Sex differences in aging are robust and ubiquitous. Demographic differences in aging generated by sex have long been recognized, but the underlying biological basis for these differences and the potential for sex-specific interventions remain understudied. To explore sex differences in the response to pro-longevity interventions, we utilized the C. elegans aging model and asked whether male lifespan and reproductive healthspan can be extended via compounds known to have pro-longevity effects in hermaphrodites. We tested seven different compounds at two concentrations each and found that lifespan was extended under all tested conditions. However, reproductive healthspan measured by mating success in late life improved under only two tested conditions, sulforaphane and metformin. These results demonstrate that lifespan and healthspan can be decoupled in C. elegans males and offer a new framework for screening pro-longevity compounds and for studying sex differences in aging in a classical aging model.

In recent decades, genome-wide association studies (GWAS) have been the major approach to understand the biological basis of individual differences in traits and diseases. However, GWAS approaches have proven to have limited predictive power to explain individual differences, particularly for complex traits and diseases in which environmental factors play a substantial role in their etiology. Indeed, individual differences persist even in genetically identical individuals, although fully separating genetic and environmental causation is difficult or impossible in most organisms. To understand the basis of individual differences in the absence of genetic differences, we measured two quantitative reproductive traits in 180 genetically identical young adult Caenorhabditis elegans roundworms in a shared environment and performed single-individual transcriptomics on each worm. We identified hundreds of genes for which expression variation was strongly associated with reproductive traits, some of which depended on prior environmental experience and some of which was random. Multiple small sets of genes together were highly predictive of reproductive traits across individuals, explaining on average over half and over a quarter of variation in the two traits. We manipulated mRNA levels of predictive genes using RNA interference to identify a set of causal genes, demonstrating the utility of this approach for both prediction and understanding underlying biology. Finally, we found that the chromatin environment of predictive genes was enriched for H3K27 trimethylation, suggesting that individual gene expression differences underlying critical traits may be driven in part by chromatin structure. Together, this work shows that individual differences in gene expression that arise independently of underlying genetic differences are both predictive and causal in shaping reproductive traits at levels that equal or exceed genetic variation.

A core facet of the National Institute on Aging’s mission is to identify pharmacological interventions that can promote human healthy aging and long life. As part of the comprehensive effort toward that goal, the NIA Division of Biology of Aging established the Caenorhabditis Intervention Testing Program (CITP) in 2013. The C. elegans model (with an 21 day lifespan) has led the field in dissection of longevity genetics and offers features that allow for relatively rapid testing and for the potential elaboration of biological mechanisms engaged by candidate geroprotectants. CITP builds on this foundation by utilizing a genetically diverse set of intervention test strains so that “subjects” represent genetic diversity akin to that that between mouse and humans. Another distinctive aspect of the CITP is a dedicated focus on reproducibility of longevity outcomes as labs at three independent test sites confirm positive outcomes. The overall goal of the Caenorhabditis Intervention Testing Program (CITP) is to identify robust and reproducible pro-longevity interventions affecting genetically diverse cohorts in the Caenorhabditis genus. A strong Data Collection Center supports data collection and dissemination. Pharmacological interventions tested by CITP can be nominated by the general public, directed by in-house screens, or supported by published scientific literature. As of December 2024, CITP tested > 75 compounds and conducted > 725,000 animal assays over 891 trials. We identified 12 compounds that confer a ≥ 20

A fundamental tenet of evolutionary genetics is that the direction and strength of selection on individual loci vary with the environment. Barcoded evolutionary lineage tracking is a powerful approach for high-throughput measurement of selection within experimental evolution that to date has largely been restricted to studies within microbial systems, largely because the random integration of barcodes within animals is limited by physical and molecular protection of the germline. Here, we use the recently developed TARDIS barcoding system in Caenorhabditis elegans to implement the first randomly inserted genomic-barcode fitness experiment within an animal model and use this system to precisely measure the influence of the concentration of the anthelmintic compound ivermectin on the strength of selection on an ivermectin resistance cassette. The combination of the trio of knockouts in neuronally expressed GluCl channels, avr-14, avr-15, and glc-1, has been previously demonstrated to provide resistance to ivermectin at high concentrations. Varying the concentration of ivermectin in liquid culture allows the strength of selection on these genes to be precisely controlled within populations of millions of individuals, with the frequency of each barcode then being measured at multiple time points via sequencing at deep coverage and used to estimate the fitness of the individual lineages in the population. The mutations display a high cost to resistance at low concentrations, rapidly losing out to wild-type genotypes, but the balance tips in their favor when the ivermectin concentration exceeds 2 nM. This trade-off in resistance is likely generated by a hindered rate of development in resistant individuals. Our results demonstrate that C. elegans can be used to generate high-precision estimates of fitness using a high-throughput barcoding approach to yield novel insights into evolutionarily and economically important traits.

The Caenorhabditis Intervention Testing Program recently characterized the longevity-promoting effects of the vitamin A derivative all-trans retinoic acid (atRA). Here, we test two atRA-related compounds, tamibarotene and bakuchiol, for longevity effects in three strains of Caenorhabditis species. Both tamibarotene, a potent RAR agonist, and bakuchiol, a meroterpene derived from Psoralea corylifolia , showed no significant increase in lifespan across a dosage range of six concentrations. Additionally, bakuchiol was broadly toxic at higher doses. These findings highlight the specificity of atRA's longevity effects and suggest that compounds related to atRA may not universally promote lifespan extension.

Traditionally, differences among individuals have been divided into genetic and environmental causes. However, both types of variation can underlie regulatory changes in gene expression — that is, epigenetic changes — that persist across cell divisions (developmental differentiation) and even across generations (transgenerational inheritance). Increasingly, epigenetic variation among individuals is recognized as an important factor in human diseases and ageing. Moreover, non-genetic inheritance can lead to evolutionary changes within populations that differ from those expected by genetic inheritance alone. Despite its importance, causally linking epigenetic variation to phenotypic differences across individuals has proven difficult, particularly when epigenetic variation operates independently of genetic variation. New genomic approaches are providing unprecedented opportunity to measure and perturb epigenetic variation, helping to elucidate the role of epigenetic variation in mediating the genotype–phenotype map. Here, we review studies that have advanced our understanding of how epigenetic variation contributes to phenotypic differences between individuals within and across generations, and provide a unifying framework that allows historical and mechanistic perspectives to more fully inform one another. Recent genomic approaches are providing unprecedented opportunity to disentangle how genotype and environment affect organismal traits. The authors review the role of epigenetic variation in mediating the genotype–phenotype map across three scales: among individuals within a generation, across one or multiple generations, and long term over evolutionary time.

Discovery of new compounds that ameliorate the negative health impacts of aging promises to be of tremendous benefit across a number of age-based comorbidities. One method to prioritize a testable subset of the nearly infinite universe of potential compounds is to use computational prediction of their likely anti-aging capacity. Here, we present a survey of longevity effects for 16 compounds suggested by a previously published computational prediction set, capitalizing upon the comprehensive, multi-species approach utilized by the Caenorhabditis Intervention Testing Program. While 11 compounds (aldosterone, arecoline, bortezomib, dasatinib, decitabine, dexamethasone, erlotinib, everolimus, gefitinib, temsirolimus, and thalidomide) either had no effect on median lifespan or were toxic, 5 compounds (all-trans retinoic acid, berberine, fisetin, propranolol, and ritonavir) extended lifespan in Caenorhabditis elegans. These computer predictions yield a remarkable positive hit rate of 30%. Deeper genetic characterization of the longevity effects of one of the most efficacious compounds, the endogenous signaling ligand all-trans retinoic acid (atRA, designated tretinoin in medical products), demonstrated a requirement for the regulatory kinases AKT-1 and AKT-2. While the canonical Akt-target FOXO/DAF-16 was largely dispensable, other conserved Akt-targets (Nrf2/SKN-1 and HSF1/HSF-1), as well as the conserved catalytic subunit of AMPK AAK-2, were all necessary for longevity extension by atRA. Our results highlight the potential of combining computational prediction of longevity interventions with the power of nematode functional genetics and underscore that the manipulation of a conserved metabolic regulatory circuit by co-opting endogenous signaling molecules is a powerful approach for discovering aging interventions.

With within-species genetic diversity estimates that span the gamut of that seen across the entirety of animals, the Caenorhabditis genus of nematodes holds unique potential to provide insights into how population size and reproductive strategies influence gene and genome organization and evolution. Our study focuses on Caenorhabditis brenneri, currently known as one of the most genetically diverse nematodes within its genus and, notably, across Metazoa. Here, we present a high-quality, gapless genome assembly and annotation for C. brenneri, revealing a common nematode chromosome arrangement characterized by gene-dense central regions and repeat-rich arms. A comparison of C. brenneri with other nematodes from the "Elegans" group revealed conserved macrosynteny but a lack of microsynteny, characterized by frequent rearrangements and low correlation of orthogroup size, indicative of high rates of gene turnover, consistent with previous studies. We also assessed genome organization within corresponding syntenic blocks in selfing and outcrossing species, affirming that selfing species predominantly experience loss of both genes and intergenic DNA. A comparison of gene structures revealed a strikingly small number of shared introns across species, yet consistent distributions of intron number and length, regardless of population size or reproductive mode, suggesting that their evolutionary dynamics are primarily reflective of functional constraints. Our study provides valuable insights into genome evolution and expands the nematode genome resources with the highly genetically diverse C. brenneri, facilitating research into various aspects of nematode biology and evolutionary processes.

The Caenorhabditis Intervention Testing Program recently characterized the longevity-promoting effects of the vitamin A derivative all-trans retinoic acid (atRA). Here, we test two atRA-related compounds, tamibarotene and bakuchiol, for longevity effects in three strains of Caenorhabditis species. Both tamibarotene, a potent RAR agonist, and bakuchiol, a meroterpene derived from Psoralea corylifolia , showed no significant increase in lifespan across a dosage range of six concentrations. Additionally, bakuchiol was broadly toxic at higher doses. These findings highlight the specificity of atRA's longevity effects and suggest that compounds related to atRA may not universally promote lifespan extension.

Sulforaphane, an organosulfur isothiocyanate derived from cruciferous vegetables, has been shown to inhibit inflammation, oxidative stress, and cancer cell growth. To explore the potential of sulforaphane as a candidate natural compound for promoting longevity more generally, we tested the dose and age-specific effects of sulforaphane on C. elegans longevity, finding that it can extend lifespan by more than 50% at the most efficacious doses, but that treatment must be initiated early in life to be effective. We then created a novel, gene-specific, transcriptional aging clock, which demonstrated that sulforaphane-treated individuals exhibited a "transcriptional age" that was approximately four days younger than age-matched controls, representing a nearly 20% reduction in biological age. The clearest transcriptional responses were detoxification pathways, which, together with the shape of the dose-response curve, indicates a likely hormetic response to sulforaphane. These results support the idea that robust longevity-extending interventions can act via global effects across the organism, as revealed by systems level changes in gene expression.

In recent decades, genome-wide association studies (GWAS) have been the major approach to understand the biological basis of individual differences in traits and diseases. However, GWAS approaches have limited predictive power to explain individual differences, particularly for complex traits and diseases in which environmental factors play a substantial role in their etiology. Indeed, individual differences persist even in genetically identical individuals, although fully separating genetic and environmental causation is difficult in most organisms. To understand the basis of individual differences in the absence of genetic differences, we measured two quantitative reproductive traits in 180 genetically identical young adult Caenorhabditis elegans roundworms in a shared environment and performed single-individual transcriptomics on each worm. We identified hundreds of genes for which expression variation was strongly associated with reproductive traits, some of which depended on individuals’ historical environments and some of which was random. Multiple small sets of genes together were highly predictive of reproductive traits, explaining on average over half and over a quarter of variation in the two traits. We manipulated mRNA levels of predictive genes to identify a set of causal genes, demonstrating the utility of this approach for both prediction and understanding underlying biology. Finally, we found that the chromatin environment of predictive genes was enriched for H3K27 trimethylation, suggesting that gene expression variation may be driven in part by chromatin structure. Together, this work shows that individual, non-genetic differences in gene expression are both highly predictive and causal in shaping reproductive traits.

Reproduction is a fundamental process that shapes the demography of every living organism yet is often difficult to assess with high precision in animals that produce large numbers of offspring. Here, we present a novel microfluidic research platform for studying Caenorhabditis elegans' egg-laying. The platform provides higher throughput than traditional solid-media assays while providing a very high degree of temporal resolution. Additionally, the environmental control enabled by microfluidic animal husbandry allows for experimental perturbations difficult to achieve with solid-media assays. We demonstrate the platform's utility by characterizing C. elegans egg-laying behavior at two commonly used temperatures, 15 and 20°C. As expected, we observed a delayed onset of egg-laying at 15°C degrees, consistent with published temperature effects on development rate. Additionally, as seen in solid media studies, egg laying output was higher under the canonical 20°C conditions. While we validated the Egg-Counter with a study of temperature effects in wild-type animals, the platform is highly adaptable to any nematode egg-laying research where throughput or environmental control needs to be maximized without sacrificing temporal resolution.

The evolution of reproductive mode is expected to have profound impacts on the genetic composition of populations. At the same time, ecological interactions can generate close associations among species, which can in turn generate a high degree of overlap in their spatial distributions. Caenorhabditis elegans is a hermaphroditic nematode that has enabled extensive advances in developmental genetics. Caenorhabditis inopinata, the sister species of C. elegans, is a gonochoristic nematode that thrives in figs and obligately disperses on fig wasps. Here, we describe patterns of genomic diversity in C. inopinata. We performed RAD-seq on individual worms isolated from the field across three Okinawan island populations. C. inopinata is about five times more diverse than C. elegans. Additionally, C. inopinata harbors greater differences in diversity among functional genomic regions (such as between genic and intergenic sequences) than C. elegans. Conversely, C. elegans harbors greater differences in diversity between high-recombining chromosome arms and low-recombining chromosome centers than C. inopinata. FST is low among island population pairs, and clear population structure could not be easily detected among islands, suggesting frequent migration of wasps between islands. These patterns of population differentiation appear comparable with those previously reported in its fig wasp vector. These results confirm many theoretical population genetic predictions regarding the evolution of reproductive mode and suggest C. inopinata population dynamics may be driven by wasp dispersal. This work sets the stage for future evolutionary genomic studies aimed at understanding the evolution of sex as well as the evolution of ecological interactions.

Abstract Ever since the discovery more than 30 years ago of mutations capable dramatically increasing lifespan, there has been an increasing push to find genetic pathways and chemical interventions that ameliorate the effects of aging and increase healthspan and longevity. But aging is not merely “getting old,” it is an accelerating rate of the decline in function and an increase in the rate of mortality with chronological age. Here, we present a new framework for understanding longevity-extending interventions by dividing the survivorship into two major parts—the onset of aging and the period of accelerated aging—and use a uniform analytical approach to derive these quantities for each of the major mortality models. We apply this new framework to a set of studies with sufficient size to estimate mortality rate, finding that for four genetic and three chemical interventions in C. elegans and five genetic and eleven chemical interventions in mice, all treatments that lead to an increase in overall lifespan do so by delaying the onset of aging and none decrease the rate of aging late in life. These effects are readily visualized using a relative hazard and relative lifespan approach. Thus, while there is great interest identify compounds and other approaches to “treat” aging, evidence to date suggests that these interventions do not actually affect aging in the formal sense. These results have important implications for the design and analysis of aging studies, as well as for the field of geroscience as a whole.

Sex-specific regulation of gene expression is the most plausible way for generating sexually differentiated phenotypes from an essentially shared genome. However, since genetic material is shared, sex-specific selection in one sex can have an indirect response in the other sex. From a gene expression perspective, this tethered response can move one sex away from their wild-type expression state and potentially impact many gene regulatory networks. Here, using experimental evolution in the model nematode Caenorhabditis elegans, we explore the coupling of direct sexual selection on males with the transcriptomic response in males and females over microevolutionary timescales to uncover the extent to which postinsemination reproductive traits share a genetic basis between the sexes. We find that differential gene expression evolved in a sex-specific manner in males, while in females, indirect selection causes an evolved response. Almost all differentially expressed genes were downregulated in both evolved males and females. Moreover, 97% of significantly differentially expressed genes in males and 69% of significantly differentially expressed genes in females have wild-type female-biased expression profile. Changes in gene expression profiles were likely driven through trans-acting pathways that are shared between the sexes. We found no evidence that the core dosage compensation machinery was impacted by experimental evolution. Together, these data suggest a defeminization of the male transcriptome and masculinization of the female transcriptome driven by direct selection on male sperm competitive ability. Our results indicate that on short evolutionary timescales, sexual selection can generate putative sexual conflict in expression space.

Engineered sites for genetic transformation have simplified transgene insertion in Caenorhabditis elegans . These strategies include our split hygromycin system ​(Stevenson et al. 2020)​ which allows for integration-specific selection of transgenes. Here we have expanded the split hygromycin selection system to include two additional chromosomal locations, both of which are permissive for germline expression, as well as engineered landing pads in three additional natural isolates. Corresponding guide and empty repair template plasmids are also available for each of these sites.

How genetic and phenotypic variation are maintained has long been one of the fundamental questions in population and quantitative genetics. A variety of factors have been implicated to explain the maintenance of genetic variation in some contexts (e.g. balancing selection), but the potential role of epigenetic regulation to influence population dynamics has been understudied. It is well recognized that epigenetic regulation, including histone methylation, small RNA expression, and DNA methylation, helps to define differences between cell types and facilitate phenotypic plasticity. In recent years, empirical studies have shown the potential for epigenetic regulation to also be heritable for at least a few generations without selection, raising the possibility that differences in epigenetic regulation can act alongside genetic variation to shape evolutionary trajectories. Heritable differences in epigenetic regulation that arise spontaneously are termed "epimutations." Epimutations differ from genetic mutations in 2 key ways-they occur at a higher rate and the loci at which they occur often revert back to their original state within a few generations. Here, we present an extension of the standard population genetic model with selection to incorporate epigenetic variation arising via epimutation. Our model assumes a diploid, sexually reproducing population with random mating. In addition to spontaneous genetic mutation, we included parameters for spontaneous epimutation and back-epimutation, allowing for 4 potential epialleles at a single locus (2 genetic alleles, each with 2 epigenetic states), each of which affect fitness. We then analyzed the conditions under which stable epialleles were maintained. Our results show that highly reversible epialleles can be maintained in long-term equilibrium under neutral conditions in a manner that depends on the epimutation and back-epimutation rates, which we term epimutation-back-epimutation equilibrium. On the other hand, epialleles that compensate for deleterious mutations cause deviations from the expectations of mutation-selection balance by a simple factor that depends on the epimutation and back-epimutation rates. We also numerically analyze several sets of fitness parameters for which large deviations from mutation-selection balance occur. Together, these results demonstrate that transient epigenetic regulation may be an important factor in the maintenance of both epigenetic and genetic variation in populations.

Body size is a fundamental trait that drives multiple evolutionary and ecological patterns. Caenorhabditis inopinata is a fig-associated nematode that is exceptionally large relative to other members of the genus, including C. elegans . We previously showed that C. inopinata is large primarily due to postembryonic cell size expansion that occurs during the larval-to-adult transition. Here, we describe gene expression patterns in C. elegans and C. inopinata throughout this developmental period to understand the transcriptional basis of body size change. We performed RNA-seq in both species across the L3, L4, and adult stages. Most genes are differentially expressed across all developmental stages, consistent with C. inopinata ’s divergent ecology and morphology. We also used a model comparison approach to identify orthologs with divergent dynamics across this developmental period between the two species. This included genes connected to neurons, behavior, stress response, developmental timing, and small RNA/chromatin regulation. Multiple hypodermal collagens were also observed to harbor divergent developmental dynamics across this period, and genes important for molting and body morphology were also detected. Genes associated with TGF-β signaling revealed idiosyncratic and unexpected transcriptional patterns given their role in body size regulation in C. elegans . Widespread transcriptional divergence between these species is unexpected and may be a signature of the ecological and morphological divergence of C. inopinata . Alternatively, transcriptional turnover may be the rule in the Caenorhabditis genus, indicative of widespread developmental system drift among species. This work lays the foundation for future functional genetic studies interrogating the bases of body size evolution in this group.

Aging is characterized by declining health that results in decreased cellular resilience and neuromuscular function. The relationship between lifespan and health, and the influence of genetic background on that relationship, has important implications in the development of pharmacological anti -aging interventions. Here we assessed swimming performance as well as survival under thermal and oxidative stress across a nematode genetic diversity test panel to evaluate health effects for three compounds previously studied in the Caenorhabditis Intervention Testing Program and thought to promote longevity in different ways - NP1 (nitrophenyl piperazine-containing compound 1), propyl gallate, and resveratrol. Overall, we find the relationships among median lifespan, oxidative stress resistance, thermotolerance, and mobility vigor to be complex. We show that oxidative stress resistance and thermotolerance vary with compound intervention, genetic background, and age. The effects of tested compounds on swimming locomotion, in contrast, are largely species -specific. In this study, thermotolerance, but not oxidative stress or swimming ability, correlates with lifespan. Notably, some compounds exert strong impact on some health measures without an equally strong impact on lifespan. Our results demonstrate the importance of assessing health and lifespan across genetic backgrounds in the effort to identify reproducible anti -aging interventions, with data underscoring how personalized treatments might be required to optimize health benefits.

AbstractReproduction is a fundamental process that shapes the demography of every living organism yet is often difficult to assess with high precision in animals that produce large numbers of offspring. Here, we present a novel microfluidic research platform for studyingCaenorhabditis elegans’egg-laying. The platform provides higher throughput than traditional solid-media assays while providing a very high degree of temporal resolution. Additionally, the environmental control enabled by microfluidic animal husbandry allows for experimental perturbations difficult to achieve with solid-media assays. We demonstrate the platform’s utility by characterizingC. elegansegg-laying behavior at two commonly used temperatures, 15 and 20°C. As expected, we observed a delayed onset of egg-laying at 15°C degrees, consistent with published temperature effects on development rate. Additionally, as seen in solid media studies, egg laying output was higher under the canonical 20°C conditions. While we validated the Egg-Counter with a study of temperature effects in wild-type animals, the platform is highly adaptable to any nematode egg-laying research where throughput or environmental control needs to be maximized without sacrificing temporal resolution.