Ecological countermeasures to prevent pathogen spillover and subsequent pandemics.

Substantial global attention is focused on how to reduce the risk of future pandemics. Reducing this risk requires investment in prevention, preparedness, and response. Although preparedness and response have received significant focus, prevention, especially the prevention of zoonotic spillover, remains largely absent from global conversations. This oversight is due in part to the lack of a clear definition of prevention and lack of guidance on how to achieve it. To address this gap, we elucidate the mechanisms linking environmental change and zoonotic spillover using spillover of viruses from bats as a case study. We identify ecological interventions that can disrupt these spillover mechanisms and propose policy frameworks for their implementation. Recognizing that pandemics originate in ecological systems, we advocate for integrating ecological approaches alongside biomedical approaches in a comprehensive and balanced pandemic prevention strategy.

Palaeoatmosphere facilitates a gliding transition to powered flight in the Eocene bat, Onychonycteris finneyi.

The evolutionary transition to powered flight remains controversial in bats, the only flying mammals. We applied aerodynamic modeling to reconstruct flight in the oldest complete fossil bat, the archaic Onychonycteris finneyi from the early Eocene of North America. Results indicate that Onychonycteris was capable of both gliding and powered flight either in a standard normodense aerial medium or in the hyperdense atmosphere that we estimate for the Eocene from two independent palaeogeochemical proxies. Aerodynamic continuity across a morphological gradient is further demonstrated by modeled intermediate forms with increasing aspect ratio (AR) produced by digital elongation based on chiropteran developmental data. Here a gliding performance gradient emerged of decreasing sink rate with increasing AR that eventually allowed applying available muscle power to achieve level flight using flapping, which is greatly facilitated in hyperdense air. This gradient strongly supports a gliding (trees-down) transition to powered flight in bats.

Geographically widespread and novel hemotropic mycoplasmas and bartonellae in Mexican free-tailed bats and sympatric North American bat species.

Bacterial pathogens remain poorly characterized in bats, especially in North America. We describe novel (and in some cases panmictic) hemoplasmas (12.9% positivity) and bartonellae (16.7% positivity) across three colonies of Mexican free-tailed bats (Tadarida brasiliensis), a partially migratory species that can seasonally travel hundreds of kilometers. Molecular analyses identified three novel Candidatus hemoplasma species most similar to another novel Candidatus species in Neotropical molossid bats. We also detected novel hemoplasmas in sympatric cave myotis (Myotis velifer) and pallid bats (Antrozous pallidus), with sequences in the latter 96.5% related to C. Mycoplasma haemohominis. We identified eight Bartonella genotypes, including those in cave myotis, with 96.7% similarity to C. Bartonella mayotimonensis. We also detected Bartonella rochalimae in migratory Tadarida brasiliensis, representing the first report of this human pathogen in bats. The seasonality and diversity of these bacteria observed here suggest that additional longitudinal, genomic, and immunological studies in bats are warranted.

Bat cellular immunity varies by year and dietary habit amidst land conversion.

Monitoring the health of wildlife populations is essential in the face of increased agricultural expansion and forest fragmentation. Loss of habitat and habitat degradation can negatively affect an animal's physiological state, possibly resulting in immunosuppression and increased morbidity or mortality. We sought to determine how land conversion may differentially impact cellular immunity and infection risk in Neotropical bats species regularly infected with bloodborne pathogens, and to evaluate how effects may vary over time and by dietary habit. We studied common vampire bats (Desmodus rotundus), northern yellow-shouldered bats (Sturnira parvidens) and Mesoamerican mustached bats (Pteronotus mesoamericanus), representing the dietary habits of sanguivory, frugivory and insectivory respectively, in northern Belize. We compared estimated total white blood cell count, leukocyte differentials, neutrophil to lymphocyte ratio and infection status with two bloodborne bacterial pathogens (Bartonella spp. and hemoplasmas) of 118 bats captured in a broadleaf, secondary forest over three years (2017-2019). During this period, tree cover decreased by 14.5% while rangeland expanded by 14.3%, indicating increasing habitat loss and fragmentation. We found evidence for bat species-specific responses of cellular immunity between years, with neutrophil counts significantly decreasing in S. parvidens from 2017 to 2018, but marginally increasing in D. rotundus. However, the odds of infection with Bartonella spp. and hemoplasmas between 2017 and 2019 did not differ between bat species, contrary to our prediction that pathogen prevalence may increase with land conversion. We conclude that each bat species invested differently in cellular immunity in ways that changed over years of increasing habitat loss and fragmentation. We recommend further research on the interactions between land conversion, immunity and infection across dietary habits of Neotropical bats for informed management and conservation.

Integrative single-cell characterization of a frugivorous and an insectivorous bat kidney and pancreas.

Frugivory evolved multiple times in mammals, including bats. However, the cellular and molecular components driving it remain largely unknown. Here, we use integrative single-cell sequencing (scRNA-seq and scATAC-seq) on insectivorous (Eptesicus fuscus; big brown bat) and frugivorous (Artibeus jamaicensis; Jamaican fruit bat) bat kidneys and pancreases and identify key cell population, gene expression and regulatory differences associated with the Jamaican fruit bat that also relate to human disease, particularly diabetes. We find a decrease in loop of Henle and an increase in collecting duct cells, and differentially active genes and regulatory elements involved in fluid and electrolyte balance in the Jamaican fruit bat kidney. The Jamaican fruit bat pancreas shows an increase in endocrine and a decrease in exocrine cells, and differences in genes and regulatory elements involved in insulin regulation. We also find that these frugivorous bats share several molecular characteristics with human diabetes. Combined, our work provides insights from a frugivorous mammal that could be leveraged for therapeutic purposes.

Expanded diversity of novel hemoplasmas in rare and undersampled Neotropical bats.

Hemotropic mycoplasmas are emerging as a model system for studying bacterial pathogens in bats, but taxonomic coverage of sampled host species remains biased. We leveraged a long-term field study in Belize to uncover novel hemoplasma diversity in bats by analyzing 80 samples from 19 species, most of which are infrequently encountered. PCR targeting the partial 16S rRNA gene found 41% of bats positive for hemoplasmas. Phylogenetic analyses found two novel host shifts of hemoplasmas, four entirely new hemoplasma genotypes, and the first hemoplasma detections in four bat species. One of these novel hemoplasmas (from Neoeptesicus furinalis) shared 97.6% identity in the partial 16S rRNA gene to a human hemoplasma (Candidatus Mycoplasma haemohominis). Additional analysis of the partial 23S rRNA gene allowed us to also designate two novel hemoplasma species, in Myotis elegans and Phyllostomus discolor, with the proposed names Candidatus Mycoplasma haematomyotis sp. nov. and Candidatus Mycoplasma haematophyllostomi sp. nov., respectively. Our analyses show that additional hemoplasma diversity in bats can be uncovered by targeting rare or undersampled host species.

Long-Read Sequencing Reveals Rapid Evolution of Immunity- and Cancer-Related Genes in Bats.

Bats are exceptional among mammals for their powered flight, extended lifespans, and robust immune systems and therefore have been of particular interest in comparative genomics. Using the Oxford Nanopore Technologies long-read platform, we sequenced the genomes of two bat species with key phylogenetic positions, the Jamaican fruit bat (Artibeus jamaicensis) and the Mesoamerican mustached bat (Pteronotus mesoamericanus), and carried out a comprehensive comparative genomic analysis with a diverse collection of bats and other mammals. The high-quality, long-read genome assemblies revealed a contraction of interferon (IFN)-α at the immunity-related type I IFN locus in bats, resulting in a shift in relative IFN-ω and IFN-α copy numbers. Contradicting previous hypotheses of constitutive expression of IFN-α being a feature of the bat immune system, three bat species lost all IFN-α genes. This shift to IFN-ω could contribute to the increased viral tolerance that has made bats a common reservoir for viruses that can be transmitted to humans. Antiviral genes stimulated by type I IFNs also showed evidence of rapid evolution, including a lineage-specific duplication of IFN-induced transmembrane genes and positive selection in IFIT2. In addition, 33 tumor suppressors and 6 DNA-repair genes showed signs of positive selection, perhaps contributing to increased longevity and reduced cancer rates in bats. The robust immune systems of bats rely on both bat-wide and lineage-specific evolution in the immune gene repertoire, suggesting diverse immune strategies. Our study provides new genomic resources for bats and sheds new light on the extraordinary molecular evolution in this critically important group of mammals.

The genome sequence of Molossusnigricans (Chiroptera, Molossidae; Miller, 1902).

We present a genome assembly from an individual male Molossus nigricans (Chordata; Mammalia; Chiroptera; Molossidae). The genome sequence is 2.41 gigabases in span. The majority of the assembly is scaffolded into 24 chromosomal pseudomolecules, with the X sex chromosome assembled.

Out of thin air: surveying tropical bat roosts through air sampling of eDNA.

Understanding roosting behaviour is essential to bat conservation and biomonitoring, often providing the most accurate methods of assessing bat population size and health. However, roosts can be challenging to survey, e.g., physically impossible to access or presenting risks for researchers. Disturbance during monitoring can also disrupt natural bat behaviour and present material risks to the population such as disrupting hibernation cycles. One solution to this is the use of non-invasive monitoring approaches. Environmental (e)DNA has proven especially effective at detecting rare and elusive species particularly in hard-to-reach locations. It has recently been demonstrated that eDNA from vertebrates is carried in air. When collected in semi-confined spaces, this airborne eDNA can provide remarkably accurate profiles of biodiversity, even in complex tropical communities. In this study, we deploy novel airborne eDNA collection for the first time in a natural setting and use this approach to survey difficult to access potential roosts in the neotropics. Using airborne eDNA, we confirmed the presence of bats in nine out of 12 roosts. The identified species matched previous records of roost use obtained from photographic and live capture methods, thus demonstrating the utility of this approach. We also detected the presence of the white-winged vampire bat (Diaemus youngi) which had never been confirmed in the area but was long suspected based on range maps. In addition to the bats, we detected several non-bat vertebrates, including the big-eared climbing rat (Ototylomys phyllotis), which has previously been observed in and around bat roosts in our study area. We also detected eDNA from other local species known to be in the vicinity. Using airborne eDNA to detect new roosts and monitor known populations, particularly when species turnover is rapid, could maximize efficiency for surveyors while minimizing disturbance to the animals. This study presents the first applied use of airborne eDNA collection for ecological analysis moving beyond proof of concept to demonstrate a clear utility for this technology in the wild.

Developmental origin underlies evolutionary rate variation across the placental skull.

The placental skull has evolved into myriad forms, from longirostrine whales to globular primates, and with a diverse array of appendages from antlers to tusks. This disparity has recently been studied from the perspective of the whole skull, but the skull is composed of numerous elements that have distinct developmental origins and varied functions. Here, we assess the evolution of the skull's major skeletal elements, decomposed into 17 individual regions. Using a high-dimensional morphometric approach for a dataset of 322 living and extinct eutherians (placental mammals and their stem relatives), we quantify patterns of variation and estimate phylogenetic, allometric and ecological signal across the skull. We further compare rates of evolution across ecological categories and ordinal-level clades and reconstruct rates of evolution along lineages and through time to assess whether developmental origin or function discriminate the evolutionary trajectories of individual cranial elements. Our results demonstrate distinct macroevolutionary patterns across cranial elements that reflect the ecological adaptations of major clades. Elements derived from neural crest show the fastest rates of evolution, but ecological signal is equally pronounced in bones derived from neural crest and paraxial mesoderm, suggesting that developmental origin may influence evolutionary tempo, but not capacity for specialisation. This article is part of the theme issue 'The mammalian skull: development, structure and function'.

The oldest known bat skeletons and their implications for Eocene chiropteran diversification.

The Fossil Lake deposits of the Green River Formation of Wyoming, a remarkable early Eocene Lagerstätte (51.98 ±0.35 Ma), have produced nearly 30 bat fossils over the last 50 years. However, diversity has thus far been limited to only two bat species. Here, we describe a new species of Icaronycteris based on two articulated skeletons discovered in the American Fossil Quarry northwest of Kemmerer, Wyoming. The relative stratigraphic position of these fossils indicates that they are the oldest bat skeletons recovered to date anywhere in the world. Phylogenetic analysis of Eocene fossil bats and living taxa places the new species within the family Icaronycteridae as sister to Icaronycteris index, and additionally indicates that the two Green River archaic bat families (Icaronycteridae and Onychonycteridae) form a clade distinct from known Old World lineages of archaic bats. Our analyses found no evidence that Icaronycteris? menui (France) nor I. sigei (India) belong to this clade; accordingly, we therefore remove them from Icaronycteridae. Taken in sum, our results indicate that Green River bats represent a separate chiropteran radiation of basal bats, and provide additional support for the hypothesis of a rapid radiation of bats on multiple continents during the early Eocene.

Integrative single-cell characterization of frugivory adaptations in the bat kidney and pancreas.

Frugivory evolved multiple times in mammals, including bats. However, the cellular and molecular components driving it remain largely unknown. Here, we used integrative single-cell sequencing on insectivorous and frugivorous bat kidneys and pancreases and identified key cell population, gene expression and regulatory element differences associated with frugivorous adaptation that also relate to human disease, particularly diabetes. We found an increase in collecting duct cells and differentially active genes and regulatory elements involved in fluid and electrolyte balance in the frugivore kidney. In the frugivorous pancreas, we observed an increase in endocrine and a decrease in exocrine cells and differences in genes and regulatory elements involved in insulin regulation. Combined, our work provides novel insights into frugivorous adaptation that also could be leveraged for therapeutic purposes.

Serum proteomics reveals a tolerant immune phenotype across multiple pathogen taxa in wild vampire bats.

Bats carry many zoonotic pathogens without showing pronounced pathology, with a few exceptions. The underlying immune tolerance mechanisms in bats remain poorly understood, although information-rich omics tools hold promise for identifying a wide range of immune markers and their relationship with infection. To evaluate the generality of immune responses to infection, we assessed the differences and similarities in serum proteomes of wild vampire bats (Desmodus rotundus) across infection status with five taxonomically distinct pathogens: bacteria (Bartonella spp., hemoplasmas), protozoa (Trypanosoma cruzi), and DNA (herpesviruses) and RNA (alphacoronaviruses) viruses. From 19 bats sampled in 2019 in Belize, we evaluated the up- and downregulated immune responses of infected versus uninfected individuals for each pathogen. Using a high-quality genome annotation for vampire bats, we identified 586 serum proteins but found no evidence for differential abundance nor differences in composition between infected and uninfected bats. However, using receiver operating characteristic curves, we identified four to 48 candidate biomarkers of infection depending on the pathogen, including seven overlapping biomarkers (DSG2, PCBP1, MGAM, APOA4, DPEP1, GOT1, and IGFALS). Enrichment analysis of these proteins revealed that our viral pathogens, but not the bacteria or protozoa studied, were associated with upregulation of extracellular and cytoplasmatic secretory vesicles (indicative of viral replication) and downregulation of complement activation and coagulation cascades. Additionally, herpesvirus infection elicited a downregulation of leukocyte-mediated immunity and defense response but an upregulation of an inflammatory and humoral immune response. In contrast to our two viral infections, we found downregulation of lipid and cholesterol homeostasis and metabolism with Bartonella spp. infection, of platelet-dense and secretory granules with hemoplasma infection, and of blood coagulation pathways with T. cruzi infection. Despite the small sample size, our results suggest that vampire bats have a similar suite of immune mechanisms for viruses distinct from responses to the other pathogen taxa, and we identify potential biomarkers that can expand our understanding of pathogenesis of these infections in bats. By applying a proteomic approach to a multi-pathogen system in wild animals, our study provides a distinct framework that could be expanded across bat species to increase our understanding of how bats tolerate pathogens.

Environmentally driven phenotypic convergence and niche conservatism accompany speciation in hoary bats.

Species that are geographically widespread may exist across environmentally heterogeneous landscapes that could influence patterns of occupation and phylogeographic structure. Previous studies have suggested that geographic range size should be positively correlated with niche breadth, allowing widespread species to sustain viable populations over diverse environmental gradients. We examined the congruence of phenotypic and phylogenetic divergence with the environmental factors that help maintain species level diversity in the geographically widespread hoary bats (Lasiurus cinereus sensu lato) across their distribution. Genetic sequences were analyzed using multiple phylogenetic and species delimitation methods, and phenotypic data were analyzed using supervised and unsupervised machine learning approaches. Spatial data from environmental, geographic, and topographic features were analyzed in a multiple regression analysis to determine their relative effect on phenotypic diversity. Ecological niches of each hoary bat species were examined in environmental space to quantify niche overlap, equivalency, and the magnitude of niche differentiation. Phylogenetic and species delimitation analyses support existence of three geographically structured species of hoary bat, each of which is phenotypically distinct. However, the Hawaiian hoary bat is morphologically more similar to the South American species than to the North American species despite a closer phylogenetic relationship to the latter. Multiple regression and niche analyses revealed higher environmental similarities between the South American and Hawaiian species. Hoary bats thus exhibit a pattern of phenotypic variation that disagrees with well-supported genetic divergences, instead indicating phenotypic convergence driven by similar environmental features and relatively conserved niches occupied in tropical latitudes.

Co-circulation of alpha- and beta-coronaviruses in Pteropus vampyrus flying foxes from Indonesia.

Bats are important reservoirs for alpha- and beta-coronaviruses. Coronaviruses (CoV) have been detected in pteropodid bats from several Southeast Asian countries, but little is known about coronaviruses in the Indonesian archipelago in proportion to its mammalian biodiversity. In this study, we screened pooled faecal samples from the Indonesian colonies of Pteropus vampyrus with unbiased next-generation sequencing. Bat CoVs related to Rousettus leschenaultii CoV HKU9 and Eidolon helvum CoV were detected. The 121 faecal samples were further screened using a conventional hemi-nested pan-coronavirus PCR assay. Three positive samples were successfully sequenced, and phylogenetic reconstruction revealed the presence of alpha- and beta-coronaviruses. CoVs belonging to the subgenera Nobecovirus, Decacovirus and Pedacovirus were detected in a single P. vampyrus roost. This study expands current knowledge of coronavirus diversity in Indonesian flying foxes, highlighting the need for longitudinal surveillance of colonies as continuing urbanization and deforestation heighten the risk of spillover events.

Attenuated evolution of mammals through the Cenozoic.

The Cenozoic diversification of placental mammals is the archetypal adaptive radiation. Yet, discrepancies between molecular divergence estimates and the fossil record fuel ongoing debate around the timing, tempo, and drivers of this radiation. Analysis of a three-dimensional skull dataset for living and extinct placental mammals demonstrates that evolutionary rates peak early and attenuate quickly. This long-term decline in tempo is punctuated by bursts of innovation that decreased in amplitude over the past 66 million years. Social, precocial, aquatic, and herbivorous species evolve fastest, especially whales, elephants, sirenians, and extinct ungulates. Slow rates in rodents and bats indicate dissociation of taxonomic and morphological diversification. Frustratingly, highly similar ancestral shape estimates for placental mammal superorders suggest that their earliest representatives may continue to elude unequivocal identification.

Ecological constraints on highly evolvable olfactory receptor genes and morphology in neotropical bats.

Although evolvability of genes and traits may promote specialization during species diversification, how ecology subsequently restricts such variation remains unclear. Chemosensation requires animals to decipher a complex chemical background to locate fitness-related resources, and thus the underlying genomic architecture and morphology must cope with constant exposure to a changing odorant landscape; detecting adaptation amidst extensive chemosensory diversity is an open challenge. In phyllostomid bats, an ecologically diverse clade that evolved plant visiting from a presumed insectivorous ancestor, the evolution of novel food detection mechanisms is suggested to be a key innovation, as plant-visiting species rely strongly on olfaction, supplementarily using echolocation. If this is true, exceptional variation in underlying olfactory genes and phenotypes may have preceded dietary diversification. We compared olfactory receptor (OR) genes sequenced from olfactory epithelium transcriptomes and olfactory epithelium surface area of bats with differing diets. Surprisingly, although OR evolution rates were quite variable and generally high, they are largely independent of diet. Olfactory epithelial surface area, however, is relatively larger in plant-visiting bats and there is an inverse relationship between OR evolution rates and surface area. Relatively larger surface areas suggest greater reliance on olfactory detection and stronger constraint on maintaining an already diverse OR repertoire. Instead of the typical case in which specialization and elaboration are coupled with rapid diversification of associated genes, here the relevant genes are already evolving so quickly that increased reliance on smell has led to stabilizing selection, presumably to maintain the ability to consistently discriminate among specific odorants-a potential ecological constraint on sensory evolution.

Feeling for food: Can rostro-mental hair arrays sense hydrodynamic cues for foraging North Atlantic right whales?

North Atlantic right whales (NARWs; Eubalaena glacialis) possess an arrangement of fine hairs on the rostrum and chin that may be used for hydrodynamic sensing during feeding. These hairs occur across mysticete species and are known to possess adequate innervation in the subdermal follicle to support their consideration as sensory hairs (vibrissae). However, the small size of the hair structure with respect to the enormous scale of the animal's body has caused doubts regarding their utility and prompted speculation that the hairs may be vestigial or minimally functional. Here we show that NARW hairs occur in abundance on the leading surface of the head in a unique and characteristic arrangement. We consider the sensory hairs in context of the fluid environment in which this species forages and argue that the size of the hair is scaled to the size of the animal's small planktonic prey, thus suggesting that the hairs play an important role in the sensory ecology of these animals.

Optimising predictive models to prioritise viral discovery in zoonotic reservoirs.

Despite the global investment in One Health disease surveillance, it remains difficult and costly to identify and monitor the wildlife reservoirs of novel zoonotic viruses. Statistical models can guide sampling target prioritisation, but the predictions from any given model might be highly uncertain; moreover, systematic model validation is rare, and the drivers of model performance are consequently under-documented. Here, we use the bat hosts of betacoronaviruses as a case study for the data-driven process of comparing and validating predictive models of probable reservoir hosts. In early 2020, we generated an ensemble of eight statistical models that predicted host-virus associations and developed priority sampling recommendations for potential bat reservoirs of betacoronaviruses and bridge hosts for SARS-CoV-2. During a time frame of more than a year, we tracked the discovery of 47 new bat hosts of betacoronaviruses, validated the initial predictions, and dynamically updated our analytical pipeline. We found that ecological trait-based models performed well at predicting these novel hosts, whereas network methods consistently performed approximately as well or worse than expected at random. These findings illustrate the importance of ensemble modelling as a buffer against mixed-model quality and highlight the value of including host ecology in predictive models. Our revised models showed an improved performance compared with the initial ensemble, and predicted more than 400 bat species globally that could be undetected betacoronavirus hosts. We show, through systematic validation, that machine learning models can help to optimise wildlife sampling for undiscovered viruses and illustrates how such approaches are best implemented through a dynamic process of prediction, data collection, validation, and updating.