protect chromosomes from damage and shortening during cell division and are suggested to relate to aging and cognitive performance. Hibernating species, such as the edible dormouse (Glis glis), exhibit remarkable telomere plasticity. It has been shown that the frequency of interbout euthermia (IBE) and the depth of torpor accelerate telomere shortening during hibernation. However, the consequence for telomeres when animals are prevented from hibernating has never been studied. In addition, we investigated whether hibernation duration or relative telomere length (RTL) affects the performance in a spatial orientation task already early in life. We studied 51 dormice from 2021 to 2024 (maximum age 3 years) and RTL in buccal mucosa cells before and after hibernation, as well as spatial cognition using a vertical maze. We observed that preventing animals from hibernating did not affect RTL. Initial telomere length (ITL) was the only consistent predictor of RTL. Further, spatial orientation was not affected by RTL. These results suggest that early life telomere dynamics in dormice are largely driven by intrinsic factors such as individual quality, rather than by hibernation performance. Our results underline the high potential for repair mechanisms in this long-lived species (up to 14 years).

This research studied the impact of various factors (including social and physiological parameters) on telomere dynamics in pet dogs. Telomeres, essential for maintaining genomic integrity, undergo shortening with each cell division, leading to cellular senescence. Previous studies in humans have linked cognitive and social factors with telomere dynamics but in animals, such associations remain understudied. This study is based on a previous study, where behavioral and cognitive changes in aging pet dogs were investigated. Together with standard variables (sex, age, body weight, diet), behavioral predictors that were assessed in the "Modified Vienna Canine Cognitive Battery" were used. This study aimed to investigate the influence of these factors on telomere dynamics in aging pet dogs. The relative telomere length of 63 dogs was measured, using a qPCR method and a model selection approach was applied to assess which variables can explain the found telomere patterns. Results revealed a strong association of the behavioral factor called trainability and telomere change. Trainability was the best predictor for telomere change over time and was the only predictor having a relative variable importance (RVI) above 0.7. This finding suggests that higher trainability positively affects telomere dynamics in aging dogs and factors like age, sex, diet, and other cognitive parameters are less important. The study sheds light on the potential role of cognitive factors in canine aging and offers insights into improving the quality of life for aging dogs, but further research is needed to comprehensively understand the interplay between behavior, cognition, and telomere dynamics in dogs.

Migratory birds must accumulate large amounts of fat prior to migration to sustain long flights. In passerines, the small body size limits the amount of energy stores that can be transported, and therefore birds undergo cycles of extreme fattening and rapid exhaustion of reserves. Research on these physiological adaptations was rattled by the discovery that birds have lost the main vertebrate regulator of fat deposition, leptin. Recent studies have thus focused on ghrelin, known as ‘hunger hormone’, a peptide secreted by the gastrointestinal tract to regulate, e.g. food intake and body mass in vertebrates. Studies on domestic species showed that, in birds, ghrelin has effects opposite to those described in mammals such as inhibiting instead of promoting food intake. Furthermore, recent studies have shown that ghrelin administration influences migratory behaviour in passerine birds. Using comparative genomics and immunoaffinity chromatography, we show that ghrelin has been lost in Eupasseres after the basic split from Acanthisitti about 50 Ma. We found that the ghrelin receptor is still conserved in passerines. The maintenance of a functional receptor system suggests that in Eupasserines, another ligand has replaced ghrelin, perhaps to bypass the feedback system that would hinder the large pre-migratory accumulation of subcutaneous fat.

BACKGROUND:Selection on or reticulate evolution of mtDNA is documented in various mammalian taxa and could lead to misleading phylogenetic conclusions if not recognized. We sequenced the MT-ND6 gene of four sympatric Mustelid species of the genus Mustela from some central European populations. We hypothesised positive selection on MT-ND6, given its functional importance and the different body sizes and life histories of the species, even though climatic differences may be unimportant for adaptation in sympatry.METHODS AND RESULTS:MT-ND6 genes were sequenced in 187 sympatric specimens of weasels, Mustela nivalis, stoats, M. erminea, polecats, M. putorius, and steppe polecats, M. eversmannii, from eastern Austria and of fourteen allopatric polecats from eastern-central Germany. Median joining networks, neighbour joining and maximum likelihood analyses as well as Bayesian inference grouped all species according to earlier published phylogenetic models. However, polecats and steppe polecats, two very closely related species, shared the same two haplotypes. We found only negative selection within the Mustela sequences, including 131 downloaded ones covering thirteen species. Positive selection was observed on three MT-ND6 codons of other mustelid genera retrieved from GenBank.CONCLUSIONS:Negative selection for MT-ND6 within the genus Mustela suggests absence of both environmental and species-specific effects of cellular energy metabolism despite large species-specific differences in body size. The presently found shared polymorphism in European polecats and steppe polecats may result from ancestral polymorphism before speciation and historical or recent introgressive hybridization; it may indicate mtDNA capture of steppe polecats by M. putorius in Europe.

Migration is one of the most extreme and energy demanding life history strategies to have evolved in the animal kingdom. In birds, champions of long-distance migrations, the seasonal emergence of the migratory phenotype is characterised by rapid physiological and metabolic remodelling, including substantial accumulation of fat stores and increases in nocturnality. The molecular underpinnings and brain adaptations to seasonal migrations remain poorly understood. Here, we exposed Common quails (Coturnix coturnix) to controlled changes in day length to simulate southward autumn migration, and then blocked the photoperiod until birds entered the non-migratory wintering phase. We first performed de novo RNA-Sequencing from selected brain samples (hypothalamus) collected from birds at a standardised time at night, either in a migratory state (when restlessness was highest and at their body mass peak), or in a non-migratory state and conducted differential gene expression and functional pathways analyses. We found that the migratory state was associated with up-regulation of a few, yet functionally well defined, gene expression networks implicated in fat trafficking, protein and carbohydrate metabolism. Further analyses that focused on candidate genes (apolipoprotein H or APOH, lysosomal associated membrane protein-2 or LAMP2) from samples collected during the day or night across the entire study population suggested differences in the expression of these genes depending on the time of the day with the largest expression levels being found in the migratory birds sampled at night. We also found that expression of APOH was positively associated with levels of nocturnal activity in the migratory birds; such an association was absent within the non-migratory birds. Our results provide novel experimental evidence revealing that hypothalamic changes in expression of apolipoprotein pathways, which regulate the circulatory transport of lipids, are likely key regulatory activators of nocturnal migratory movements. Our study paves the way for performing deeper functional investigations on seasonal molecular remodelling underlying the development of the migratory phenotype.

Genome size varies greatly across the tree of life and transposable elements are an important contributor to this variation. Among vertebrates, amphibians display the greatest variation in genome size, making them ideal models to explore the causes and consequences of genome size variation. However, high-quality genome assemblies for amphibians have, until recently, been rare. Here, we generate a high-quality genome assembly for the dyeing poison frog, Dendrobates tinctorius. We compare this assembly to publicly available frog genomes and find evidence for both large-scale conserved synteny and widespread rearrangements between frog lineages. Comparing conserved orthologs annotated in these genomes revealed a strong correlation between genome size and gene size. To explore the cause of gene-size variation, we quantified the location of transposable elements relative to gene features and find that the accumulation of transposable elements in introns has played an important role in the evolution of gene size in D. tinctorius, while estimates of insertion times suggest that many insertion events are recent and species-specific. Finally, we carry out population-scale mobile-element sequencing and show that the diversity and abundance of transposable elements in poison frog genomes can complicate genotyping from repetitive element sequence anchors. Our results show that transposable elements have clearly played an important role in the evolution of large genome size in D. tinctorius. Future studies are needed to fully understand the dynamics of transposable element evolution and to optimize primer or bait design for cost-effective population-level genotyping in species with large, repetitive genomes.

Oxidative stress is thought to be one of the main causes of ageing as it progressively damages cell components throughout life, eventually causing cellular failure and apoptosis. In many organisms, telomeres shorten throughout life under the effect of, amongst other factors, oxidative stress, and are therefore commonly used as marker of biological ageing. However, hibernators, which are regularly exposed to acute oxidative stress when rewarming from torpor, are unexpectedly long-lived. In this review, we explore the causes of oxidative stress associated with hibernation and its impact on telomere dynamics in different taxa, focussing on hibernating rodents. We then speculate on the adaptive mechanisms of hibernators to compensate for the effects of oxidative stress, which may explain their increased longevity. Because winter hibernation appears to be associated with high oxidative stress, hibernators, particularly rodents, may periodically invest in repair mechanisms and antioxidant defences, resulting in seasonal variations in telomere lengths. This research shows how species with a slow life-history strategy deal with large changes in oxidative stress, unifying evolutionary and physiological theories of ageing. Because of the marked seasonal variation in telomere length, we also draw attention when using telomeres as markers for biological aging in seasonal heterotherms and possibly in other highly seasonal species.

Genetic differentiation between populations inhabiting ecologically different habitats might appear because of limited dispersal and gene flow, which may lead to patterns of phenotypic divergence and local adaptation. In this study, we use dispersal, genotypic (24 microsatellite loci) and phenotypic (body size and clutch size) data to analyse patterns of genetic structuring and phenotypic divergence in a blue tit (Cyanistes caeruleus) population inhabiting a continuous and heterogeneous woodland along a valley. The two slopes of the valley differ in their forest formations and environmental conditions. Findings showed that most blue tits reproduced within their natal slope. Accordingly, microsatellite analyses revealed that populations of blue tits established in the two slopes show subtle genetic differentiation. The two genetic populations diverged in clutch size, exceeding the level of differentiation expected based on genetic drift, hence suggesting divergent selection (or other processes promoting divergence) on this life-history trait. Our findings reveal that restricted dispersal and spatial heterogeneity may lead to genetic differentiation among bird populations at a surprisingly small scale. In this respect, it is worth highlighting that such differentiation occurs for an organism with high dispersal capacity and within a continuous woodland. Moreover, we show that small-scale ecological differences, together with limited gene flow, can result in selection favouring different phenotypes even within the same continuum population.

Amphibians globally suffer from emerging infectious diseases like chytridiomycosis caused by the continuously spreading chytrid fungi. One is Batrachochytrium salamandrivorans (Bsal) and its disease ‒ the ‘salamander plague’ ‒ which is lethal to several caudate taxa. Recently introduced into Western Europe, long distance dispersal of Bsal, likely through human mediation, has been reported. Herein we study if Alpine salamanders (Salamandra atra and S. lanzai) are yet affected by the salamander plague in the wild. Members of the genus Salamandra are highly susceptible to Bsal leading to the lethal disease. Moreover, ecological modelling has shown that the Alps and Dinarides, where Alpine salamanders occur, are generally suitable for Bsal. We analysed skin swabs of 818 individuals of Alpine salamanders and syntopic amphibians at 40 sites between 2017 to 2022. Further, we compiled those with published data from 319 individuals from 13 sites concluding that Bsal infections were not detected. Our results suggest that the salamander plague so far is absent from the geographic ranges of Alpine salamanders. That means that there is still a chance to timely implement surveillance strategies. Among others, we recommend prevention measures, citizen science approaches, and ex situ conservation breeding of endemic salamandrid lineages.

For behavioural tests such as the cognitive judgement bias task (JBT), animals usually undergo weeks of training involving various elements, such as handling, social isolation and learning the task. These may influence their affective state and other welfare indicators. Here, we investigated the effects of training for a JBT on fear-related behaviour in laying hens and a potential welfare indicator - telomere shortening. Hens were trained for a JBT (N = 16, across 2 batches), or kept as controls (N = 16, across 2 batches) that received no training or trainingrelated handling. Hens that reached the learning criterion (N = 14) were tested in six JBT test sessions. Before and after the trained hens received JBT training, blood was collected from all hens, and they were assessed for fear-related behaviour by being subjected to open field, human approach, and tonic immobility tests. Relative telomere lengths (RTL; i.e., ratio of telomeric repeats versus a single copy control) were obtained from blood samples with a quantitative real-time polymerase chain reaction (PCR) method. As anticipation of the JBT sessions could affect the JBT outcome, we also recorded behavioural indicators of anticipation prior to each JBT test session. Except for an increased latency to first step in the open field test after training in the trained hens (t = 3.1, df=28, P = 0.012; conditional R2=0.59), no other fear-related behaviour significantly differed in either group (P > 0.05) and RTL was not affected. In addition, none of the anticipation indicators predicted the JBT test outcome. Contra to our hypotheses, welfare-related aspects may not be strongly affected by the JBT training. However, the enriched housing of the hens from both treatments might have minimised the effect of training on fear and telomere shortening, or training may have affected indicators other than the ones we investigated. We conclude that because JBT training and testing does not affect fear assessment and telomere shortening outcomes in hens housed in enriched housing conditions, the same individuals can be used in welfare assessments involving similar assessments. Our results also suggest that the JBT itself may be robust against the temporary affective states associated with anticipation.

The Sicilian wolf remained isolated in Sicily from the end of the Pleistocene until its extermination in the 1930s-1960s. Given its long-term isolation on the island and distinctive morphology, the genetic origin of the Sicilian wolf remains debated. We sequenced four nuclear genomes and five mitogenomes from the seven existing museum specimens to investigate the Sicilian wolf ancestry, rela-tionships with extant and extinct wolves and dogs, and diversity. Our results show that the Sicilian wolf is most closely related to the Italian wolf but carries ancestry from a lineage related to European Eneolithic and Bronze Age dogs. The average nucleotide diversity of the Sicilian wolf was half of the Italian wolf, with 37-50% of its genome contained in runs of homozygosity. Overall, we show that, by the time it went extinct, the Sicilian wolf had high inbreeding and low-genetic diversity, consistent with a population in an insular environment.

Telomere dynamics in hibernating species are known to reflect seasonal changes in somatic maintenance. Throughout hibernation, the periodic states of rewarming, known as inter-bout euthermia or arousals, are associated with high metabolic costs including shortening of telomeres. In the active season, if high energetic resources are available, telomere length can be restored in preparation for the upcoming winter. The mechanism for telomere elongation has not been clearly demonstrated, although the action of the ribonucleoprotein complex, telomerase, has been implicated in many species. Here we tested for levels of telomerase activity in the garden dormouse (Eliomys quercinus) at different seasonal time points throughout the year and across ages from liver tissues of male juveniles to adults. We found that telomerase is active at high levels across seasons (during torpor and inter-bout euthermia, plus in the active season) but that there was a substantial decrease in activity in the month prior to hibernation. Telomerase levels were consistent across age groups and were independent of feeding regime and time of birth (early or late born). The changes in activity levels that we detected were broadly associated with changes in telomere lengths measured in the same tissues. We hypothesise that i) telomerase is the mechanism used by garden dormice for maintenance of telomeres and that ii) activity is kept at high levels throughout the year until pre-hibernation when resources are diverted to increasing fat reserves for overwintering. We found no evidence for a decrease in telomerase activity with age or a final increase in telomere length which has been detected in other hibernating rodents.

Background Torpor is an energy saving strategy achieved by substantial reductions of metabolic rate and body temperature that enables animals to survive periods of low resource availability. During hibernation (multiday torpor), the frequency of periodic rewarming—characterised by high levels of oxidative stress—is associated with shortening of telomeres, a marker of somatic maintenance. Objectives In this study, we determined the impact of ambient temperature on feeding behaviour and telomere dynamics in hibernating garden dormice ( Eliomys quercinus ) over winter. This obligate hibernator prepares for hibernation by accumulating fat stores but can also feed during hibernation. Methodology Food intake, torpor pattern, changes in telomere length, and body mass change were assessed in animals housed at experimentally controlled temperatures of either 14 °C (i.e., a mild winter) or 3 °C (i.e., a cold winter) over 6 months. Results When hibernating at 14 °C, dormice experienced 1.7-fold more frequent and 2.4-fold longer inter-bout euthermia, and spent significantly less time torpid, compared to animals hibernating at 3 °C. Higher food intake enabled individuals to compensate for increased energetic costs when hibernating at milder temperatures (14 °C vs. 3 °C), to buffer body mass loss and thus increase winter survival. Interestingly, we observed a significant increase of telomere length over the entire hibernation period, irrespective of temperature treatment. Conclusion We conclude that higher temperatures during winter, if associated with sufficient food availability, can have a positive effect on the individual’s energy balance and somatic maintenance. These results suggest that winter food availability might be a crucial determinant for the survival of the garden dormouse in the context of ever-increasing environmental temperatures.

We found major seasonal changes of polyunsaturated fatty acids (PUFAs) in muscular phospholipids (PL) in a large non-hibernating mammal, the red deer (Cervus elaphus). Dietary supply of essential linoleic acid (LA) and α-linolenic acid (ALA) had no, or only weak influence, respectively. We further found correlations of PL PUFA concentrations with the activity of key metabolic enzymes, independent of higher winter expression. Activity of the sarcoplasmic reticulum (SR) Ca++-ATPase increased with SR PL concentrations of n-6 PUFA, and of cytochrome c oxidase and citrate synthase, indicators of ATP-production, with concentrations of eicosapentaenoic acid in mitochondrial PL. All detected cyclic molecular changes were controlled by photoperiod and are likely of general relevance for mammals living in seasonal environments, including humans. During winter, these changes at the molecular level presumably compensate for Arrhenius effects in the colder peripheral body parts and thus enable a thrifty life at lower body temperature.

Filarioid nematodes (commonly known as filarial worms) are known to impact human and domestic animal health, but studies examining their ecological relevance and impacts on wildlife are still underrepresented. In the case of birds, microfilariae are typically found at low prevalence, but they may negatively affect some fitness-related traits. Here, we study the prevalence and associations of microfilariae in a wild population of blue tits (Cyanistes caeruleus) inhabiting a woodland comprising different forestry formations. In addition, we characterize the filarioid lineages through the cytochrome c oxidase subunit I (COI) gene sequence. We found a moderate prevalence of microfilariae in the blue tit population (9.4%) and that the presence of such parasites was negatively associated with host body mass. Neither forest type nor host sex influenced microfilariae presence. Phylogenetic analyses revealed the presence of five filarioid lineages clustered in the Onchocercidae family-four out of five lineages clustered in the Splendidofilaria clade, while the remaining lineage could not be clearly assigned to a genus. In addition, this is the first study examining the filarioid lineages infecting the blue tit. Our results suggest that hosts in poorer body condition, in terms of lower body mass, are more susceptible to be parasitized by filarioid nematodes and call for further genetic studies of these parasites.

In wild boar, Sus scrofa, from Europe, domestic pig-typical ancestry is traced at varying levels. We hypothesised wild boar with pig-typical gene pool characteristics, i.e., “introgression”, congregate more in peri-urban habitats, because of less shyness and better adaptation to anthropogenic stress. We used 16 microsatellites to study introgression levels of 375 wild boar from peri-urban Vienna, Austria, and rural regions in comparison to commercial slaughter pigs, Mangaliza, and Turopolje pigs. We also expected more introgression in locations of warmer climates and lower precipitation. Despite discrimination of wild boar and pigs with 99.73% and 97.87% probability, respectively, all wild boars exhibited pig-typical gene pool characteristics, mostly at a very low level. Recent hybridisation was suspected in only 0.53% of wild boar, corresponding to the current largely indoor pig breeding/rearing in the region, with no chance of natural gene exchange between pigs and wild boar. Rather, pig ancestry in wild boar stems from incomplete gene pool differentiation during domestication and/or historical introgressions, when free-ranging pig farming was common. Individual introgression levels were lower in wild boar from peri-urban habitats, possibly reflecting the largely historical absence of pig farms there. Moreover, a marginal precipitation effect, but no temperature effect on introgression was observed. The latter, however, needs to be explored further by a more comprehensive data set.

Brown hares (Lepus europaeus) from Türkiye exhibit substantial phenotypic variation, assumingly connected to regional climate differences. We used general linear models (GLMs) of linear measurements and geometric morphometric data of dorsal and ventral skull and lateral mandible landmarks of 160 adult individuals from 75 locations across Türkiye, to specifically test for climate-related effects on skull size and shape, while accounting for potentially confounding effects of sex, geographic location, and population genetic assignment. We found a moderate positive correlation between individual condylobasal lengths (CBL) and centroid size scores (CSS) and used them separately as dependent skull size variables in our GLMs. Skull shape factors were obtained from principal component analyses (PCAs), separately for transformed linear morphometric and landmark data; they were also used as dependent variables in our GLMs, along with sex, geographic sampling coordinates, CBL or CSS, as well as temperature and precipitation factors from PCAs of local climate data as fixed factors. Our multivariate GLMs returned no climate effect on skull size and only one minor precipitation effect on geometric skull shape. Rather, skull size and shape were predominantly and significantly affected by the assignment of the hares to the genetic populations as determined by an earlier microsatellite study. Our results correspond to microphylogeographic differentiation of skull size and shape of the studied hares, largely indepedent of specific adaptation to local or regional climate.

The genes of the major histocompatibility complex (MHC) are among the best candidates for studying adaptive variation driven often by pathogen-mediated selection. However, diversity of these genes might be differently shaped by selective and non-selective evolutionary mechanisms. In this study, we assessed the spatial distribution of MHC class II DRB exon 2 gene diversity in hares from Tunisia (Lepus capensis) across a steep ecological gradient and tried to disentangle the driving mechanisms shaping the observed diversity. We also compared the current data with previous results from the MHC class II DQA and DQB loci of the same individuals. We found a relatively high level of diversity and spatial differentiation similar to that observed earlier for DQA and DQB. We also detected clear evidence of positive selection acting on the DRB gene. Nevertheless, recombination was considered as a more important process shaping the evolution of the DRB locus. Our information theory-based models revealed statistically meaningful climate effects on the occurrence of some alleles (i.e., protein variants), regardless of their geographical distribution patterns, supposedly reflecting adaptation to spatial pathogen variation.