The Ubiquitin Ligase Adaptor NDFIP1 Interacts with TRESK and Negatively Regulates the Background K+ Current.

The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the Xenopus oocyte expression system by two-electrode voltage clamp and biochemical methods. We showed that the coexpression of NDFIP1 abolished the TRESK current under the condition where the other K+ channels were not affected. Mutations in the three PPxY motifs of NDFIP1, which are responsible for the interaction with the Nedd4 ubiquitin ligase, prevented a reduction in the TRESK current. Furthermore, the overexpression of a dominant-negative Nedd4 construct in the oocytes coexpressing TRESK with NDFIP1 partially reversed the down-modulating effect of the adaptor protein on the K+ current. The biochemical data were also consistent with the functional results. An interaction between epitope-tagged versions of TRESK and NDFIP1 was verified by co-immunoprecipitation experiments. The coexpression of NDFIP1 with TRESK induced the ubiquitination of the channel protein. Altogether, the results suggest that TRESK is directly controlled by and highly sensitive to the activation of the NDFIP1-Nedd4 system. The NDFIP1-mediated reduction in the TRESK component may induce depolarization, increase excitability, and attenuate the calcium dependence of the membrane potential by reducing the calcineurin-activated fraction in the ensemble background K+ current.

Life-history stage influences immune investment and oxidative stress in response to environmental heterogeneity in Antarctic fur seals.

Immune defenses are crucial for survival but costly to develop and maintain. Increased immune investment is therefore hypothesized to trade-off with other life-history traits. Here, we examined innate and adaptive immune responses to environmental heterogeneity in wild Antarctic fur seals. In a fully crossed, repeated measures design, we sampled 100 pups and their mothers from colonies of contrasting density during seasons of contrasting food availability. Biometric and cortisol data as well as blood for the analysis of 13 immune and oxidative status markers were collected at two key life-history stages. We show that immune responses of pups are more responsive than adults to variation in food availability, but not population density, and are modulated by cortisol and condition. Immune investment is associated with different oxidative status markers in pups and mothers. Our results suggest that early life stages show greater sensitivity to extrinsic and intrinsic effectors, and that immunity may be a strong target for natural selection even in low-pathogen environments such as Antarctica.

Spatial Variation in the Inflammatory Response of House Sparrows in their Native Range.

Characterizing spatial differences in wildlife immunity is the first step to identify environmental drivers of host defense and disease risks. The house sparrow (Passer domesticus) is a model system for ecoimmunology, but spatial differences in immunity have been largely restricted to the invasive range of this global species. We provide an initial test of spatial variation in immune response to phytohemagglutinin in the native range, finding that birds from Romania have greater inflammatory responses than birds from Egypt. Future broad surveys across the house sparrow native range could contextualize these differences and determine underlying drivers.

Oxidative damage varies in response to bacterial, fungal and viral antigen challenges in bats.

The immune system plays an important role in defending against pathogens and regulating physiological homeostasis, but the strength of the immune responses depends on the type of pathogen. The immune system of bats shows a high variability in responsiveness towards various pathogens; they can safely harbor certain pathogens that are highly lethal to other mammals. Oxidative stress may act as a pathophysiological cellular mechanism mediating the immunological function of bats because of its potentially detrimental effects on physiological homeostasis, fertility and longevity. By experimentally exposing greater mouse-eared bats (Myotis myotis) to three antigens, it was previously shown that animals reacted immunologically most strongly to bacterial and viral antigens, but not to fungal ones. As a follow up, in this study we observed that both bacterial and fungal antigens induced a significant increase of plasma oxidative damage, whereas viral antigens did not cause any increase of plasma oxidative damage at all albeit the mild immune response. Thus, experimental bats were able to avoid oxidative stress only in the face of a viral antigen, possibly by dampening inflammatory signalling. Bats may be able to handle viral infections and live well beyond expectations by reducing the detrimental effects of molecular oxidation.

In sickness and in health: the dynamics of the fruit bat gut microbiota under a bacterial antigen challenge and its association with the immune response.

Introduction: Interactions between the gut microbiome (GM) and the immune system influence host health and fitness. However, few studies have investigated this link and GM dynamics during disease in wild species. Bats (Mammalia: Chiroptera) have an exceptional ability to cope with intracellular pathogens and a unique GM adapted to powered flight. Yet, the contribution of the GM to bat health, especially immunity, or how it is affected by disease, remains unknown. Methods: Here, we examined the dynamics of the Egyptian fruit bats' (Rousettus aegyptiacus) GM during health and disease. We provoked an inflammatory response in bats using lipopolysaccharides (LPS), an endotoxin of Gram-negative bacteria. We then measured the inflammatory marker haptoglobin, a major acute phase protein in bats, and analyzed the GM (anal swabs) of control and challenged bats using high-throughput 16S rRNA sequencing, before the challenge, 24h and 48h post challenge. Results: We revealed that the antigen challenge causes a shift in the composition of the bat GM (e.g., Weissella, Escherichia, Streptococcus). This shift was significantly correlated with haptoglobin concentration, but more strongly with sampling time. Eleven bacterial sequences were correlated with haptoglobin concentration and nine were found to be potential predictors of the strength of the immune response, and implicit of infection severity, notably Weissella and Escherichia. The bat GM showed high resilience, regaining the colony's group GM composition rapidly, as bats resumed foraging and social activities. Conclusion: Our results demonstrate a tight link between bat immune response and changes in their GM, and emphasize the importance of integrating microbial ecology in ecoimmunological studies of wild species. The resilience of the GM may provide this species with an adaptive advantage to cope with infections and maintain colony health.

Critical contribution of the intracellular C-terminal region to TRESK channel activity is revealed by the epithelial Na+ current ratio method.

TRESK (K2P18.1) possesses unique structural proportions within the K2P background potassium channel family. The previously described TRESK regulatory mechanisms are based on the long intracellular loop between the second and the third transmembrane segments (TMS). However, the functional significance of the exceptionally short intracellular C-terminal region (iCtr) following the fourth TMS has not yet been examined. In the present study, we investigated TRESK constructs modified at the iCtr by two-electrode voltage clamp and the newly developed epithelial sodium current ratio (ENaR) method in Xenopus oocytes. The ENaR method allowed the evaluation of channel activity by exclusively using electrophysiology and provided data that are otherwise not readily available under whole-cell conditions. TRESK homodimer was connected with two ENaC (epithelial Na+ channel) heterotrimers, and the Na+ current was measured as an internal reference, proportional to the number of channels in the plasma membrane. Modifications of TRESK iCtr resulted in diverse functional effects, indicating a complex contribution of this region to K+ channel activity. Mutations of positive residues in proximal iCtr locked TRESK in low activity, calcineurin-insensitive state, although this phosphatase binds to distant motifs in the loop region. Accordingly, mutations in proximal iCtr may prevent the transmission of modulation to the gating machinery. Replacing distal iCtr with a sequence designed to interact with the inner surface of the plasma membrane increased the activity of the channel to unprecedented levels, as indicated by ENaR and single channel measurements. In conclusion, the distal iCtr is a major positive determinant of TRESK function.

Bacterial killing activity and lysozymes: A stable defence mechanism in stallion seminal plasma?

During insemination, bacterial contamination of the ejaculate can lead to reduced sperm quality and transmission of pathogens to the female, thus should be avoided. The semen of a variety of animal taxa possess antimicrobial properties against a wide range of bacterial species through antimicrobial molecules, such as lysozyme, but their variance and the factors influencing it are unknown for most species. In this study, the antibacterial defence (bacterial killing activity (BKA) against Escherichia (E.) coli and Staphylococcus (S.) aureus as well as lysozyme concentration) was studied in seminal fluid from two consecutive ejaculates of 18 stallions. All ejaculates showed BKA against the tested bacteria, which correlated between the two consecutive ejaculates (rS  = 0.526, p = .025 for E. coli and rS  = 0.656, p = .003 for S. aureus) and appeared to be stable over the tested period. The lysozyme concentration (LC) showed no significant correlation between the consecutive ejaculates (rS  = 0.161, p = .681). However, LC had a positive correlation to the ratio of apoptotic spermatozoa within the ejaculates (rS  = 0.426, p = .019). In contrast to other livestock (e.g., boar, bull), the BKA in stallion semen did not correlate significantly with the age of the animal nor sperm quality characteristics.

Seasonal challenges of tropical bats in temperate zones.

To examine the challenges faced by free-ranging Rousettus aegyptiacus living at the northern edge of their distribution, we performed a retrospective analysis of 2196 clinical cases reported by a bat rescue NGO over a period of 36 months, from throughout Israel. All cases of injured bats were evaluated and categorized according to date, place, sex, age, and etiology of the morbidity. The data analysis revealed an increase in all types of morbidity during the wintertime, with more than two-fold the number of cases per week compared to in the summer, over three consecutive years. Moreover, we found that the number of abandoned pups peaked during spring and summer, when adult morbidity is minimal. We characterized two prominent types of previously undescribed morbidities in R. aegyptiacus. We also employed GPS tracking to monitor the movement and foraging of dozens of bats, and to examine the potential correlates of elevated winter morbidity. Our results suggest that it is mainly harsh weather that drives the observed winter morbidity, with food limitations playing a minor-role. We hypothesize that R. aegyptiacus, of tropical origin, is facing major seasonal survival difficulties near the northern edge of its distribution, probably limiting its spread further northwards still.

Differences in acute phase response to bacterial, fungal and viral antigens in greater mouse-eared bats (Myotis myotis).

The acute phase response (APR) is an evolutionarily well-conserved part of the innate immune defense against pathogens. However, recent studies in bats yielded surprisingly diverse results compared to previous APR studies on both vertebrate and invertebrate species. This is especially interesting due to the known role of bats as reservoirs for viruses and other intracellular pathogens, while being susceptible to extracellular microorganisms such as some bacteria and fungi. To better understand these discrepancies and the reservoir-competence of bats, we mimicked bacterial, viral and fungal infections in greater mouse-eared bats (Myotis myotis) and quantified different aspects of the APR over a two-day period. Individuals reacted most strongly to a viral (PolyI:C) and a bacterial (LPS) antigen, reflected by an increase of haptoglobin levels (LPS) and an increase of the neutrophil-to-lymphocyte-ratio (PolyI:C and LPS). We did not detect fever, leukocytosis, body mass loss, or a change in the overall functioning of the innate immunity upon challenge with any antigen. We add evidence that bats respond selectively with APR to specific pathogens and that the activation of different parts of the immune system is species-specific.

First Steps towards the Development of Epigenetic Biomarkers in Female Cheetahs (Acinonyx jubatus).

Free-ranging cheetahs (Acinonyx jubatus) are generally healthy, whereas cheetahs under human care, such as those in zoological gardens, suffer from ill-defined infectious and degenerative pathologies. These differences are only partially explained by husbandry management programs because both groups share low genetic diversity. However, mounting evidence suggests that physiological differences between populations in different environments can be tracked down to differences in epigenetic signatures. Here, we identified differentially methylated regions (DMRs) between free-ranging cheetahs and conspecifics in zoological gardens and prospect putative links to pathways relevant to immunity, energy balance and homeostasis. Comparing epigenomic DNA methylation profiles obtained from peripheral blood mononuclear cells (PBMCs) from eight free-ranging female cheetahs from Namibia and seven female cheetahs living in zoological gardens within Europe, we identified DMRs of which 22 were hypermethylated and 23 hypomethylated. Hypermethylated regions in cheetahs under human care were located in the promoter region of a gene involved in host-pathogen interactions (KLC1) and in an intron of a transcription factor relevant for the development of pancreatic ß-cells, liver, and kidney (GLIS3). The most canonical mechanism of DNA methylation in promoter regions is assumed to repress gene transcription. Taken together, this could indicate that hypermethylation at the promoter region of KLC1 is involved in the reduced immunity in cheetahs under human care. This approach can be generalized to characterize DNA methylation profiles in larger cheetah populations under human care with a more granular longitudinal data collection, which, in the future, could be used to monitor the early onset of pathologies, and ultimately translate into the development of biomarkers with prophylactic and/or therapeutic potential.

Induced bacterial sickness causes inflammation but not blood oxidative stress in Egyptian fruit bats (Rousettus aegyptiacus).

Bats are particularly interesting vertebrates in their response to pathogens owing to extremes in terms of tolerance and resistance. Oxidation is often a by-product of processes involved in the acute phase response, which may result in antimicrobial or self-damaging effects. We measured the immunological and oxidative status responses of Egyptian fruit bats (Rousettus aegyptiacus) to a simulated bacterial infection using lipopolysaccharide injection. As expected, experimental bats exhibited increases in two humoral immunological markers. However, they surprisingly did not show any effects across two markers of oxidative damage and four antioxidant markers. We propose that this lack of effects on oxidative status may be due to a reduction in cell metabolism through sickness behaviours or given life history traits, such as a long lifespan and a frugivorous diet. Finally, the consistency in the pattern of elevation in haptoglobin and lysozyme between current and previous findings highlights their utility as diagnostic markers for extracellular infections in bats.

An assessment of mercury and its dietary drivers in fur of Arctic wolves from Greenland and High Arctic Canada.

Mercury has become a ubiquitous hazardous element even ending up in pristine areas such as the Arctic, where it biomagnifies and leaves especially top predators vulnerable to potential health effects. Here we investigate total mercury (THg) concentrations and dietary proxies for trophic position and habitat foraging (δ15N and δ13C, respectively) in fur of 30 Arctic wolves collected during 1869-1998 in the Canadian High Arctic and Greenland. Fur THg concentrations (mean ± SD) of 1.46 ± 1.39 µg g -1 dry weight are within the range of earlier reported values for other Arctic terrestrial species. Based on putative thresholds for Hg-mediated toxic health effects, the studied Arctic wolves have most likely not been at compromised health. Dietary proxies show high dietary plasticity among Arctic wolves deriving nutrition from both marine and terrestrial food sources at various trophic positions. Variability in THg concentrations seem to be related to the wolves' trophic position rather than to different carbon sources or regional differences (East Greenland, the Foxe Basin and Baffin Bay area, respectively). Although the present study remains limited due to the scarce, yet unique historic study material and small sample size, it provides novel information on temporal and spatial variation in Hg pollution of remote Arctic species.

Serological Evidence That SARS-CoV-2 Has Not Emerged in Deer in Germany or Austria during the COVID-19 Pandemic.

Spillover of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) to North American white-tailed deer (Odocoileus virginianus) has been documented. However, it is unclear if this is a phenomenon specific to North American deer or is a broader problem. We evaluated pre and pandemic exposure of German and Austrian deer species using a SARS-CoV-2 pseudoneutralization assay. In stark contrast to North American white-tailed deer, we found no evidence of SARS-CoV-2 exposure.

Differences in constitutive innate immunity between divergent Australian marsupials.

Understanding immunity in wildlife populations is important from both One Health and conservation perspectives. The constitutive innate immune system is the first line of defence against pathogens, and comparisons among taxa can test the impact of evolution and life history on immune function. We investigated serum bacterial killing ability (BKA) of five marsupial species that employ varying life history strategies, demonstrated to influence immunity in other vertebrates. The brushtail possum and eastern grey kangaroo had the greatest BKA, while ringtail possums and koalas had the least. These differences were independent of social structure, captivity status and phylogeny, but were associated with diet and body size. Sex and disease status had no effect on BKA in koalas, however potential for differences between wild and captive koalas warrants further investigation. The current study has provided a foundation for future investigations into how adaptive and innate immunity interact in marsupials from an eco-evolutionary perspective.

Sexual dimorphism in immune function and oxidative physiology across birds: The role of sexual selection.

Sex-specific physiology is commonly reported in animals, often indicating lower immune indices and higher oxidative stress in males than in females. Sexual selection is argued to explain these differences, but empirical evidence is limited. Here, we explore sex differences in immunity, oxidative physiology and packed cell volume of wild, adult, breeding birds (97 species, 1997 individuals, 14 230 physiological measurements). We show that higher female immune indices are most common across birds (when bias is present), but oxidative physiology shows no general sex-bias and packed cell volume is generally male-biased. In contrast with predictions based on sexual selection, male-biased sexual size dimorphism is associated with male-biased immune measures. Sexual dichromatism, mating system and parental roles had no effect on sex-specificity in physiology. Importantly, female-biased immunity remained after accounting for sexual selection indices. We conclude that cross-species differences in physiological sex-bias are largely unrelated to sexual selection and alternative explanations should be explored.

Translocation of TMEM175 Lysosomal Potassium Channel to the Plasma Membrane by Dynasore Compounds.

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson's disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.

Applied ecoimmunology: using immunological tools to improve conservation efforts in a changing world.

Ecoimmunology is a rapidly developing field that explores how the environment shapes immune function, which in turn influences host-parasite relationships and disease outcomes. Host immune defence is a key fitness determinant because it underlies the capacity of animals to resist or tolerate potential infections. Importantly, immune function can be suppressed, depressed, reconfigured or stimulated by exposure to rapidly changing environmental drivers like temperature, pollutants and food availability. Thus, hosts may experience trade-offs resulting from altered investment in immune function under environmental stressors. As such, approaches in ecoimmunology can provide powerful tools to assist in the conservation of wildlife. Here, we provide case studies that explore the diverse ways that ecoimmunology can inform and advance conservation efforts, from understanding how Galapagos finches will fare with introduced parasites, to using methods from human oncology to design vaccines against a transmissible cancer in Tasmanian devils. In addition, we discuss the future of ecoimmunology and present 10 questions that can help guide this emerging field to better inform conservation decisions and biodiversity protection. From better linking changes in immune function to disease outcomes under different environmental conditions, to understanding how individual variation contributes to disease dynamics in wild populations, there is immense potential for ecoimmunology to inform the conservation of imperilled hosts in the face of new and re-emerging pathogens, in addition to improving the detection and management of emerging potential zoonoses.

GPS Telemetry Reveals a Zebra With Anthrax as Putative Cause of Death for Three Cheetahs in the Namib Desert.

Anthrax is a bacterial disease caused by Bacillus anthracis that affects wildlife, livestock and also humans in different parts of the world. It is endemic in some parts of Africa, including Namibia, with species differing in their susceptibility to the disease. Carnivores are typically less susceptible to anthrax than herbivores. Most carnivore species survive infection and have high seroprevalence against anthrax, whereas most herbivore species have low seroprevalence and typically die quickly when infected. Several reports have shown that cheetahs, unlike most other large carnivores, are susceptible to anthrax leading to a sudden death. This finding was suggested to be linked to the low genetic variability of cheetahs which might reduce an adequate immune response and thus explain such a high susceptibility to the disease. Here, we report an incidence of three free-ranging cheetahs that died within 24 h after feeding on a mountain zebra that tested positive for anthrax in the Namib Desert. We were able to reconstruct this incidence with the data recorded in the GPS (Global Positioning System) collar worn by one of the cheetahs and retrieved in the field. It is very likely that the cheetahs died from anthrax, although Bacillus anthracis could not be isolated from tissue and soil samples by bacterial culturing. The mountain zebra is the first described case of a wild animal that tested positive for anthrax in this arid area in southwestern of Namibia. We discuss the negative laboratory results of the cheetahs in the light of new insights of their immune system and its potential to mount a response against this bacteria.

Negative Influence by the Force: Mechanically Induced Hyperpolarization via K2P Background Potassium Channels.

The two-pore domain K2P subunits form background (leak) potassium channels, which are characterized by constitutive, although not necessarily constant activity, at all membrane potential values. Among the fifteen pore-forming K2P subunits encoded by the KCNK genes, the three members of the TREK subfamily, TREK-1, TREK-2, and TRAAK are mechanosensitive ion channels. Mechanically induced opening of these channels generally results in outward K+ current under physiological conditions, with consequent hyperpolarization and inhibition of membrane potential-dependent cellular functions. In the past decade, great advances have been made in the investigation of the molecular determinants of mechanosensation, and members of the TREK subfamily have emerged among the best-understood examples of mammalian ion channels directly influenced by the tension of the phospholipid bilayer. In parallel, the crucial contribution of mechano-gated TREK channels to the regulation of membrane potential in several cell types has been reported. In this review, we summarize the general principles underlying the mechanical activation of K2P channels, and focus on the physiological roles of mechanically induced hyperpolarization.