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Exposure to early life adversity is linked to detrimental fitness outcomes across taxa. Owing to the challenges of collecting longitudinal data, direct evidence for long-term fitness effects of early life adversity from long-lived species remains relatively scarce. Here, we test the effects of early life adversity on male and female longevity in a free-ranging population of rhesus macaques ( Macaca mulatta ) on Cayo Santiago, Puerto Rico. We leveraged six decades of data to quantify the relative importance of 10 forms of early life adversity for 6599 macaques. Individuals that experienced more early life adversity died earlier than those that experienced less adversity. Mortality risk was highest during early life, defined as birth to 4 years old, but heightened mortality risk was also present in macaques that survived to adulthood. Females and males were affected differently by some forms of adversity, and these differences might be driven by varying energetic demands and dispersal patterns. Our results show that the fitness consequences of early life adversity are not uniform across individuals but vary as a function of the type of adversity, timing and social context, and thus contribute to our limited but growing understanding of the evolution of early life sensitivities.This article is part of the discussion meeting issue 'Understanding age and society using natural populations'.

Phenotypic aging is ubiquitous across mammalian species, suggesting shared underlying mechanisms of aging. Aging is linked to molecular changes to DNA methylation and gene expression, and environmental factors, such as severe external challenges or adversities, can moderate these age-related changes. Yet, it remains unclear whether environmental adversities affect gene regulation via the same molecular pathways as chronological, or 'primary', aging. Investigating molecular aging in naturalistic animal populations can fill this gap by providing insight into shared molecular mechanisms of aging and the effects of a greater diversity of environmental adversities - particularly those that can be challenging to study in humans or laboratory organisms. Here, we characterised molecular aging - specifically, CpG methylation - in a sample of free-ranging rhesus macaques living off the coast of Puerto Rico (n samples = 571, n individuals = 499), which endured a major hurricane during our study. Age was associated with methylation at 78,661 sites (31% of all sites tested). Age-associated hypermethylation occurred more frequently in areas of active gene regulation, while hypomethylation was enriched in regions that show less activity in immune cells, suggesting these regions may become de-repressed in older individuals. Age-associated hypomethylation also co-occurred with increased chromatin accessibility while hypermethylation showed the opposite trend, hinting at a coordinated, multi-level loss of epigenetic stability during aging. We detected 32,048 CpG sites significantly associated with exposure to a hurricane, and these sites overlapped age-associated sites, most strongly in regulatory regions and most weakly in quiescent regions. Together, our results suggest that environmental adversity may contribute to aging-related molecular phenotypes in regions of active gene transcription, but that primary aging has specific signatures in non-regulatory regions., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Characterizing DNA methylation patterns is important for addressing key questions in evolutionary biology, geroscience, and medical genomics. While costs are decreasing, whole-genome DNA methylation profiling remains prohibitively expensive for most population-scale studies, creating a need for cost-effective, reduced representation approaches (i.e., assays that rely on microarrays, enzyme digests, or sequence capture to target a subset of the genome). Most common whole genome and reduced representation techniques rely on bisulfite conversion, which can damage DNA resulting in DNA loss and sequencing biases. Enzymatic methyl sequencing (EM-seq) was recently proposed to overcome these issues, but thorough benchmarking of EM-seq combined with cost-effective, reduced representation strategies has not yet been performed. To do so, we optimized Targeted Methylation Sequencing protocol (TMS)-which profiles ∼4 million CpG sites-for miniaturization, flexibility, and multispecies use at a cost of ∼$80. First, we tested modifications to increase throughput and reduce cost, including increasing multiplexing, decreasing DNA input, and using enzymatic rather than mechanical fragmentation to prepare DNA. Second, we compared our optimized TMS protocol to commonly used techniques, specifically the Infinium MethylationEPIC BeadChip (n=55 paired samples) and whole genome bisulfite sequencing (n=6 paired samples). In both cases, we found strong agreement between technologies (R² = 0.97 and 0.99, respectively). Third, we tested the optimized TMS protocol in three non-human primate species (rhesus macaques, geladas, and capuchins). We captured a high percentage (mean=77.1%) of targeted CpG sites and produced methylation level estimates that agreed with those generated from reduced representation bisulfite sequencing (R² = 0.98). Finally, we applied our protocol to profile age-associated DNA methylation variation in two subsistence-level populations-the Tsimane of lowland Bolivia and the Orang Asli of Peninsular Malaysia-and found age-methylation patterns that were strikingly similar to those reported in high income cohorts, despite known differences in age-health relationships between lifestyle contexts. Altogether, our optimized TMS protocol will enable cost-effective, population-scale studies of genome-wide DNA methylation levels across human and non-human primate species.

A major goal in evolutionary biology and biomedicine is to understand the complex interactions between genetic variants, the epigenome, and gene expression. However, the causal relationships between these factors remain poorly understood. mSTARR-seq, a methylation-sensitive massively parallel reporter assay, is capable of identifying methylation-dependent regulatory activity at many thousands of genomic regions simultaneously, and allows for the testing of causal relationships between DNA methylation and gene expression on a region-by-region basis. Here, we developed a multiplexed mSTARR-seq protocol to assay naturally occurring human genetic variation from 25 individuals sampled from 10 localities in Europe and Africa. We identified 6,957 regulatory elements in either the unmethylated or methylated state, and this set was enriched for enhancer and promoter annotations, as expected. The expression of 58% of these regulatory elements was modulated by methylation, which was generally associated with decreased RNA expression. Within our set of regulatory elements, we used allele-specific expression analyses to identify 8,020 sites with genetic effects on gene regulation; further, we found that 42.3% of these genetic effects varied between methylated and unmethylated states. Sites exhibiting methylation-dependent genetic effects were enriched for GWAS and EWAS annotations, implicating them in human disease. Compared to datasets that assay DNA from a single European individual, our multiplexed assay uncovers dramatically more genetic effects and methylation-dependent genetic effects, highlighting the importance of including diverse individuals in assays which aim to understand gene regulatory processes.

Understanding how genetic variation impacts gene expression is a major goal of genomics; however, only a fraction of disease-associated loci have been demonstrated to impact gene expression when cells are in an unperturbed "steady state." In this issue of Cell Genomics, Lin et al. 1 investigate how exposure to a particular cellular context (i.e., a high-cholesterol, high-fat diet) can enhance our ability to identify new regulatory variants through longitudinal sampling of three tissue types in the baboon., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Social adversity can increase the age-associated risk of disease and death, yet the biological mechanisms that link social adversities to aging remain poorly understood. Long-term naturalistic studies of nonhuman animals are crucial for integrating observations of social behavior throughout an individual's life with detailed anatomical, physiological, and molecular measurements. Here, we synthesize the body of research from one such naturalistic study system, Cayo Santiago, which is home to the world's longest continuously monitored free-ranging population of rhesus macaques (Macaca mulatta). We review recent studies of age-related variation in morphology, gene regulation, microbiome composition, and immune function. We also discuss ecological and social modifiers of age-markers in this population. In particular, we summarize how a major natural disaster, Hurricane Maria, affected rhesus macaque physiology and social structure and highlight the context-dependent and domain-specific nature of aging modifiers. Finally, we conclude by providing directions for future study, on Cayo Santiago and elsewhere, that will further our understanding of aging across different domains and how social adversity modifies aging processes., Competing Interests: Competing interests The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Adverse experiences in early life are associated with aging-related disease risk and mortality across many species. In humans, confounding factors, as well as the difficulty of directly measuring experiences and outcomes from birth till death, make it challenging to identify how early life adversity impacts aging and health. These challenges can be mitigated, in part, through the study of non-human animals, which are exposed to parallel forms of adversity and can age similarly to humans. Furthermore, studying the links between early life adversity and aging in natural populations of non-human animals provides an excellent opportunity to better understand the social and ecological pressures that shaped the evolution of early life sensitivities. Here, we highlight ongoing and future research directions that we believe will most effectively contribute to our understanding of the evolution of early life sensitivities and their repercussions., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Exposure to adversity during early life is linked to lasting detrimental effects on evolutionary fitness across many taxa. However, due to the challenges of collecting longitudinal data, especially in species where one sex disperses, direct evidence from long-lived species remains relatively scarce. Here we test the effects of early life adversity on male and female longevity in a free-ranging population of rhesus macaques ( Macaca mulatta ) at Cayo Santiago, Puerto Rico. We leveraged six decades of data to quantify the relative importance of ten forms of early life adversity for 6,599 macaques (3,230 male, 3,369 female), with a smaller sample size (N=299) for one form of adversity (maternal social isolation) which required high-resolution behavioral data. We found that individuals who experienced more early life adversity died earlier than those who experienced less adversity. Mortality risk was highest during early life, defined as birth to four years old, suggesting acute survival effects of adversity, but heightened mortality risk was also present in macaques who survived to adulthood. Females and males were affected differently by some forms of adversity, and these differences might be driven by varying energetic demands, female philopatry, and male dispersal. By leveraging data on thousands of macaques collected over decades, our results show that the fitness consequences of early life adversity are not uniform across individuals but vary as a function of the type of adversity, timing, and social context, and thus contribute to our limited but growing understanding of the evolution of early life sensitivities in long-lived species.

Social adversity can increase the age-associated risk of disease and death, yet the biological mechanisms that link social adversities to aging remain poorly understood. Long-term naturalistic studies of nonhuman animals are crucial for integrating observations of social behavior throughout an individual's life with detailed anatomical, physiological, and molecular measurements. Here, we synthesize the body of research from one such naturalistic study system, Cayo Santiago Island, which is home to the world's longest continuously monitored free-ranging population of rhesus macaques. We review recent studies of age-related variation in morphology, gene regulation, microbiome composition, and immune function. We also discuss ecological and social modifiers of age-markers in this population. In particular, we summarize how a major natural disaster, Hurricane Maria, affected rhesus macaque physiology and social structure and highlight the context-dependent and domain-specific nature of aging modifiers. Finally, we conclude by providing directions for future study, on Cayo Santiago and elsewhere, that will further our understanding of aging across different domains and how social adversity modifies aging processes.

Although mate choice is expected to favor partners with advantageous genetic properties, the relative importance of genome-wide characteristics, such as overall heterozygosity or kinship, versus specific loci, is unknown. To disentangle genome-wide and locus-specific targets of mate choice, we must first understand congruence in global and local variation within the same individual. This study compares genetic diversity, both absolute and relative to other individuals (i.e., complementarity), assessed across the genome to that found at the major histocompatibility complex (MHC), a hyper-variable gene family integral to immune system function and implicated in mate choice across species. Using DNA from 22 captive olive baboons ( Papio anubis ), we conducted double digest restriction site-associated DNA sequencing to estimate genome-wide heterozygosity and kinship, and sequenced two class I and two class II MHC loci. We found that genome-wide diversity was not associated with MHC diversity, and that diversity at class I MHC loci was not correlated with diversity at class II loci. Additionally, kinship was a significant predictor of the number of MHC alleles shared between dyads at class II loci. Our results provide further evidence of the strong selective pressures maintaining genetic diversity at the MHC in comparison to other randomly selected sites throughout the genome. Furthermore, our results indicate that class II MHC disassortative mate choice may mediate inbreeding avoidance in this population. Our study suggests that mate choice favoring genome-wide genetic diversity is not always synonymous with mate choice favoring MHC diversity, and highlights the importance of controlling for kinship when investigating MHC-associated mate choice., Competing Interests: The authors have no conflicts of interest to disclose., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

A large brain is a defining feature of modern humans, and much work has been dedicated to exploring the molecular underpinnings of this trait. Although numerous studies have focused on genes associated with human microcephaly, no studies have explicitly focused on genes associated with megalencephaly. Here, we investigate 16 candidate genes that have been linked to megalencephaly to determine if: (1) megalencephaly-associated genes evolved under positive selection across primates; and (2) selection pressure on megalencephaly-associated genes is linked to primate brain size. We found evidence for positive selection for only one gene, OFD1, with 1.8% of the sites estimated to have dN/dS values greater than 1; however, we did not detect a relationship between selection pressure on this gene and brain size across species, suggesting that selection for changes to non-brain size traits drove evolutionary changes to this gene. In fact, our primary analyses did not identify significant associations between selection pressure and brain size for any candidate genes. While we did detect positive associations for two genes (GPC3 and TBC1D7) when two phyletic dwarfs (i.e., species that underwent recent evolutionary decreases in brain size) were excluded, these associations did not withstand FDR correction. Overall, these results suggest that sequence alterations to megalencephaly-associated genes may have played little to no role in primate brain size evolution, possibly due to the highly pleiotropic effects of these genes. Future comparative studies of gene expression levels may provide further insights. This study enhances our understanding of the genetic underpinnings of brain size evolution in primates and identifies candidate genes that merit further exploration., (© 2022. The Author(s).)

Two mycobacteriophage genomes were newly sequenced and annotated. Duggie and Hocus were discovered, enriched, and isolated from soil using Mycobacterium smegmatis mc 2 155. The bacteriophages are lytic Siphoviridae and belong to the B1 subcluster. The Hocus and Duggie genomes are highly similar to one another in both nucleotide sequence and gene content., (Copyright © 2020 Doyle et al.)

The relationship between male mating opportunities, stress, and glucocorticoid concentrations is complicated by the fact that physiological stress and glucocorticoid concentrations can be influenced by dominance rank, group size, and the stability of the male dominance hierarchy, along with ecological factors. We studied the three highest-ranking males in nine different social groups within the same free-ranging population of rhesus macaques on Cayo Santiago, Puerto Rico, during the mating season, to examine variation in glucocorticoid concentrations in relation to number of females that conceived each month, alpha status, number of adult males in a group, and male rank hierarchy stability. We found that glucocorticoid concentrations were highest in the early mating season period when more females conceived in each group and declined linearly as the mating season progressed and the number of conceptive females decreased. Alpha males had significantly higher mean monthly glucocorticoid concentrations than other high-ranking males throughout the study period. Male age, number of adult males in a group, and hierarchy stability were not significantly associated with glucocorticoid concentrations. Our findings suggest that alpha males may experience significantly higher levels of physiological stress than their immediate subordinates and that this stress coincides with the period of the mating season when most conceptions occur., (Copyright © 2017 Elsevier Inc. All rights reserved.)

One of the hallmarks of the tumour microenvironment is hypoxia resulting from increased oxygen consumption by proliferative cancer cells and altered vasculature. Hypoxic tension initiates various cellular signals and can drive epithelial to mesenchymal transition (EMT), a process important in cancer progression. In this study, using the antioxidant N-acetylcysteine (NAC), we show that hypoxia-induced reactive oxygen species (ROS) in MDA-MB-468 breast cancer cells, selectively regulate hypoxia-induced increases in N-cadherin and SERPINE1, two proteins involved in cell adhesion. Treatment of cells with NAC also attenuated hypoxia-mediated activation of EGFR, but did not have any effect on hypoxia-mediated induction of HIF1α. Exogenous hydrogen peroxide phenocopied the effects of hypoxia on N-cadherin and SERPINE1 expression and EGFR activation, suggesting its possible involvement in these hypoxia-mediated events. Reflective of their effect on cell adhesion proteins and EGFR (associated with migratory phenotypes), NAC also reduced cell migration under hypoxic conditions, a crucial event in metastasis. Our findings suggest a selective role for redox signalling in the regulation of specific components of the responses to hypoxia and induction of EMT in breast cancer cells. This study provides new evidence supporting the potential of targeting ROS as a therapeutic strategy for the control of breast cancer metastasis.

Following increasing reinfestation with Triatoma infestans after insecticide spraying, the household incidence of infection with Trypanosoma cruzi in children was positively related to the domestic abundance of infected T. infestans and the presence or proportion of infected dogs or cats in Amamá, a rural village in northwestern Argentina. Seven (12.1%) children seronegative for antibodies to T. cruzi at baseline, with no history of travel or blood transfusion, seroconverted after three years. Six incident cases lived in houses heavily infested with T. infestans, with high proportions of bugs infected with T. cruzi and having fed on humans or dogs. The remaining incident case occurred under a very light domestic infestation detected only at the endpoint, and most bugs had fed on humans. Dogs had a 17 times greater force of infection than children (4.3% per year). Sustained vector surveillance is crucially needed in high-risk areas for Chagas disease such as the Gran Chaco.

The association between the nutritional state of mongrel dogs naturally infected with Trypanosoma cruzi and their infectivity to Triatoma infestans bugs and immune response to Trypanosoma cruzi were studied in the rural village of Amamá, northwestern Argentina. All of the 97 evaluated dogs were classified into one of three categories of external clinical aspect (ECA) based on the degree of muscle development, external evidence of bone structures, state of the hair of the coat, existence of fatty deposits, and facial expression. ECA was significantly associated with two nutritional indicators, hematocrit and skin-fold thickness, but not with total serum proteins. For all dogs, hematocrit was significantly correlated with skin-fold thickness. The 2-year survival probability decreased significantly from 60.7% for dogs with good ECA to 45.9% and 31.2% for those with regular and bad ECA, respectively. The age-adjusted relative odds of infection for Triatoma infestans xeno-diagnosis nymphs that fed once on a dog seroreactive for Trypanosoma cruzi decreased significantly as ECA improved, when tested by multiple logistic regression analysis. A delayed hypersensitivity reaction was observed in all of the seroreactive dogs with good ECA but only in 45-50% of those with regular or bad ECA. Dogs with bad ECA had a 2.6 and 6.3 times greater probability of infecting triatomines after a single full blood meal than dogs with regular or good ECA, respectively. Our study shows that the reservoir competence of dogs for Trypanosoma cruzi was associated with ECA, which is a surrogate and valid index of nutritional state.

The risk of domestic reinfestation by Triatoma infestans after a single community-wide application of delta-methrin (2.5% suspension concentrate at 25 mg active ingredient/m2) was studied in Amamá, north-west Argentina, where no insecticide spraying had been done by official control services. The percentage of infested houses fell from 88% before spraying in 1985 to nil during the 6 months after spraying, and thereafter increased from 5% in 1986 to 96% before a second treatment in 1992, fitting closely to a logistic model (r2 = 0.997). Significant risk factors associated with domestic reinfestation determined from stepwise logistic regression and one-factor analysis were the density of T. infestans in bedrooms just before spraying and the surface structure of indoor walls. Peak densities of bugs in 1988-1989 significantly differed between levels of both risk factors. Our study suggests the existence of stable determinants of infestation linked to the household which, in the absence of effective control measures, would also determine the speed of house recolonization and the ensuing bug densities. Plastering of mud walls before application of insecticides to all domestic and peridomestic structures is supported by the study.

The association between Trypanosoma cruzi parasitaemia in dogs and cats and Tryp. cruzi infection rates in domestic Triatoma infestans was studied in a cross-sectional survey of 31 houses (89%) in the rural villages of Trinidad and Mercedes, north-west Argentina, where no spraying of insecticides had ever been done. Similar prevalence rates of parasitaemia, determined by xenodiagnosis, were recorded among 68 dogs (41.2%) and 28 cats (39.3%). Bug infection rates were significantly associated with the presence of infected cats (those with positive xenodiagnosis) stratified by the number of infected dogs (relative risk = RR = 1.90; 95% confidence interval = CI = 1.51-2.38), and with the number of infected dogs stratified by the presence of infected cats (RR = 2.71; CI = 1.81-4.07). The percentage of infected bugs in houses with and without children stratified by the presence of infected dogs or cats was not significantly different (RR = 0.69; CI = 0.45-1.05). The combined effect of infected dogs and infected cats on bug infection rates fitted closely with an additive transmission model. Bug infection rates were significantly higher when infected dogs shared the sleeping areas of people than when they did not (RR = 1.79; CI = 1.1-2.91). Our study showed that infected dogs and infected cats increase the risk of domestic transmission of Tryp. cruzi to T. infestans.

The influence of reinfections on the infectivity to Triatoma infestans of dogs seropositive to Trypanosoma cruzi was investigated in Amamá, north-west Argentina, before (1984) and after (1986-1987) elimination of domestic bugs with residual insecticides in 1985. Infectivity was quantified by the percentage of infected bugs in each xenodiagnosis of a seropositive dog, i.e. the infective force (IF). Exposure to reinfection was represented by the domiciliary density of infected bugs and by an index of the potentially-infective contacts (IPIC) experienced by each dog (calculated as the product of the relative density of bugs per dog, the proportion of infected bugs, and the proportion of bugs taking blood meals from the dog). IF was unrelated to the dog's age or sex, or the level of specific antibodies to T. cruzi. Before vector elimination, the IF of seropositive dogs was significantly associated with both the density of infected bugs and the IPIC. After vector elimination, seropositive dogs resident at houses which had 10-70 infected bugs before spraying showed a higher median IF (67-75%) than those living in houses with zero to eight infected bugs before spraying (47-55%). The age-specific IF of seropositive dogs aged one year or older, recorded before (45-64%) and after (52-56%) the elimination of domestic bugs, did not differ significantly. Our data suggest that the infectivity of seropositive dogs to bugs is modified by previous or current exposure to bug-mediated reinfections, and that this relationship might be irreversible in the short term.

The association between household seroreactivity to Trypanosoma cruzi in dogs and children and T. cruzi infection rates in domestic Triatoma infestans was investigated in 1988-1989 in the rural community of Amamá, north-west Argentina, where house spraying with residual pyrethroids was carried out in 1985. Three years after spraying, a greater reduction of the average T. cruzi prevalence rate in dogs (from 83% to 40%) than in children (from 48% to 30%) was accompanied by a substantial decrease in vector infection rates from 51%-63% to 21%. At a household level, in homes with or without seroreactive children, the percentage of infected T. infestans was 4.5-4.7 times higher when seroreactive dogs were present (27.1%-34.8%) than when they were not (5.8%-7.7%; stratified relative risk [RR] = 4.58). The contribution of seroreactive children to bug infection rates was not significant (RR = 1.29). The combined effect of both seroreactive dogs and seroreactive children fitted equally well with additive or multiplicative transmission models. Bug infection rates showed an increasing trend with the number of seroreactive dogs and an inverse association with the age of the youngest seroreactive dog. Our study supports the hypothesis of a causal association between the presence and number of infected dogs and increased levels of T. cruzi transmission to domestic T. infestans.

A three-year demographic and seroparasitological follow-up of the canine population of a rural area of Argentina endemic for Chagas' disease was carried out in order to (a) describe the population dynamics of domestic dogs, and (b) predict the decrease in the prevalence of Trypanosoma cruzi among these reservoirs after indoor spraying of houses with residual insecticides. For the latter project we designed an age-structured model with a discrete time scale. We assumed a time-dependent exponential decay in the frequency of infected hosts proportional to the host mortality rate, and also assumed and that no differential mortality existed between infected and non-infected dogs. Validation of the model was carried out, and yielded an extremely close fit between observed and theoretical values. The relevance of the model as an aid to designing strategies for the control of animal domestic reservoirs of T. cruzi, and the role of dogs as efficient sentinels of re-infestation of an area, are discussed.