A taste of wilderness: supplementary feeding of red deer (Cervus elaphus) increases individual bacterial microbiota diversity but lowers abundance of important gut symbionts.

The gut microbiome plays a crucial role in the health and well-being of animals. It is especially critical for ruminants that depend on this bacterial community for digesting their food. In this study, we investigated the effects of management conditions and supplemental feeding on the gut bacterial microbiota of red deer (Cervus elaphus) in the Bavarian Forest National Park, Germany. Fecal samples were collected from free-ranging deer, deer within winter enclosures, and deer in permanent enclosures. The samples were analyzed by high-throughput sequencing of the 16 S rRNA gene. The results showed that the gut bacterial microbiota differed in diversity, abundance, and heterogeneity within and between the various management groups. Free-ranging deer exhibited lower alpha diversity compared with deer in enclosures, probably because of the food supplementation available to the animals within the enclosures. Free-living individuals also showed the highest beta diversity, indicating greater variability in foraging grounds and plant species selection. Moreover, free-ranging deer had the lowest abundance of potentially pathogenic bacterial taxa, suggesting a healthier gut microbiome. Winter-gated deer, which spent some time in enclosures, exhibited intermediate characteristics between free-ranging and all-year-gated deer. These findings suggest that the winter enclosure management strategy, including supplementary feeding with processed plants and crops, has a significant impact on the gut microbiome composition of red deer. Overall, this study provides important insights into the effects of management conditions, particularly winter enclosure practices, on the gut microbiome of red deer. Understanding these effects is crucial for assessing the potential health implications of management strategies and highlights the value of microbiota investigations as health marker.

Bat species assemblage predicts coronavirus prevalence.

Anthropogenic disturbances and the subsequent loss of biodiversity are altering species abundances and communities. Since species vary in their pathogen competence, spatio-temporal changes in host assemblages may lead to changes in disease dynamics. We explore how longitudinal changes in bat species assemblages affect the disease dynamics of coronaviruses (CoVs) in more than 2300 cave-dwelling bats captured over two years from five caves in Ghana. This reveals uneven CoV infection patterns between closely related species, with the alpha-CoV 229E-like and SARS-related beta-CoV 2b emerging as multi-host pathogens. Prevalence and infection likelihood for both phylogenetically distinct CoVs is influenced by the abundance of competent species and naïve subadults. Broadly, bat species vary in CoV competence, and highly competent species are more common in less diverse communities, leading to increased CoV prevalence in less diverse bat assemblages. In line with the One Health framework, our work supports the notion that biodiversity conservation may be the most proactive measure to prevent the spread of pathogens with zoonotic potential.

MHC-I alleles mediate clearance and antibody response to the zoonotic Lassa virus in Mastomys rodent reservoirs.

West African Mastomys rodents are the primary reservoir of the zoonotic Lassa virus (LASV). The virus causes haemorrhagic Lassa fever and considerable mortality in humans. To date, the role of Mastomys immunogenetics in resistance to, and persistence of, LASV infections is largely unknown. Here, we investigated the role of Major Histocompatibility Complex class I (MHC-I) on LASV infection status (i.e., active vs. cleared infection, determined via PCR and an immunofluorescence assay on IgG antibodies, respectively) in Mastomys natalensis and M. erythroleucus sampled within southwestern Nigeria. We identified more than 190 and 90 MHC-I alleles by Illumina high throughput-sequencing in M. natalensis and M. erythroleucus, respectively, with different MHC allele compositions and frequencies between LASV endemic and non-endemic sites. In M. natalensis, the MHC allele ManaMHC-I*006 was negatively associated with active infections (PCR-positive) and positively associated with cleared infections (IgG-positive) simultaneously, suggesting efficient immune responses that facilitate LASV clearance in animals carrying this allele. Contrarily, alleles ManaMHC-I*008 and ManaMHC-I*021 in M. natalensis, and MaerMHC-I*008 in M. erythroleucus, were positively associated with active infection, implying susceptibility. Alleles associated with susceptibility shared a glutamic acid at the positively selected codon 57, while ManaMHC-I*006 featured an arginine. There was no link between number of MHC alleles per Mastomys individual and LASV prevalence. Thus, specific alleles, but not MHC diversity per se, seem to mediate antibody responses to viremia. We conclude that co-evolution with LASV likely shaped the MHC-I diversity of the main LASV reservoirs in southwestern Nigeria, and that information on reservoir immunogenetics may hold insights into transmission dynamics and zoonotic spillover risks.

Immunogenetic-pathogen networks shrink in Tome's spiny rat, a generalist rodent inhabiting disturbed landscapes.

Anthropogenic disturbance may increase the emergence of zoonoses. Especially generalists that cope with disturbance and live in close contact with humans and livestock may become reservoirs of zoonotic pathogens. Yet, whether anthropogenic disturbance modifies host-pathogen co-evolutionary relationships in generalists is unknown. We assessed pathogen diversity, neutral genome-wide diversity (SNPs) and adaptive MHC class II diversity in a rodent generalist inhabiting three lowland rainforest landscapes with varying anthropogenic disturbance, and determined which MHC alleles co-occurred more frequently with 13 gastrointestinal nematodes, blood trypanosomes, and four viruses. Pathogen-specific selection pressures varied between landscapes. Genome-wide diversity declined with the degree of disturbance, while MHC diversity was only reduced in the most disturbed landscape. Furthermore, pristine forest landscapes had more functional important MHC-pathogen associations when compared to disturbed forests. We show co-evolutionary links between host and pathogens impoverished in human-disturbed landscapes. This underscores that parasite-mediated selection might change even in generalist species following human disturbance which in turn may facilitate host switching and the emergence of zoonoses.

Immunogenetics, sylvatic plague and its vectors: insights from the pathogen reservoir Mastomys natalensis in Tanzania.

Yersinia pestis is a historically important vector-borne pathogen causing plague in humans and other mammals. Contemporary zoonotic infections with Y. pestis still occur in sub-Saharan Africa, including Tanzania and Madagascar, but receive relatively little attention. Thus, the role of wildlife reservoirs in maintaining sylvatic plague and spillover risks to humans is largely unknown. The multimammate rodent Mastomys natalensis is the most abundant and widespread rodent in peri-domestic areas in Tanzania, where it plays a major role as a Y. pestis reservoir in endemic foci. Yet, how M. natalensis' immunogenetics contributes to the maintenance of plague has not been investigated to date. Here, we surveyed wild M. natalensis for Y. pestis vectors, i.e., fleas, and tested for the presence of antibodies against Y. pestis using enzyme-linked immunosorbent assays (ELISA) in areas known to be endemic or without previous records of Y. pestis in Tanzania. We characterized the allelic and functional (i.e., supertype) diversity of the major histocompatibility complex (MHC class II) of M. natalensis and investigated links to Y. pestis vectors and infections. We detected antibodies against Y. pestis in rodents inhabiting both endemic areas and areas considered non-endemic. Of the 111 nucleotide MHC alleles, only DRB*016 was associated with an increased infestation with the flea Xenopsylla. Surprisingly, we found no link between MHC alleles or supertypes and antibodies of Y. pestis. Our findings hint, however, at local adaptations towards Y. pestis vectors, an observation that more exhaustive sampling could unwind in the future.

Climate change drives loss of bacterial gut mutualists at the expense of host survival in wild meerkats.

Climate change and climate-driven increases in infectious disease threaten wildlife populations globally. Gut microbial responses are predicted to either buffer or exacerbate the negative impacts of these twin pressures on host populations. However, examples that document how gut microbial communities respond to long-term shifts in climate and associated disease risk, and the consequences for host survival, are rare. Over the past two decades, wild meerkats inhabiting the Kalahari have experienced rapidly rising temperatures, which is linked to the spread of tuberculosis (TB). We show that over the same period, the faecal microbiota of this population has become enriched in Bacteroidia and impoverished in lactic acid bacteria (LAB), a group of bacteria including Lactococcus and Lactobacillus that are considered gut mutualists. These shifts occurred within individuals yet were compounded over generations, and were better explained by mean maximum temperatures than mean rainfall over the previous year. Enriched Bacteroidia were additionally associated with TB exposure and disease, the dry season and poorer body condition, factors that were all directly linked to reduced future survival. Lastly, abundances of LAB taxa were independently and positively linked to future survival, while enriched taxa did not predict survival. Together, these results point towards extreme temperatures driving an expansion of a disease-associated pathobiome and loss of beneficial taxa. Our study provides the first evidence from a longitudinally sampled population that climate change is restructuring wildlife gut microbiota, and that these changes may amplify the negative impacts of climate change through the loss of gut mutualists. While the plastic response of host-associated microbiotas is key for host adaptation under normal environmental fluctuations, extreme temperature increases might lead to a breakdown of coevolved host-mutualist relationships.

MHC class II genes mediate susceptibility and resistance to coronavirus infections in bats.

Understanding the immunogenetic basis of coronavirus (CoV) susceptibility in major pathogen reservoirs, such as bats, is central to inferring their zoonotic potential. Members of the cryptic Hipposideros bat species complex differ in CoV susceptibility, but the underlying mechanisms remain unclear. The genes of the major histocompatibility complex (MHC) are the best understood genetic basis of pathogen resistance, and differences in MHC diversity are one possible reason for asymmetrical infection patterns among closely related species. Here, we aimed to link asymmetries in observed CoV (CoV-229E, CoV-2B and CoV-2Bbasal) susceptibility to immunogenetic differences amongst four Hipposideros bat species. From the 2072 bats assigned to their respective species using the mtDNA cytochrome b gene, members of the most numerous and ubiquitous species, Hipposideros caffer D, were most infected with CoV-229E and SARS-related CoV-2B. Using a subset of 569 bats, we determined that much of the existent allelic and functional (i.e. supertype) MHC DRB class II diversity originated from common ancestry. One MHC supertype shared amongst all species, ST12, was consistently linked to susceptibility with CoV-229E, which is closely related to the common cold agent HCoV-229E, and infected bats and those carrying ST12 had a lower body condition. The same MHC supertype was connected to resistance to CoV-2B, and bats with ST12 were less likely be co-infected with CoV-229E and CoV-2B. Our work suggests a role of immunogenetics in determining CoV susceptibility in bats. We advocate for the preservation of functional genetic and species diversity in reservoirs as a means of mitigating the risk of disease spillover.

Wildlife gut microbiomes of sympatric generalist species respond differently to anthropogenic landscape disturbances.

BACKGROUND: Human encroachment into nature and the accompanying environmental changes are a big concern for wildlife biodiversity and health. While changes on the macroecological scale, i.e. species community and abundance pattern, are well documented, impacts on the microecological scale, such as the host's microbial community, remain understudied. Particularly, it is unclear if impacts of anthropogenic landscape modification on wildlife gut microbiomes are species-specific. Of special interest are sympatric, generalist species, assumed to be more resilient to environmental changes and which often are well-known pathogen reservoirs and drivers of spill-over events. Here, we analyzed the gut microbiome of three such sympatric, generalist species, one rodent (Proechimys semispinosus) and two marsupials (Didelphis marsupialis and Philander opossum), captured in 28 study sites in four different landscapes in Panama characterized by different degrees of anthropogenic disturbance. RESULTS: Our results show species-specific gut microbial responses to the same landscape disturbances. The gut microbiome of P. semispinosus was less diverse and more heterogeneous in landscapes with close contact with humans, where it contained bacterial taxa associated with humans, their domesticated animals, and potential pathogens. The gut microbiome of D. marsupialis showed similar patterns, but only in the most disturbed landscape. P. opossum, in contrast, showed little gut microbial changes, however, this species' absence in the most fragmented landscapes indicates its sensitivity to long-term isolation. CONCLUSION: These results demonstrate that wildlife gut microbiomes even in generalist species with a large ecological plasticity are impacted by human encroachment into nature, but differ in resilience which can have critical implications on conservation efforts and One Health strategies.

Disentangling direct from indirect effects of habitat disturbance on multiple components of biodiversity.

Human habitat disturbance affects both species diversity and intraspecific genetic diversity, leading to correlations between these two components of biodiversity (termed species-genetic diversity correlation, SGDC). However, whether SGDC predictions extend to host-associated communities, such as the intestinal parasite and gut microbial diversity, remains largely unexplored. Additionally, the role of dominant generalist species is often neglected despite their importance in shaping the environment experienced by other members of the ecological community, and their role as source, reservoir and vector of zoonotic diseases. New analytical approaches (e.g. structural equation modelling, SEM) can be used to assess SGDC relationships and distinguish among direct and indirect effects of habitat characteristics and disturbance on the various components of biodiversity. With six concrete and biologically sound models in mind, we collected habitat characteristics of 22 study sites from four distinct landscapes located in central Panama. Each landscape differed in the degree of human disturbance and fragmentation measured by several quantitative variables, such as canopy cover, canopy height and understorey density. In terms of biodiversity, we estimated on the one hand, (a) small mammal species diversity, and, on the other hand, (b) genome-wide diversity, (c) intestinal parasite diversity and (d) gut microbial heterogeneity of the most dominant generalist species (Tome's spiny rat, Proechimys semispinosus). We used SEMs to assess the links between habitat characteristics and biological diversity measures. The best supported SEM suggested that habitat characteristics directly and positively affect the richness of small mammals, the genetic diversity of P. semispinosus and its gut microbial heterogeneity. Habitat characteristics did not, however, directly impact intestinal parasite diversity. We also detected indirect, positive effects of habitat characteristics on both host-associated assemblages via small mammal richness. For microbes, this is likely linked to cross species transmission, particularly in shared and/or anthropogenically altered habitats, whereas host diversity mitigates parasite infections. The SEM revealed an additional indirect but negative effect on intestinal parasite diversity via host genetic diversity. Our study showcases that habitat alterations not only affect species diversity and host genetic diversity in parallel, but also species diversity of host-associated assemblages. The impacts from human disturbance are therefore expected to ripple through entire ecosystems with far reaching effects felt even by generalist species.

Las perturbaciones antropogénicas sobre los hábitats naturales pueden afectar tanto a la diversidad de las especies como a la diversidad genética intraespecífica, dando lugar a correlaciones entre estos dos elementos de la biodiversidad (denominados correlación de la diversidad genética de las especies, SGDC por sus siglas en inglés). Sin embargo, todavía queda sin explorar si las predicciones de la SGDC afectan a las comunidades de parásitos y microorganismos intestinales asociadas al hospedador. Adicionalmente, el rol que juegan las especies generalistas, especialmente aquéllas dominantes, suele ser descuidado, a pesar de la importancia de control que ejercen sobre la estructura de la comunidad, y su rol como fuente, reservorio y vector de enfermedades zoonóticas. Para poder evaluar las relaciones de SGDC y distinguir entre los efectos directos e indirectos que tienen las características del hábitat y las perturbaciones sobre los distintos componentes de la biodiversidad, se pueden utilizar nuevos enfoques analíticos como por ejemplo los modelos de ecuaciones estructurales (SEM, por sus siglas en inglés). Considerando seis modelos específicos y biológicamente sólidos, recopilamos las características del hábitat de 22 sitios ubicados en cuatro paisajes distintos situados en el centro de Panamá. Cada paisaje difería en el grado de perturbación antropogénica y fragmentación, medido por diferentes variables cuantitativas, como la cobertura del dosel, la altura del dosel y la densidad del sotobosque. En términos de biodiversidad, por un lado estimamos (1) la diversidad de especies de pequeños mamíferos y, por otro lado (2) la diversidad del genoma completo, (3) la diversidad de parásitos intestinales, y (4) la heterogeneidad de las comunidades microbianas del intestino de la especie generalista más dominante, la rata espinosa de Tomes Proechimys semispinosus. Para evaluar los vínculos entre las características del hábitat y las medidas de diversidad biológica se utilizó el modelado SEM. El SEM mejor apoyado sugirió que las características del hábitat afectan directa y positivamente a la abundancia de pequeños mamíferos, a la diversidad genética de P. semispinosus y a la heterogeneidad microbiana intestinal. Sin embargo, se observó que las características del hábitat no tienen un efecto directo en la diversidad de parásitos intestinales. Aparte de estos efectos directos, detectamos efectos indirectos y positivos de las características del hábitat en ambos conjuntos asociados al hospedador (diversidad de parásitos y microorganismos intestinales) a través de la abundancia de pequeños mamíferos. En el caso de las comunidades microbianas, esto está probablemente relacionado con la transmisión interespecífica, especialmente en hábitats compartidos y/o antropogénicamente alterados; mientras que la diversidad de hospedadores mitiga las infecciones de parásitos. El SEM reveló un efecto indirecto adicional pero negativo sobre la diversidad de parásitos intestinales a través de la diversidad genética de los hospedadores. Nuestro estudio muestra que los patrones de SGDC se filtran a través de las varias capas de diversidad biológica, añadiendo los ensamblajes asociados al hospedador como componentes biológicos afectados por las alteraciones del hábitat.

Gut microbiota individuality is contingent on temporal scale and age in wild meerkats.

Inter-individual differences in gut microbiota composition are hypothesized to generate variation in host fitness-a premise for the evolution of host-gut microbe symbioses. However, recent evidence suggests that gut microbial communities are highly dynamic, challenging the notion that individuals harbour unique gut microbial phenotypes. Leveraging a long-term dataset of wild meerkats, we reconcile these concepts by demonstrating that the relative importance of identity for shaping gut microbiota phenotypes depends on the temporal scale. Across meerkat lifespan, year-to-year variation overshadowed the effects of identity and social group in predicting gut microbiota composition, with identity explaining on average less than 2% of variation. However, identity was the strongest predictor of microbial phenotypes over short sampling intervals (less than two months), predicting on average 20% of variation. The effect of identity was also dependent on meerkat age, with the gut microbiota becoming more individualized and stable as meerkats aged. Nevertheless, while the predictive power of identity was negligible after two months, gut microbiota composition remained weakly individualized compared to that of other meerkats for up to 1 year. These findings illuminate the degree to which individualized gut microbial signatures can be expected, with important implications for the time frames over which gut microbial phenotypes may mediate host physiology, behaviour and fitness in natural populations.

A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth.

Angiogenesis, the process by which endothelial cells (ECs) form new blood vessels from existing ones, is intimately linked to the tissue's metabolic milieu and often occurs at nutrient-deficient sites. However, ECs rely on sufficient metabolic resources to support growth and proliferation. How endothelial nutrient acquisition and usage are regulated is unknown. Here we show that these processes are instructed by Yes-associated protein 1 (YAP)/WW domain-containing transcription regulator 1 (WWTR1/TAZ)-transcriptional enhanced associate domain (TEAD): a transcriptional module whose function is highly responsive to changes in the tissue environment. ECs lacking YAP/TAZ or their transcriptional partners, TEAD1, 2 and 4 fail to divide, resulting in stunted vascular growth in mice. Conversely, activation of TAZ, the more abundant paralogue in ECs, boosts proliferation, leading to vascular hyperplasia. We find that YAP/TAZ promote angiogenesis by fuelling nutrient-dependent mTORC1 signalling. By orchestrating the transcription of a repertoire of cell-surface transporters, including the large neutral amino acid transporter SLC7A5, YAP/TAZ-TEAD stimulate the import of amino acids and other essential nutrients, thereby enabling mTORC1 activation. Dissociating mTORC1 from these nutrient inputs-elicited by the loss of Rag GTPases-inhibits mTORC1 activity and prevents YAP/TAZ-dependent vascular growth. Together, these findings define a pivotal role for YAP/TAZ-TEAD in controlling endothelial mTORC1 and illustrate the essentiality of coordinated nutrient fluxes in the vasculature.

Transcending sea turtles: First report of hatching failure in eggs of an Amazonian freshwater turtle with symptoms of the fungal emerging disease fusariosis.

During the last few decades, fungal pathogens have caused devastating population declines across a broad range of taxa. A newly emerging fungal disease, sea turtle egg fusariosis, caused by members of the Fusarium solani species complex (FSSC), has been reported to be responsible for hatching failure in sea turtles worldwide. However, this has not been detected in fresh water turtle species. Here, using relocated clutches and artificial incubation, we report high hatching failure in eggs symptomatic of fusariosis in the yellow-spotted Amazon River turtle (Podocnemis unifilis) inhabiting a pristine environment in the Ecuadorian Amazon. In 2020, we screened 680 eggs of the yellow-spotted Amazon River turtle, relocated from wild nesting areas to artificial nests, for visual symptoms of fusariosis and to estimate hatchability despite infection. We selected 68 eggs sampled in 2019 to confirm Fusarium infection by PCR amplification of the TEF-1α gene and sequenced seven of those amplicons on an Illumina Miseq to assess FSSC membership. We observed fusariosis symptoms in 42% of the 680 eggs. The proportion of symptomatic eggs within nests was negatively linked to the proportion of eggs that hatched. Hatchability was 8% for symptomatic eggs compared with 72% of asymptomatic eggs. Through PCR testing, 58% of symptomatic and 8% of asymptomatic eggs sampled in 2019 tested positive for Fusarium spp., and sequencing revealed that nine sequence variants from three asymptomatic and four symptomatic eggs corresponded to F. keratoplasticum, F. solani and F. falciforme, the three major FSSC pathogens reported in sea turtle egg fusariosis. Our study suggests that hatching failure in eggs linked to symptoms of fusariosis appears to be partially caused by Fusarium pathogens within FSSC in a freshwater turtle. Thus, fusariosis is more widespread among the Testudines than previously reported and is not limited to sea environments, findings of particular conservation concern.

Egg microbiota is the starting point of hatchling gut microbiota in the endangered yellow-spotted Amazon river turtle.

Establishment and development of gut microbiota during vertebrates' early life are likely to be important predictors of health and fitness. Host-parental and host-environment interactions are essential to these processes. In oviparous reptiles whose nests represent a source of the parent's microbial inocula, the relative role of host-selection and stochastic environmental factors during gut microbial assemblage remains unknown. We sampled eggs incubated in artificial nests as well as hatchlings and juveniles (up to 30 days old) of the yellow-spotted Amazon river turtle (Podocnemis unifilis) developing in tubs filled with river water. We examined the relative role of the internal egg microbiota and the abiotic environment on hatchling and juvenile turtle's cloacal microbiota assemblages during the first 30 days of development. A mean of 71% of ASVs in hatched eggs could be traced to the nest environmental microbiota and in turn a mean of 77% of hatchlings' cloacal ASVs were traced to hatched eggs. Between day 5 and 20 of juvenile turtle's development, the river water environment plays a key role in the establishment of the gut microbiota (accounting for a mean of 13%-34.6% of cloacal ASVs) and strongly influences shifts in microbial diversity and abundance. After day 20, shifts in gut microbiota composition were mainly driven by host-selection processes. Therefore, colonization by environmental microbiota is key in the initial stages of establishing the host's gut microbiota which is subsequently shaped by host-selection processes. Our study provides a novel quantitative understanding of the host-environment interactions during gut microbial assemblage of oviparous reptiles.

CHARACTERIZING TUBERCULOSIS PROGRESSION IN WILD MEERKATS (SURICATA SURICATTA) FROM FECAL SAMPLES AND CLINICAL SIGNS.

Tuberculosis (TB) is an increasing threat to wildlife, yet tracking its spread is challenging because infections often appear to be asymptomatic, and diagnostic tools such as blood tests can be invasive and resource intensive. Our understanding of TB biology in wildlife is therefore limited to a small number of well-studied species. Testing of fecal samples using PCR is a noninvasive method that has been used to detect Mycobacterium bovis shedding amongst badgers, yet its utility more broadly for TB monitoring in wildlife is unclear. We combined observation data of clinical signs with PCR testing of 388 fecal samples to characterize longitudinal dynamics of TB progression in 66 wild meerkats (Suricata suricatta) socially exposed to Mycobacterium suricattae between 2000 and 2018. Our specific objectives were 1) to test whether meerkat fecal samples can be used to monitor TB; 2) to characterize TB progression between three infection states (PCR-negative exposed, PCR-positive asymptomatic, and PCR positive with clinical signs); and 3) estimate individual heterogeneity in TB susceptibility, defined here as the time between TB exposure and detection, and survival after TB detection. We found that the TB detection probability once meerkats developed clinical signs was 13% (95% confidence interval 3-46%). Nevertheless, with an adapted test protocol of 10 PCR replicates per sample we detected hidden TB infections in 59% of meerkats before the onset of clinical signs. Meerkats became PCR positive approximately 14 mo after initial exposure, developed clinical signs approximately 1 yr after becoming PCR positive, and died within 5 mo of developing clinical signs. Individual variation in disease progression was high, with meerkats developing clinical signs from immediately after exposure to 3.4 yr later. Overall, our study generates novel insights into wildlife TB progression, and may help guide adapted management strategies for TB-susceptible wildlife populations.

A Faithful Gut: Core Features of Gastrointestinal Microbiota of Long-Distance Migratory Bats Remain Stable despite Dietary Shifts Driving Differences in Specific Bacterial Taxa.

Migratory animals live in a world of constant change. Animals undergo many physiological changes preparing themselves for the migration. Although this field has been studied extensively over the last decades, we know relatively little about the seasonal changes that occur in the microbial communities that these animals carry in their guts. Here, we assessed the V4 region of the 16S rRNA high-throughput sequencing data as a proxy to estimate microbiome diversity of tequila bats from fecal pellets and evaluate how the natural process of migration shapes the microbiome composition and diversity. We collected samples from individual bats at two localities in the dry forest biome (Chamela and Coquimatlán) and one site at the endpoint of the migration in the Sonoran Desert (Pinacate). We found that the gut microbiome of the tequila bats is dominated largely by Firmicutes and Proteobacteria. Our data also provide insights on how microbiome diversity shifts at the same site in consecutive years. Our study has demonstrated that both locality and year-to-year variation contribute to shaping the composition, overall diversity, and "uniqueness" of the gut microbiome of migratory nectar-feeding female bats, with localities from the dry forest biome looking more like each other compared to those from the desert biome. In terms of beta diversity, our data show a stratified effect in which the samples' locality was the strongest factor influencing the gut microbiome but with significant variation between consecutive years at the same locality. IMPORTANCE Migratory animals live in a world of constant change. The whole-body ecosystem needs a strong adapting capacity to thrive despite the changes. Our study used next-generation sequencing to determine how gut microbial change along the migratory path of the nectar-feeding tequila bats. The study of the gut microbiome is a great tool that can provide important insights that are relevant not just for management and conservation but also an initial investigation of the extent of the adaptation and preparedness of the individual animals, with respect not just to their current environment but also to all the environments involved in their yearly cycle.

Diurnal oscillations in gut bacterial load and composition eclipse seasonal and lifetime dynamics in wild meerkats.

Circadian rhythms in gut microbiota composition are crucial for metabolic function, yet the extent to which they govern microbial dynamics compared to seasonal and lifetime processes remains unknown. Here, we investigate gut bacterial dynamics in wild meerkats (Suricata suricatta) over a 20-year period to compare diurnal, seasonal, and lifetime processes in concert, applying ratios of absolute abundance. We found that diurnal oscillations in bacterial load and composition eclipsed seasonal and lifetime dynamics. Diurnal oscillations were characterised by a peak in Clostridium abundance at dawn, were associated with temperature-constrained foraging schedules, and did not decay with age. Some genera exhibited seasonal fluctuations, whilst others developed with age, although we found little support for microbial senescence in very old meerkats. Strong microbial circadian rhythms in this species may reflect the extreme daily temperature fluctuations typical of arid-zone climates. Our findings demonstrate that accounting for circadian rhythms is essential for future gut microbiome research.

Human encroachment into wildlife gut microbiomes.

In the Anthropocene, humans, domesticated animals, wildlife, and their environments are interconnected, especially as humans advance further into wildlife habitats. Wildlife gut microbiomes play a vital role in host health. Changes to wildlife gut microbiomes due to anthropogenic disturbances, such as habitat fragmentation, can disrupt natural gut microbiota homeostasis and make animals vulnerable to infections that may become zoonotic. However, it remains unclear whether the disruption to wildlife gut microbiomes is caused by habitat fragmentation per se or the combination of habitat fragmentation with additional anthropogenic disturbances, such as contact with humans, domesticated animals, invasive species, and their pathogens. Here, we show that habitat fragmentation per se does not impact the gut microbiome of a generalist rodent species native to Central America, Tome's spiny rat Proechimys semispinosus, but additional anthropogenic disturbances do. Indeed, compared to protected continuous and fragmented forest landscapes that are largely untouched by other human activities, the gut microbiomes of spiny rats inhabiting human-disturbed fragmented landscapes revealed a reduced alpha diversity and a shifted and more dispersed beta diversity. Their microbiomes contained more taxa associated with domesticated animals and their potential pathogens, suggesting a shift in potential metagenome functions. On the one hand, the compositional shift could indicate a degree of gut microbial adaption known as metagenomic plasticity. On the other hand, the greater variation in community structure and reduced alpha diversity may signal a decline in beneficial microbial functions and illustrate that gut adaption may not catch up with anthropogenic disturbances, even in a generalist species with large phenotypic plasticity, with potentially harmful consequences to both wildlife and human health.

Control of endothelial quiescence by FOXO-regulated metabolites.

Endothelial cells (ECs) adapt their metabolism to enable the growth of new blood vessels, but little is known how ECs regulate metabolism to adopt a quiescent state. Here, we show that the metabolite S-2-hydroxyglutarate (S-2HG) plays a crucial role in the regulation of endothelial quiescence. We find that S-2HG is produced in ECs after activation of the transcription factor forkhead box O1 (FOXO1), where it limits cell cycle progression, metabolic activity and vascular expansion. FOXO1 stimulates S-2HG production by inhibiting the mitochondrial enzyme 2-oxoglutarate dehydrogenase. This inhibition relies on branched-chain amino acid catabolites such as 3-methyl-2-oxovalerate, which increase in ECs with activated FOXO1. Treatment of ECs with 3-methyl-2-oxovalerate elicits S-2HG production and suppresses proliferation, causing vascular rarefaction in mice. Our findings identify a metabolic programme that promotes the acquisition of a quiescent endothelial state and highlight the role of metabolites as signalling molecules in the endothelium.

Jumping the green wall: The use of PNA-DNA clamps to enhance microbiome sampling depth in wildlife microbiome research.

As microbiome research moves away from model organisms to wildlife, new challenges for microbiome high-throughput sequencing arise caused by the variety of wildlife diets. High levels of contamination are commonly observed emanating from the host (mitochondria) or diet (chloroplast). Such high contamination levels affect the overall sequencing depth of wildlife samples thus decreasing statistical power and leading to poor performance in downstream analysis. We developed an amplification protocol utilizing PNA-DNA clamps to maximize the use of resources and to increase the sampling depth of true microbiome sequences in samples with high levels of plastid contamination. We chose two study organisms, a bat (Leptonyteris yerbabuenae) and a bird (Mimus parvulus), both relying on heavy plant-based diets that sometimes lead to traces of plant-based fecal material producing high contamination signals from chloroplasts and mitochondria. On average, our protocol yielded a 13-fold increase in bacterial sequence amplification compared with the standard protocol (Earth Microbiome Protocol) used in wildlife research. For both focal species, we were able to increase significantly the percentage of sequences available for downstream analyses after the filtering of plastids and mitochondria. Our study presents the first results obtained by using PNA-DNA clamps to block the PCR amplification of chloroplast and mitochondrial DNA from the diet in the gut microbiome of wildlife. The method involves a cost-effective molecular technique instead of the filtering out of unwanted sequencing reads. As 33% and 26% of birds and bats, respectively, have a plant-based diet, the tool that we present here will optimize the sequencing and analysis of wild microbiomes.

Involvement of NDPK-B in Glucose Metabolism-Mediated Endothelial Damage via Activation of the Hexosamine Biosynthesis Pathway and Suppression of O-GlcNAcase Activity.

Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.