Communication hubs of an asocial cat are the source of a human-carnivore conflict and key to its solution.

Human-wildlife conflicts occur worldwide. Although many nonlethal mitigation solutions are available, they rarely use the behavioral ecology of the conflict species to derive effective and long-lasting solutions. Here, we use a long-term study with 106 GPS-collared free-ranging cheetahs (Acinonyx jubatus) to demonstrate how new insights into the socio-spatial organization of this species provide the key for such a solution. GPS-collared territory holders marked and defended communication hubs (CHs) in the core area of their territories. The CHs/territories were distributed in a regular pattern across the landscape such that they were not contiguous with each other but separated by a surrounding matrix. They were kept in this way by successive territory holders, thus maintaining this overdispersed distribution. The CHs were also visited by nonterritorial cheetah males and females for information exchange, thus forming hotspots of cheetah activity and presence. We hypothesized that the CHs pose an increased predation risk to young calves for cattle farmers in Namibia. In an experimental approach, farmers shifted cattle herds away from the CHs during the calving season. This drastically reduced their calf losses by cheetahs because cheetahs did not follow the herds but instead preyed on naturally occurring local wildlife prey in the CHs. This implies that in the cheetah system, there are "problem areas," the CHs, rather than "problem individuals." The incorporation of the behavioral ecology of conflict species opens promising areas to search for solutions in other conflict species with nonhomogenous space use.

Intravital Dynamic and Correlative Imaging of Mouse Livers Reveals Diffusion-Dominated Canalicular and Flow-Augmented Ductular Bile Flux.

BACKGROUND AND AIMS: Small-molecule flux in tissue microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods. We developed two independent techniques that allow the quantification of advection (flow) and diffusion in individual bile canaliculi and in interlobular bile ducts of intact livers in living mice, namely fluorescence loss after photoactivation and intravital arbitrary region image correlation spectroscopy. APPROACH AND RESULTS: The results challenge the prevailing "mechano-osmotic" theory of canalicular bile flow. After active transport across hepatocyte membranes, bile acids are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts is diffusion augmented by regulatable advection. Photoactivation of fluorescein bis-(5-carboxymethoxy-2-nitrobenzyl)-ether in entire lobules demonstrated the establishment of diffusive gradients in the bile canalicular network and the sink function of interlobular ducts. In contrast to the bile canalicular network, vectorial transport was detected and quantified in the mesh of interlobular bile ducts. CONCLUSIONS: The liver consists of a diffusion-dominated canalicular domain, where hepatocytes secrete small molecules and generate a concentration gradient and a flow-augmented ductular domain, where regulated water influx creates unidirectional advection that augments the diffusive flux.

Early-life adversity predicts performance and fitness in a wild social carnivore.

Studies on humans indicate that encountering multiple sources of adversity in childhood increases the risk of poor long-term health and premature death. Far less is known about cumulative effects of adversity during early life in wildlife. Focusing on the spotted hyena Crocuta crocuta, a social mammal with small litters, extensive maternal care, slow development and access to resources determined by social rank, we determined the contribution of ecological, maternal, social and demographic factors during early life on performance and fitness, and tested whether the impact of early-life adversity is cumulative. Using longitudinal data from 666 female hyenas in the Serengeti National Park, we determined the early growth rate, survival to adulthood, age at first reproduction (AFR), lifetime reproductive success (LRS) and longevity. We fitted multivariate models in which we tested the effects of environmental factors on these performance measures. We then constructed a cumulative adversity index and fitted models to test the effect of this index on each performance measure. Finally, the value of cumulative adversity models was tested by comparing them to multivariate and single-effect models in which the effect of each environmental factor was considered separately. High maternal rank decreased the AFR of daughters. Singleton and dominant cubs had higher growth rate than subordinate cubs, and singletons also had a higher survival chance to adulthood than subordinates. Daughters of prime age mothers had a higher growth rate, longevity and LRS. Little and heavy rainfall decreased survival to adulthood. Increasing numbers of lactating female clan members decreased growth rate, survival to adulthood and LRS. Cumulative adversity negatively affected short-term performance and LRS. Multivariate models outperformed cumulative adversity and single-effect models for all measures except for AFR and longevity, for which single-effect models performed better. Our results suggest that in some wildlife populations the combination of specific conditions in early life may matter more than the accumulation of adverse conditions as such.

Des études menées chez l'homme indiquent que le fait de rencontrer de multiples sources d'adversité pendant l'enfance augmente le risque de troubles de la santé à long terme et de décès prématuré. On en sait beaucoup moins sur les effets cumulatifs de l'adversité en début de vie chez les animaux sauvages. En nous concentrant sur la hyène tachetée Crocuta crocuta, un mammifère social avec des portées de petite taille, des soins maternels intensifs, un développement lent et un accès aux ressources déterminé par le rang social, nous avons déterminé la contribution des facteurs écologiques, maternels, sociaux et démographiques en début de vie sur la performance et le succès reproducteur, et nous avons testé si l'adversité précoce a un effet cumulatif. En utilisant les données longitudinales de 666 hyènes femelles dans le parc national du Serengeti, nous avons calculé le taux de croissance précoce, la survie à l'âge adulte, l'âge à la première reproduction, le succès reproducteur et la longévité. Nous avons utilisé des modèles multivariés dans lesquels nous avons testé les effets des facteurs environnementaux sur ces mesures de performance. Nous avons ensuite construit un indice de risque cumulatif et utilisé des modèles pour tester l'effet de cet indice sur chaque mesure de performance. Enfin, nous avons testé la valeur de ces modèles en les comparant aux modèles multivariés et simples dans lesquels l'effet de chaque facteur environnemental était considéré séparément. Un rang maternel élevé diminue l'âge à la première reproduction des filles. Les femelles sans frère ou sœur ou dominantes dans une portée de jumeaux ont un taux de croissance plus élevé que les femelles subordonnées, et les femelles sans frère ou sœur ont également une plus grande chance de survie à l'âge adulte que les subordonnées. Les filles de mères dans la fleur de l'âge montrent de meilleurs taux de croissance, longévité et succès reproducteur. Des précipitations faibles ou fortes réduisent la survie à l'âge adulte. L'augmentation du nombre de femelles allaitantes dans le clan réduit le taux de croissance, la survie à l'âge adulte et le succès reproducteur. L'adversité précoce cumulée a un effet négatif sur la performance à court terme et le succès reproducteur. Les modèles multivariés sont toutefois plus performants que les modèles avec l'indice de risque cumulatif et plus performants que les modèles simples pour toutes les mesures, à l'exception de l'âge à la première reproduction et de la longévité, pour lesquelles les modèles simples sont plus performants. Nos résultats suggèrent que chez certaines populations sauvages, la combinaison de conditions spécifiques rencontrées en début de vie peut avoir plus d'importance que l'accumulation de conditions défavorables en tant que telles.

The virus-host interface: Molecular interactions of Alphacoronavirus-1 variants from wild and domestic hosts with mammalian aminopeptidase N.

The Alphacoronavirus-1 species include viruses that infect numerous mammalian species. To better understand the wide host range of these viruses, better knowledge on the molecular determinants of virus-host cell entry mechanisms in wildlife hosts is essential. We investigated Alphacoronavirus-1 infection in carnivores using long-term data on Serengeti spotted hyenas (Crocuta crocuta) and molecular analyses guided by the tertiary structure of the viral spike (S) attachment protein's interface with the host receptor aminopeptidase N (APN). We sequenced the complete 3'-end region of the genome of nine variants from wild African carnivores, plus the APN gene of 15 wild carnivore species. Our results revealed two outbreaks of Alphacoronavirus-1 infection in spotted hyenas associated with genetically distinct canine coronavirus type II (CCoVII) variants. Within the receptor binding domain (RBD) of the S gene the residues that directly bind to the APN receptor were conserved in all variants studied, even those infecting phylogenetically diverse host taxa. We identified a variable region within RBD located next to a region that directly interacts with the APN receptor. Two residues within this variable region were under positive selection in hyena variants, indicating that both sites were associated with adaptation of CCoVII to spotted hyena APN. Analysis of APN sequences revealed that most residues that interact with the S protein are conserved in wild carnivores, whereas some adjacent residues are highly variable. Of the variable residues, four that are critical for virus-host binding were under positive selection and may modulate the efficiency of virus attachment to carnivore APN.

'Keeping the kids at home' can limit the persistence of contagious pathogens in social animals.

Social networks are considered to be 'highly modular' when individuals within one module are more connected to each other than they are to individuals in other modules. It is currently unclear how highly modular social networks influence the persistence of contagious pathogens that generate lifelong immunity in their hosts when between-group interactions are age dependent. This trait occurs in social species with communal nurseries, where juveniles are reared together for a substantial period in burrows or similar forms of containment and are thus in isolation from contact with individuals in other social groups. Our main objective was to determine whether, and to what extent, such age-dependent patterns of between-group interactions consistently increased the fade-out probability of contagious pathogens that generate lifelong immunity in their hosts. We hypothesised that in populations of species where juveniles are raised in communal nurseries, a high proportion of recovered adults in a group would form a 'protective barrier' around susceptible juveniles against pathogen transmission, thereby increasing the probability of epidemic fade-out in the population. To test this idea, we implemented a spatially implicit individual-based susceptible-infected-recovered (SIR) model for a large range of generic host and pathogen traits. Our results indicated that (a) the probability of epidemic fade-out was consistently higher in populations with communal nurseries, especially for highly contagious pathogens (high basic reproduction number, R0 ) and (b) communal nurseries can counteract the cost of group living in terms of infection risk to a greater extent than variation in other traits. We discuss our findings in relation to herd immunity and outline the importance of considering the network structure of a given host population before implementing management measures such as vaccinations, since interventions focused on individuals with high between-group contact should be particularly effective for controlling pathogen spread in hosts with communal nurseries.

Beyond the landscape: Resistance modelling infers physical and behavioural gene flow barriers to a mobile carnivore across a metropolitan area.

Urbanization affects key aspects of wildlife ecology. Dispersal in urban wildlife species may be impacted by geographical barriers but also by a species' inherent behavioural variability. There are no functional connectivity analyses using continuous individual-based sampling across an urban-rural continuum that would allow a thorough assessment of the relative importance of physical and behavioural dispersal barriers. We used 16 microsatellite loci to genotype 374 red foxes (Vulpes vulpes) from the city of Berlin and surrounding rural regions in Brandenburg in order to study genetic structure and dispersal behaviour of a mobile carnivore across the urban-rural landscape. We assessed functional connectivity by applying an individual-based landscape genetic optimization procedure. Three commonly used genetic distance measures yielded different model selection results, with only the results of an eigenvector-based multivariate analysis reasonably explaining genetic differentiation patterns. Genetic clustering methods and landscape resistance modelling supported the presence of an urban population with reduced dispersal across the city border. Artificial structures (railways, motorways) served as main dispersal corridors within the cityscape, yet urban foxes avoided densely built-up areas. We show that despite their ubiquitous presence in urban areas, their mobility and behavioural plasticity, foxes were affected in their dispersal by anthropogenic presence. Distinguishing between man-made structures and sites of human activity, rather than between natural and artificial structures, is thus essential for better understanding urban fox dispersal. This differentiation may also help to understand dispersal of other urban wildlife and to predict how behaviour can shape population genetic structure beyond physical barriers.

Concurrent Proteomic Fingerprinting and Molecular Analysis of Cyathostomins.

Rapid, cost-effective, efficient, and reliable helminth species identification is of considerable importance to understand host-parasite interactions, clinical disease, and drug resistance. Cyathostomins (Nematoda: Strongylidae) are considered to be the most important equine parasites, yet research on this group is hampered by the large number of 50 morphologically differentiated species, their occurrence in mixed infections with often more than 10 species and the difficulties associated with conventional identification methods. Here, MALDI-TOF MS, previously successfully applied to identify numerous organisms, is evaluated and compared with conventional and molecular genetic approaches. A simple and robust protocol for protein extraction and subsequent DNA isolation allowing molecular confirmation of proteomic findings is developed, showing that MALDI-TOF MS can discriminate adult stages of the two closely related cyathostomin species Cylicostephanus longibursatus and Cylicostephanus minutus. Intraspecific variability of proteomic profiles within morphospecies demonstrated an identification of morphospecies with an accuracy of close to 100%. In contrast, three genospecies within C. minutus and sex-specific profiles within both morphospecies could not be reliably discriminated using MALDI-TOF MS. In conclusion, MALDI-TOF MS complemented by the molecular protocol is a reliable and efficient approach for cyathostomin species identification.

Canine distemper virus in the Serengeti ecosystem: molecular adaptation to different carnivore species.

Was the 1993/1994 fatal canine distemper virus (CDV) epidemic in lions and spotted hyaenas in the Serengeti ecosystem caused by the recent spillover of a virulent domestic dog strain or one well adapted to these noncanids? We examine this question using sequence data from 13 'Serengeti' strains including five complete genomes obtained between 1993 and 2011. Phylogenetic and haplotype network analyses reveal that strains from noncanids during the epidemic were more closely related to each other than to those from domestic or wild canids. All noncanid 'Serengeti' strains during the epidemic encoded: (1) one novel substitution G134S in the CDV-V protein; and (2) the rare amino acid combination 519I/549H at two sites under positive selection in the region of the CDV-H protein that binds to SLAM (CD 150) host cell receptors. Worldwide, only a few noncanid strains in the America II lineage encode CDV-H 519I/549H. All canid 'Serengeti' strains during the epidemic coded CDV-V 134G, and CDV-H 519R/549Y, or 519R/549H. A functional assay of cell entry revealed the highest performance by CDV-H proteins encoding 519I/549H in cells expressing lion SLAM receptors, and the highest performance by proteins encoding 519R/549Y, typical of dog strains worldwide, in cells expressing dog SLAM receptors. Our findings are consistent with an epidemic in lions and hyaenas caused by CDV variants better adapted to noncanids than canids, but not with the recent spillover of a dog strain. Our study reveals a greater complexity of CDV molecular epidemiology in multihost environments than previously thought.

Colonization history shaped the immunity of the western house mouse.

The general development of immune response in the short and long term is a product of the antigenic environment in which a species resides. Colonization of a novel antigenic environment by a species would be expected to alter the immune system. Animals that successfully adapt their immune responses will successfully colonize new locations. However, founder events associated with colonization by limited numbers of individuals from a source population will constrain adaptability. How these contradicting forces shape immunity in widely distributed species is unknown. The western house mouse (Mus musculus domesticus) spread globally from the Indo-Pakistani cradle, often in association with human migration and settlement. In the present study, we tested the hypothesis that wild-derived outbred laboratory populations of house mice from their original range (Iran) and historically recent European invasive populations (from France and Germany) present differences in immune functional diversity corresponding to recent historical founder events in Europe and movement to novel antigenic environments. We found that (1) European mice had lower total white blood cell (WBC) counts but higher immunoglobulin E concentrations than their Iranian counterparts, and (2) there were no significant differences in the measured immunological parameters among European populations. The results indicate that founder events in European mice and selection pressure exerted by the composition of local parasitic helminth communities underlie the observed patterns.

Epigenetic signatures of social status in wild female spotted hyenas (Crocuta crocuta).

In mammalian societies, dominance hierarchies translate into inequalities in health, reproductive performance and survival. DNA methylation is thought to mediate the effects of social status on gene expression and phenotypic outcomes, yet a study of social status-specific DNA methylation profiles in different age classes in a wild social mammal is missing. We tested for social status signatures in DNA methylation profiles in wild female spotted hyenas (Crocuta crocuta), cubs and adults, using non-invasively collected gut epithelium samples. In spotted hyena clans, female social status influences access to resources, foraging behavior, health, reproductive performance and survival. We identified 149 differentially methylated regions between 42 high- and low-ranking female spotted hyenas (cubs and adults). Differentially methylated genes were associated with energy conversion, immune function, glutamate receptor signalling and ion transport. Our results provide evidence that socio-environmental inequalities are reflected at the molecular level in cubs and adults in a wild social mammal.

Sibling competition and hunger increase allostatic load in spotted hyaenas.

Allostatis is the process of maintaining homeostatis through behavioural or physiological responses to challenges, and its cumulative energetic cost is termed allostatic load. The allostatic load hypothesis predicts that hunger and the mechanisms that establish and maintain social dominance should have a strong impact on allostatic load. In spotted hyaenas, dominance between twin siblings emerges during intense early competition for maternal milk and involves trained winner/loser effects. Conflict over access to teats declines with age as behavioural dominance conventions are established. In young litters, the allostatic load of subordinates measured in terms of faecal glucocorticoid metabolite concentrations (fGMCs) should be higher than that of dominants. When low milk provisioning threatens survival, hungry subordinates are more assertive, particularly when competing against a dominant sister. Dominants challenged by assertive subordinates should have allostatic loads and fGMCs above those of dominants with subordinates that adhere to dominance conventions. We show that in young litters, subordinates had significantly higher fGMCs than dominants, and dominant sisters had significantly higher fGMCs than dominant brothers. When hungry, both dominants and subordinates had significantly higher fGMCs than when fed. Our results provide evidence that hunger and sibling competition affect allostatic load in spotted hyaenas.

Coronavirus genotype diversity and prevalence of infection in wild carnivores in the Serengeti National Park, Tanzania.

Knowledge of coronaviruses in wild carnivores is limited. This report describes coronavirus genetic diversity, species specificity and infection prevalence in three wild African carnivores. Coronavirus RNA was recovered from fresh feces from spotted hyena and silver-backed jackal, but not bat-eared fox. Analysis of sequences of membrane (M) and spike (S) gene fragments revealed strains in the genus Alphacoronavirus, including three distinct strains in hyenas and one distinct strain in a jackal. Coronavirus RNA prevalence was higher in feces from younger (17 %) than older (3 %) hyenas, highlighting the importance of young animals for coronavirus transmission in wild carnivores.

Pup Recruitment in a Eusocial Mammal-Which Factors Influence Early Pup Survival in Naked Mole-Rats?

In eusocial insects, offspring survival strongly depends on the quality and quantity of non-breeders. In contrast, the influence of social factors on offspring survival is more variable in cooperatively breeding mammals since maternal traits also play an important role. This difference between cooperative insects and mammals is generally attributed to the difference in the level of sociality. Examining offspring survival in eusocial mammals should, therefore, clarify to what extent social organization and taxonomic differences determine the relative contribution of non-breeders and maternal effects to offspring survival. Here, we present the first in-depth and long-term study on the influence of individual, maternal, social and environmental characteristics on early offspring survival in a eusocial breeding mammal, the naked mole-rat (Heterocephalus glaber). Similarly to other mammals, pup birth mass and maternal characteristics such as body mass and the number of mammae significantly affected early pup survival. In this eusocial species, the number of non-breeders had a significant influence on early pup survival, but this influence was negative-potentially an artifact of captivity. By contrasting our findings with known determinants of survival in eusocial insects we contribute to a better understanding of the origin and maintenance of eusociality in mammals.

ORCA-SPY enables killer whale sound source simulation, detection, classification and localization using an integrated deep learning-based segmentation.

Acoustic identification of vocalizing individuals opens up new and deeper insights into animal communications, such as individual-/group-specific dialects, turn-taking events, and dialogs. However, establishing an association between an individual animal and its emitted signal is usually non-trivial, especially for animals underwater. Consequently, a collection of marine species-, array-, and position-specific ground truth localization data is extremely challenging, which strongly limits possibilities to evaluate localization methods beforehand or at all. This study presents ORCA-SPY, a fully-automated sound source simulation, classification and localization framework for passive killer whale (Orcinus orca) acoustic monitoring that is embedded into PAMGuard, a widely used bioacoustic software toolkit. ORCA-SPY enables array- and position-specific multichannel audio stream generation to simulate real-world ground truth killer whale localization data and provides a hybrid sound source identification approach integrating ANIMAL-SPOT, a state-of-the-art deep learning-based orca detection network, followed by downstream Time-Difference-Of-Arrival localization. ORCA-SPY was evaluated on simulated multichannel underwater audio streams including various killer whale vocalization events within a large-scale experimental setup benefiting from previous real-world fieldwork experience. Across all 58,320 embedded vocalizing killer whale events, subject to various hydrophone array geometries, call types, distances, and noise conditions responsible for a signal-to-noise ratio varying from [Formula: see text] dB to 3 dB, a detection rate of 94.0 % was achieved with an average localization error of 7.01[Formula: see text]. ORCA-SPY was field-tested on Lake Stechlin in Brandenburg Germany under laboratory conditions with a focus on localization. During the field test, 3889 localization events were observed with an average error of 29.19[Formula: see text] and a median error of 17.54[Formula: see text]. ORCA-SPY was deployed successfully during the DeepAL fieldwork 2022 expedition (DLFW22) in Northern British Columbia, with a mean average error of 20.01[Formula: see text] and a median error of 11.01[Formula: see text] across 503 localization events. ORCA-SPY is an open-source and publicly available software framework, which can be adapted to various recording conditions as well as animal species.

Sibling rivalry: training effects, emergence of dominance and incomplete control.

Within-brood or -litter dominance provides fitness-related benefits if dominant siblings selfishly skew access to food provided by parents in their favour. Models of facultative siblicide assume that dominants exert complete control over their subordinate sibling's access to food and that control is maintained, irrespective of the subordinate's hunger level. By contrast, a recent functional hypothesis suggests that subordinates should contest access to food when the cost of not doing so is high. Here, we show that within spotted hyena (Crocuta crocuta) twin litters, dominants most effectively skew access to maternal milk in their favour when their aggression prompts a highly submissive response. When hungry, subordinates were less submissive in response to aggression, thereby decreasing lost suckling time and increasing suckling time lost by dominants. In a species where adult females socially dominate adult males, juvenile females were more often dominant than males in mixed-sex litters, and subordinate sisters used more effective counter-tactics against dominant brothers than subordinate brothers against dominant sisters. Our results provide, to our knowledge, the first evidence in a mammal that dominant offspring in twin litters do not exert complete control over their sibling's access to resources (milk), and that sibling dominance relationships are influenced by sibling sex and training effects.

The impact of a pathogenic bacterium on a social carnivore population.

1. The long-term ecological impact of pathogens on group-living, large mammal populations is largely unknown. We evaluated the impact of a pathogenic bacterium, Streptococcus equi ruminatorum, and other key ecological factors on the dynamics of the spotted hyena Crocuta crocuta population in the Ngorongoro Crater, Tanzania. 2. We compared key demographic parameters during two years when external signs of bacterial infection were prevalent ('outbreak') and periods of five years before and after the outbreak when such signs were absent or rare. We also tested for density dependence and calculated the basic reproductive rate R(0) of the bacterium. 3. During the five pre-outbreak years, the mean annual hyena mortality rate was 0.088, and annual population growth was relatively high (13.6%). During the outbreak, mortality increased by 78% to a rate of 0.156, resulting in an annual population decline of 4.3%. After the outbreak, population size increased moderately (5.1%) during the first three post-outbreak years before resuming a growth similar to pre-outbreak levels (13.9%). We found no evidence that these demographic changes were driven by density dependence or other ecological factors. 4. Most hyenas showed signs of infection when prey abundance in their territory was low. During the outbreak, mortality increased among adult males and yearlings, but not among adult females - the socially dominant group members. These results suggest that infection and mortality were modulated by factors linked to low social status and poor nutrition. During the outbreak, we estimated R(0) for the bacterium to be 2.7, indicating relatively fast transmission. 5. Our results suggest that the short-term 'top-down' impact of S. equi ruminatorum during the outbreak was driven by 'bottom-up' effects on nutritionally disadvantaged age-sex classes, whereas the longer-term post-outbreak reduction in population growth was caused by poor survival of juveniles during the outbreak and subsequent poor recruitment of breeding females. These results suggest synergistic effects of 'bottom-up' and 'top-down' processes on host population dynamics.

Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in spotted hyenas (Crocuta crocuta).

The use of enzyme immunoassays (EIAs) to measure faecal glucocorticoid metabolites (fGCM) is a useful non-invasive technique to monitor adrenocortical activity in vertebrates. The first objective of this study was to validate an 'in-house' EIA (cortisol-3-CMO) for the measurement of fGCM concentrations in spotted hyenas. High-performance liquid chromatography (HPLC) was used to characterise fGCM in samples from a captive hyena that received an i.v. injection of [(3)H] cortisol. All HPLC fractions were analysed with the EIA for the presence and quantities of radiolabelled fGCM. Radiolabelled fGCM consisted of substances with a higher polarity than cortisol and substances of lower polarity that eluted between cortisol and corticosterone. Authentic radiolabelled cortisol was not detected. The EIA measured substantial amounts of immunoreactivity corresponding to the radioactive peaks. It also detected a significant increase in fGCMs after an adrenocorticotropic hormone (ACTH) challenge in two other captive animals and a significant increase in fGCMs in a fourth captive animal after anaesthesia. The second objective was to investigate an age effect on fGCM: we conducted pairwise comparisons of fGCM concentrations in individual free-ranging juvenile spotted hyenas when less than 6 months of age and when between 6 and 24 months of age. We expected juveniles to experience a more unpredictable and therefore more stressful environment when younger than when older. When younger, juveniles had significantly higher fGCM concentrations than when they were older. Our results demonstrate that our assay can be used to assess adrenocortical activity in spotted hyenas.

Female and male Eurasian lynx have distinct spatial tactics at different life-history stages in a high-density population.

Knowledge regarding the spatial behavior of the Eurasian lynx is mainly inferred from populations in Europe. We used GPS telemetry to record the spatial behavior of nine individuals in northwestern Anatolia obtaining eleven home ranges (HRs). Analyses revealed the smallest mean HR sizes (nHR ♀  = 4) at 57 km2 (95% kernel utilization distribution, KUD) and 56 km2 (95% minimum convex polygon, MCP), ever reported for adult female Eurasian lynx. Adult males either occupied small permanent territories (nHR♂.T = 2), with a mean of 176 km2 (95% KUD) and 150 km2 (95% MCP), or were residents without territories (floaters, nHR♂. F  = 2) roaming across large, stable HRs with a mean size of 2,419 km2 (95% KUD) and 1,888 km2 (95% MCP), comparable to HR sizes of Scandinavian lynx populations. Three disperser subadult males did not hold stable HRs (mean 95% KUD = 203 km2, mean 95% MCP = 272 km2). At 4.9 individuals per 100 km2, population density was one of the highest recorded, suggesting that the presence of adult male floaters was a consequence of a landscape fully occupied by territorials and revealing a flexibility of spatial behavior of Eurasian lynx not previously recognized. Such a high population density, small HRs, and behavioral flexibility may have been aided by the legal protection from and apparent low levels of poaching of this population. The observed spatial tactics are unlikely to be seen in most of the previously studied Eurasian lynx populations, as they either suffer medium to high levels of human-caused mortality or were unlikely to be at carrying capacity. For effective and appropriate conservation planning, data from felid populations in a reasonably natural state such as ours, where space, density, prey, and pathogens are likely to be the key drivers of spatial dynamics, are therefore essential.

Noninvasively measured immune responses reflect current parasite infections in a wild carnivore and are linked to longevity.

Host immune defenses are important components of host-parasite interactions that affect the outcome of infection and may have fitness consequences for hosts when increased allocation of resources to immune responses undermines other essential life processes. Research on host-parasite interactions in large free-ranging wild mammals is currently hampered by a lack of verified noninvasive assays. We successfully adapted existing assays to measure innate and adaptive immune responses produced by the gastrointestinal mucosa in spotted hyena (Crocuta crocuta) feces, including enzyme-linked immunosorbent assays (ELISAs), to quantify fecal immunoglobulins (total IgA, total IgG) and total fecal O-linked oligosaccharides (mucin). We investigated the effect of infection load by an energetically costly hookworm (Ancylostoma), parasite richness, host age, sex, year of sampling, and clan membership on immune responses and asked whether high investment in immune responses during early life affects longevity in individually known spotted hyenas in the Serengeti National Park, Tanzania. Fecal concentrations of IgA, IgG, and mucin increased with Ancylostoma egg load and were higher in juveniles than in adults. Females had higher mucin concentrations than males. Juvenile females had higher IgG concentrations than juvenile males, whereas adult females had lower IgG concentrations than adult males. High IgA concentrations during the first year of life were linked to reduced longevity after controlling for age at sampling and Ancylostoma egg load. Our study demonstrates that the use of noninvasive methods can increase knowledge on the complex relationship between gastrointestinal parasites and host local immune responses in wild large mammals and reveal fitness-relevant effects of these responses.

Mitogenome Phylogeny Including Data from Additional Subspecies Provides New Insights into the Historical Biogeography of the Eurasian lynx Lynx lynx.

Previous molecular studies of the wide-ranging Eurasian lynx Lynx lynx focused mainly on its northern Palearctic populations, with the consequence that the reconstruction of this species' evolutionary history did not include genetic variation present in its southern Palearctic distribution. We sampled a previously not considered Asian subspecies (L. l. dinniki), added published data from another Asian subspecies (L. l. isabellinus), and reassessed the Eurasian lynx mtDNA phylogeny along with previously published data from northern Palearctic populations. Our mitogenome-based analyses revealed the existence of three major clades (A: Central Asia, B: SE Europe/SW Asia, C: Europe and Northern Asia) and at least five lineages, with diversification in Lynx lynx commencing at least 28kyr earlier than hitherto estimated. The subspecies L. l. isabellinus harbors the most basal matriline, consistent with the origin of Lynx lynx in this subspecies' current range. L. l. dinniki harbors the second most basal matriline, which is related to, and may be the source of, the mtDNA diversity of the critically endangered Balkan lynx L. l. balcanicus. Our results suggest that the Anatolian peninsula was a glacial refugium for Eurasian lynx, with previously unconsidered implications for the colonization of Europe by this species.