Rachel A. Johnston

Communities living in biodiversity hotspots are at increased risk of pathogen exposure and spread due to insufficient access to nutrition, clean water, and sanitation. Viruses, in particular, have high epidemic and spillover potential, and widespread health impacts, yet, viral diversity and dynamics remain understudied in many high-risk regions. In Madagascar, a hotspot with unparalleled biodiversity, socioeconomic constraints, and a heavy burden of infectious diseases, ongoing healthcare initiatives provide opportunities to survey circulating viral diversity. Here, we used a metagenomic workflow to characterize the viromes of oral/nasal swabs from febrile individuals visiting a mobile healthcare clinic in southeastern Madagascar, of which 49% were malaria-positive ( Plasmodium falciparum ). We characterized sequences of human-infecting viruses from twelve families (Adenoviridae, Anelloviridae, Orthoherpesviridae, Papillomaviridae, Polyomaviridae , Astroviridae, Coronaviridae, Matonaviridae, Orthomyxoviridae, Paramyxoviridae, Picornaviridae, and Sedoreoviridae ). These viral sequences generally showed high similarity to known lineages circulating globally, but multiple viruses considered rare or emerging were identified (e.g. enterovirus C105, enterovirus C117). While we did not detect a significant association between malaria status and pathogenic virus presence, younger individuals had higher odds of having a pathogenic virus. Nonpathogenic viruses in Anelloviridae were prevalent and diverse with richness ranging from 0 to 70. Individuals with P. falciparum or a pathogenic virus tended to share more anelloviruses. We also detected viruses that are protist-associated, plant-infecting, or with unknown hosts. Overall, our work documents extensive viral diversity in Madagascar, highlighting how metagenomic workflows can be used to build viral genomic resources for pathogen surveillance in regions with high infectious disease burden.

Genomic tools are increasingly important for conservation and wildlife health surveillance, yet their use is often constrained by limited resources, sample quality, and the need for minimally invasive approaches. Here we show that untargeted low-coverage sequencing applied to scale-clip DNA can jointly address population structure, genotype–phenotype associations, and pathogen surveillance in timber rattlesnakes (Crotalus horridus), a species threatened by habitat fragmentation, human encroachment, and the emerging fungal pathogen Ophidiomyces ophidiicola. We sequenced 107 snakes sampled from eight populations in the Appalachian Mountains of the northeastern United States to a mean depth of 0.94x per individual. Population differentiation is pronounced and only weakly correlated with geographic distance, and inbreeding coefficients in some populations are high enough to suggest fitness consequences. Genome-wide association analysis identifies a locus for the black-to-yellow color morph containing ALDH4A1, a member of a gene family implicated in vertebrate pigmentation but distinct from melanin pathway pigmentation genes. Our untargeted sequencing method also captures host-associated microbes: O. ophidiicola loads are higher in snakes with clinical signs of snake fungal disease, though some asymptomatic individuals carry substantial loads, and skin-associated microbial communities are disrupted in snakes with higher fungal burdens. That all of these inferences derive from the same minimally invasive sampling and sequencing workflow highlights the scalability of this approach for biodiversity conservation.

With dramatic advancements in biological data generation, genetic rescue and reproductive technologies, and inter‐institutional coordination of care across entire animal populations, zoos, aquariums, and their collaborators are uniquely positioned to lead population‐wide research benefiting animal wellbeing and species survival. However, procedural and inter‐institutional barriers make it exceedingly difficult to access existing zoological biospecimens and data at scale. To address this, the Zoonomics Working Group, representing diverse roles across three zoological associations (AZA, EAZA, WAZA), proposes a biodiversity biobank alliance that develops and delivers shared resources to support the collection, storage, and sharing of biological samples and associated data across the zoological and conservation community. By biobank alliance, we mean a community‐guided effort that develops shared resources, standards, ethos, and practices for collecting, storing, and sharing biological samples and associated data voluntarily through transparent processes, consistent with professional accreditation standards and international best practices. While initially focused on addressing the needs and regulatory landscape of U.S. institutions, the alliance is designed to create frameworks that are adaptable and adoptable for international expansion. Such a framework would help the zoological community navigate the ethical, legal, and practical challenges of managing biospecimen collections, making access more efficient, reliable, and robust. Achieving this vision requires collective agreement on ethical principles such as reciprocity, transparency, and data stewardship, ensuring that research is both feasible and proactively supported. Such coordination will drive advances in fundamental biology and accelerate progress in animal health, welfare, management, and biodiversity conservation.

Given that some DNA viruses have been found to exhibit virus–host co-evolution and establish lifelong infection, mammals with unique evolutionary histories in island ecosystems likely host exceptionally diverse viruses. Madagascar is inhabited by endemic non-human primate and rodent lineages interacting with expansive populations of introduced non-native rodents across the island. Using a viral metagenomic workflow on 189 oral swabs of lemurs and rodents in southeastern Madagascar, we characterized genomic sequences of DNA viruses in the families Adenoviridae , Circoviridae , Orthoherpesviridae , Papillomaviridae , Parvoviridae and Polyomaviridae and assessed their phylogenetic relationships to known viruses. Endemic lemurs and tufted-tailed rats displayed particularly novel DNA viral diversity mirroring the geographic isolation and subsequently rich evolutionary history of their hosts. Notably, we provide the first coding-complete sequences in lemurs of herpesviruses, polyomaviruses, adeno-associated viruses and circoviruses. In contrast, the DNA viral communities of black rats in Madagascar were similar to those found in globally distributed black and brown rat populations, given their broad geographic spread and relatively recent introduction to the island. Given the scarcity of viral research in natural populations of lemurs and rodents in Madagascar despite the island’s exceptional biodiversity and escalating anthropogenic pressures, this study provides a genomic and phylogenetic foundation for DNA viruses infecting Malagasy lemurs and rodents.

Anelloviruses are circular, negative-sense single-stranded DNA viruses that have remarkable diversity and ubiquity across mammals. However, few studies have attempted to determine anellovirus diversity and dynamics across a biodiverse landscape. Madagascar offers a unique opportunity to study anellovirus diversity, with speciose, endemic mammalian lineages that have evolved in geographic isolation for millions of years. These endemic animals frequently interact with more recently introduced populations of non-native small mammals. From oral swab samples taken from natural populations of lemurs, rodents and shrews in the Manombo Special Reserve and surrounding area in southeastern Madagascar, we determined the complete genomes of anelloviruses from black rats ( n =647 genomes), Webb’s tufted-tailed rats ( n =2), mouse lemurs ( n =4), a woolly lemur ( n =1) and a house shrew ( n =3). We observed distinct anellovirus lineages in the endemic lemurs and tufted-tailed rats, which we infer to be the result of their long-term geographic isolation in Madagascar. Lemur-infecting anelloviruses, in particular, do not cluster with other primate-infecting anelloviruses. In contrast, anellovirus diversity in widespread, non-native rodents (i.e. black rats) was similar to that of closely related, globally dispersed rodent species, concordant with their later introduction to Madagascar. With our large anellovirus dataset from black rats, we examined anellovirus alpha- and beta-diversity and viral co-occurrence networks. Black rat anellovirus populations showed high intra-individual variation, but the overall pool of circulating anelloviruses was consistent across age classes and sexes. Adult relative to juvenile/subadult black rats harboured richer anellovirus populations and were more connected within the co-occurrence network. Proximity between individuals and greater intra-individual viral diversity were also linked to more virus-sharing between black rats.

The early-life environment can profoundly shape the trajectory of an animal’s life, even years or decades later. One mechanism proposed to contribute to these early-life effects is DNA methylation. However, the frequency and functional importance of DNA methylation in shaping early-life effects on adult outcomes is poorly understood, especially in natural populations. Here, we integrate prospectively collected data on fitness-associated variation in the early environment with DNA methylation estimates at 477,270 CpG sites in 256 wild baboons. We find highly heterogeneous relationships between the early-life environment and DNA methylation in adulthood: aspects of the environment linked to resource limitation (e.g., low-quality habitat, early-life drought) are associated with many more CpG sites than other types of environmental stressors (e.g., low maternal social status). Sites associated with early resource limitation are enriched in gene bodies and putative enhancers, suggesting they are functionally relevant. Indeed, by deploying a baboon-specific, massively parallel reporter assay, we show that a subset of windows containing these sites are capable of regulatory activity, and that, for 88% of early drought-associated sites in these regulatory windows, enhancer activity is DNA methylation-dependent. Together, our results support the idea that DNA methylation patterns contain a persistent signature of the early-life environment. However, they also indicate that not all environmental exposures leave an equivalent mark and suggest that socioenvironmental variation at the time of sampling is more likely to be functionally important. Thus, multiple mechanisms must converge to explain early-life effects on fitness-related traits.

Previously, we showed that a massively parallel reporter assay, mSTARR-seq, could be used to simultaneously test for both enhancer-like activity and DNA methylation-dependent enhancer activity for millions of loci in a single experiment (Lea et al., 2018). Here, we apply mSTARR-seq to query nearly the entire human genome, including almost all CpG sites profiled either on the commonly used Illumina Infinium MethylationEPIC array or via reduced representation bisulfite sequencing. We show that fragments containing these sites are enriched for regulatory capacity, and that methylation-dependent regulatory activity is in turn sensitive to the cellular environment. In particular, regulatory responses to interferon alpha (IFNA) stimulation are strongly attenuated by methyl marks, indicating widespread DNA methylation-environment interactions. In agreement, methylation-dependent responses to IFNA identified via mSTARR-seq predict methylation-dependent transcriptional responses to challenge with influenza virus in human macrophages. Our observations support the idea that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures—one of the tenets of biological embedding. However, we also find that, on average, sites previously associated with early life adversity are not more likely to functionally influence gene regulation than expected by chance.

Previously we showed that a massively parallel reporter assay, mSTARR-seq, could be used to simultaneously test for both enhancer-like activity and DNA methylation-dependent enhancer activity for millions of loci in a single experiment (Lea et al., 2018). Here we apply mSTARR-seq to query nearly the entire human genome, including almost all CpG sites profiled either on the commonly used Illumina Infinium MethylationEPIC array or via reduced representation bisulfite sequencing. We show that fragments containing these sites are enriched for regulatory capacity, and that methylation-dependent regulatory activity is in turn sensitive to the cellular environment. In particular, regulatory responses to interferon alpha (IFNA) stimulation are strongly attenuated by methyl marks, indicating widespread DNA methylation-environment interactions. In agreement, methylation-dependent responses to IFNA identified via mSTARR-seq predict methylation-dependent transcriptional responses to challenge with influenza virus in human macrophages. Our observations support the idea that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures—one of the tenets of biological embedding. However, we also find that, on average, sites previously associated with early life adversity are not more likely to functionally influence gene regulation than expected by chance.

The early life environment can profoundly shape the trajectory of an animal’s life, even years or decades later. One mechanism proposed to contribute to these early life effects is DNA methylation. However, the frequency and functional importance of DNA methylation in shaping early life effects on adult outcomes is poorly understood, especially in natural populations. Here, we integrate prospectively collected data on fitness-associated variation in the early environment with DNA methylation estimates at 477,270 CpG sites in 256 wild baboons. We find highly heterogeneous relationships between the early life environment and DNA methylation in adulthood: aspects of the environment linked to resource limitation (e.g., low-quality habitat, early life drought) are associated with many more CpG sites than other types of environmental stressors (e.g., low maternal social status). Sites associated with early resource limitation are enriched in gene bodies and putative enhancers, suggesting they are functionally relevant. Indeed, by deploying a baboon-specific, massively parallel reporter assay, we show that a subset of windows containing these sites are capable of regulatory activity, and that, for 88% of early drought-associated sites in these regulatory windows, enhancer activity is DNA methylation-dependent. Together, our results support the idea that DNA methylation patterns contain a persistent signature of the early life environment. However, they also indicate that not all environmental exposures leave an equivalent mark and suggest that socioenvironmental variation at the time of sampling is more likely to be functionally important. Thus, multiple mechanisms must converge to explain early life effects on fitness-related traits. Significance statement: The environment animals face when young can affect how they function throughout life. Long-lasting changes in DNA methylation—a chemical mark deposited on DNA that can affect gene activity—have been hypothesized to contribute to early life effects. But evidence for persistent, early environment-associated differences in DNA methylation is lacking in wild animals. Here, we show that early life adversity in wild baboons predicts DNA methylation levels in adulthood, especially for animals born in low resource environments and drought conditions. We also show that some of the changes we observe in DNA methylation have the capacity to influence gene activity levels. Together, our results support the idea that early experiences can become biologically embedded in the genomes of wild animals.

Previously we showed that a massively parallel reporter assay, mSTARR-seq, could be used to simultaneously test for both enhancer-like activity and DNA methylation-dependent enhancer activity for millions of loci in a single experiment (Lea et al ., 2018). Here we apply mSTARR-seq to query nearly the entire human genome, including almost all CpG sites profiled either on the commonly used Illumina Infinium MethylationEPIC array or via reduced representation bisulfite sequencing. We show that fragments containing these sites are enriched for regulatory capacity, and that methylation-dependent regulatory activity is in turn sensitive to the cellular environment. In particular, regulatory responses to interferon alpha (IFNA) stimulation are strongly attenuated by methyl marks, indicating widespread DNA methylation-environment interactions. In agreement, methylation-dependent responses to IFNA identified via mSTARR-seq predict methylation-dependent transcriptional responses to challenge with influenza virus in human macrophages. Our observations support the idea that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures—one of the tenets of biological embedding. However, we also find that, on average, sites previously associated with early life adversity are not more likely to functionally influence gene regulation than expected by chance.