Immune aging in annual killifish.

Turquoise killifish (Nothobranchius furzeri) evolved a naturally short lifespan of about six months and exhibit aging hallmarks that affect multiple organs. These hallmarks include protein aggregation, telomere shortening, cellular senescence, and systemic inflammation. Turquoise killifish possess the full spectrum of vertebrate-specific innate and adaptive immune system. However, during their recent evolutionary history, they lost subsets of mucosal-specific antibody isoforms that are present in other teleosts. As they age, the immune system of turquoise killifish undergoes dramatic cellular and systemic changes. These changes involve increased inflammation, reduced antibody diversity, an increased prevalence of pathogenic microbes in the intestine, and extensive DNA damage in immune progenitor cell clusters. Collectively, the wide array of age-related changes occurring in turquoise killifish suggest that, despite an evolutionary separation spanning hundreds of millions of years, teleosts and mammals share common features of immune system aging. Hence, the spontaneous aging observed in the killifish immune system offers an excellent opportunity for discovering fundamental and conserved aspects associated with immune system aging across vertebrates. Additionally, the species' naturally short lifespan of only a few months, along with its experimental accessibility, offers a robust platform for testing interventions to improve age-related dysfunctions in the whole organism and potentially inform the development of immune-based therapies for human aging-related diseases.

Dietary restriction mitigates the age-associated decline in mouse B cell receptor repertoire diversity.

Aging impairs the capacity to respond to novel antigens, reducing immune protection against pathogens and vaccine efficacy. Dietary restriction (DR) extends life- and health span in diverse animals. However, little is known about the capacity of DR to combat the decline in immune function. Here, we study the changes in B cell receptor (BCR) repertoire during aging in DR and control mice. By sequencing the variable region of the BCR heavy chain in the spleen, we show that DR preserves diversity and attenuates the increase in clonal expansions throughout aging. Remarkably, mice starting DR in mid-life have repertoire diversity and clonal expansion rates indistinguishable from chronic DR mice. In contrast, in the intestine, these traits are unaffected by either age or DR. Reduced within-individual B cell repertoire diversity and increased clonal expansions are correlated with higher morbidity, suggesting a potential contribution of B cell repertoire dynamics to health during aging.

Taxonomic Profiling of Fecal and Intestinal Microbiota in the African Turquoise Killifish Nothobranchius furzeri.

Killifish are emerging as a new laboratory system in which to study a range of questions, from the genetic basis of embryo dormancy to life history trait evolution, age-dependent neurodegeneration, and the connection between microbial community structure and biology of aging. Over the past decade, advances in high-throughput sequencing have helped uncover the vast diversity of microbial communities present in environmental samples and on host epithelia. Here, we describe an optimized protocol to study the taxonomic composition of intestinal and fecal microbiota in laboratory-raised as well as natural killifish populations and provide comprehensive step-by-step instructions for tissue sampling, high-throughput genomic DNA extraction, and the generation of 16S V3V4 rRNA and 16S V4 rRNA gene libraries.

Noninvasive Ejaculate Collection from the African Turquoise Killifish Nothobranchius furzeri.

Ejaculate collection from live animals is a fundamental technique used to perform repeated measures on the same individual-for instance, to test the effect of different treatments/conditions on the same animal or to conduct longitudinal studies. Moreover, collecting the ejaculate allows one to perform in vitro fertilizations (IVFs) or sperm cryopreservation from valuable individuals (e.g., mutant, transgenic animals) without the need to kill the animals. We developed a noninvasive protocol based on a stripping procedure for collecting the ejaculate from live males of the African turquoise killifish (Nothobranchius furzeri). The proposed procedure allows the retrieval of mature sperm along with the seminal fluid. The ejaculate can be used within 1 h of the collection to perform in vitro fertilizations and/or sperm quality measurements. Contamination by mucus, water, urine, or feces can activate the sperm and/or negatively affect sperm quality, so special care is needed to avoid contamination and thus prolong sperm viability. Since in small fish the amount of ejaculate collected is limited to a few microliters, in the present protocol we also provide recommendations on housing conditions to maximize the probability to obtain ejaculate samples.

Sequencing the Immunoglobulin Heavy-Chain Locus (IgH) in Turquoise Killifish (Nothobranchius furzeri).

B cells undergo a process of somatic recombination leading to the synthesis of a staggering diversity of transmembrane and secreted antibodies. This process dates back to the evolution of jawed vertebrates and is pivotal to the origin of lymphocyte-based adaptive immune responses. Here, we optimized a sequencing-based protocol to characterize B-cell-specific IgH transcripts from bulk RNA in the African turquoise killifish, an emerging model organism characterized by naturally short life span and by a vast range of age-dependent dysfunctions.

Resilience integrates concepts in aging research.

Aging research is unparalleled in the breadth of disciplines it encompasses, from evolutionary studies examining the forces that shape aging to molecular studies uncovering the underlying mechanisms of age-related functional decline. Despite a common focus to advance our understanding of aging, these disciplines have proceeded along distinct paths with little cross-talk. We propose that the concept of resilience can bridge this gap. Resilience describes the ability of a system to respond to perturbations by returning to its original state. Although resilience has been applied in a few individual disciplines in aging research such as frailty and cognitive decline, it has not been explored as a unifying conceptual framework that is able to connect distinct research fields. We argue that because a resilience-based framework can cross broad physiological levels and time scales it can provide the missing links that connect these diverse disciplines. The resulting framework will facilitate predictive modeling and validation and influence targets and directions in research on the biology of aging.

Extensive age-dependent loss of antibody diversity in naturally short-lived turquoise killifish.

Aging individuals exhibit a pervasive decline in adaptive immune function, with important implications for health and lifespan. Previous studies have found a pervasive loss of immune-repertoire diversity in human peripheral blood during aging; however, little is known about repertoire aging in other immune compartments, or in species other than humans. Here, we perform the first study of immune-repertoire aging in an emerging model of vertebrate aging, the African turquoise killifish (Nothobranchius furzeri). Despite their extremely short lifespans, these killifish exhibit complex and individualized heavy-chain repertoires, with a generative process capable of producing millions of distinct productive sequences. Whole-body killifish repertoires decline rapidly in within-individual diversity with age, while between-individual variability increases. Large, expanded B-cell clones exhibit far greater diversity loss with age than small clones, suggesting important differences in how age affects different B-cell populations. The immune repertoires of isolated intestinal samples exhibit especially dramatic age-related diversity loss, related to an elevated prevalence of expanded clones. Lower intestinal repertoire diversity was also associated with transcriptomic signatures of reduced B-cell activity, supporting a functional role for diversity changes in killifish immunosenescence. Our results highlight important differences in systemic vs. organ-specific aging dynamics in the adaptive immune system.

Large-scale characterization of sex pheromone communication systems in Drosophila.

Insects use sex pheromones as a reproductive isolating mechanism to attract conspecifics and repel heterospecifics. Despite the profound knowledge of sex pheromones, little is known about the coevolutionary mechanisms and constraints on their production and detection. Using whole-genome sequences to infer the kinship among 99 drosophilids, we investigate how phylogenetic and chemical traits have interacted at a wide evolutionary timescale. Through a series of chemical syntheses and electrophysiological recordings, we identify 52 sex-specific compounds, many of which are detected via olfaction. Behavioral analyses reveal that many of the 43 male-specific compounds are transferred to the female during copulation and mediate female receptivity and/or male courtship inhibition. Measurement of phylogenetic signals demonstrates that sex pheromones and their cognate olfactory channels evolve rapidly and independently over evolutionary time to guarantee efficient intra- and inter-specific communication systems. Our results show how sexual isolation barriers between species can be reinforced by species-specific olfactory signals.

Ancestral transoceanic colonization and recent population reduction in a nonannual killifish from the Seychelles archipelago.

Whether freshwater fish colonize remote islands following tectonic or transoceanic dispersal remains an evolutionary puzzle. Integrating dating of known tectonic events with phylogenomics and current species distribution, we find that killifish species distribution is not explained by species dispersal by tectonic drift only. Investigating the colonization of a nonannual killifish (golden panchax, Pachypanchax playfairii) on the Seychelle islands, we found genetic support for transoceanic dispersal and experimentally discovered an adaptation to complete tolerance to seawater. At the macroevolutionary scale, despite their long-lasting isolation, nonannual golden panchax show stronger genome-wide purifying selection than annual killifishes from continental Africa. However, progressive decline in effective population size over a more recent timescale has probably led to the segregation of slightly deleterious mutations across golden panchax populations, which represents a potential threat for species preservation in the long term.

Intra-species differences in population size shape life history and genome evolution.

The evolutionary forces shaping life history divergence within species are largely unknown. Turquoise killifish display differences in lifespan among wild populations, representing an ideal natural experiment in evolution and diversification of life history. By combining genome sequencing and population genetics, we investigate the evolutionary forces shaping lifespan among wild turquoise killifish populations. We generate an improved reference genome assembly and identify genes under positive and purifying selection, as well as those evolving neutrally. Short-lived populations from the outer margin of the species range have small population size and accumulate deleterious mutations in genes significantly enriched in the WNT signaling pathway, neurodegeneration, cancer and the mTOR pathway. We propose that limited population size due to habitat fragmentation and repeated population bottlenecks, by increasing the genome-wide mutation load, exacerbates the effects of mutation accumulation and cumulatively contribute to the short adult lifespan.

Microbiota-host interactions shape ageing dynamics.

Occupying the interface between host and environment, host-associated microbes play fundamental roles in nutrient absorption, essential metabolite synthesis, development of the immune system, defence against pathogens and pathogenesis. Microbiota composition and function is rather stable during adulthood, while it dramatically changes during early development, frailty and disease. Ageing is associated with progressive decrease of homeostasis, often resulting in disruption of the physiological balance between host and commensal microbes, ultimately leading to dysbiosis and host demise. Generally, high microbial diversity is associated with health and a youthful state, while low individual microbial diversity and larger inter-individual microbial diversity is associated with ageing and disease states. Different species are equipped with species-specific commensal, symbiotic and pathogenic microbial communities. How and whether the specific host-microbiota consortia co-evolved with host physiology to ensure homeostasis and promote individual fitness remains an open question. In this essay, we propose that the evolution of vertebrate-specific immune adaptations may have enabled the establishment of highly diverse, species-specific commensal microbial communities. We discuss how the maintenance of intact immune surveillance mechanisms, which allow discrimination between commensal and pathogenic bacteria, fail during ageing and lead to the onset of known ageing-related diseases. We discuss how host-microbiota interactions are key to maintaining homeostasis despite external perturbations, but also how they affect a range of host-specific ageing-related phenotypes. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Extreme genomic volatility characterizes the evolution of the immunoglobulin heavy chain locus in cyprinodontiform fishes.

The evolution of the adaptive immune system has provided vertebrates with a uniquely sophisticated immune toolkit, enabling them to mount precise immune responses against a staggeringly diverse range of antigens. Like other vertebrates, teleost fishes possess a complex and functional adaptive immune system; however, our knowledge of the complex antigen-receptor genes underlying its functionality has been restricted to a small number of experimental and agricultural species, preventing systematic investigation into how these crucial gene loci evolve. Here, we analyse the genomic structure of the immunoglobulin heavy chain (IGH) gene loci in the cyprinodontiforms, a diverse and important group of teleosts present in many different habitats across the world. We reconstruct the complete IGH loci of the turquoise killifish (Nothobranchius furzeri) and the southern platyfish (Xiphophorus maculatus) and analyse their in vivo gene expression, revealing the presence of species-specific splice isoforms of transmembrane IGHM. We further characterize the IGH constant regions of 10 additional cyprinodontiform species, including guppy, Amazon molly, mummichog and mangrove killifish. Phylogenetic analysis of these constant regions suggests multiple independent rounds of duplication and deletion of the teleost-specific antibody class IGHZ in the cyprinodontiform lineage, demonstrating the extreme volatility of IGH evolution. Focusing on the cyprinodontiforms as a model taxon for comparative evolutionary immunology, this work provides novel genomic resources for studying adaptive immunity and sheds light on the evolutionary history of the adaptive immune system.

Cell cycle dynamics during diapause entry and exit in an annual killifish revealed by FUCCI technology.

BACKGROUND: Annual killifishes are adapted to surviving and reproducing over alternating dry and wet seasons. During the dry season, all adults die and desiccation-resistant embryos remain encased in dry mud for months or years in a state of diapause where their development is halted in anticipation of the months that have to elapse before their habitats are flooded again. Embryonic development of annual killifishes deviates from canonical teleost development. Epiblast cells disperse during epiboly, and a "dispersed phase" precedes gastrulation. In addition, annual fish have the ability to enter diapause and block embryonic development at the dispersed phase (diapause I), mid-somitogenesis (diapause II) and the final phase of development (diapause III). Developmental transitions associated with diapause entry and exit can be linked with cell cycle events. Here we set to image this transition in living embryos. RESULTS: To visibly explore cell cycle dynamics during killifish development in depth, we created a stable transgenic line in Nothobranchius furzeri that expresses two fluorescent reporters, one for the G1 phase and one for the S/G2 phases of the cell cycle, respectively (Fluorescent Ubiquitination-based Cell Cycle Indicator, FUCCI). Using this tool, we observed that, during epiboly, epiblast cells progressively become quiescent and exit the cell cycle. All embryos transit through a phase where dispersed cells migrate, without showing any mitotic activity, possibly blocked in the G1 phase (diapause I). Thereafter, exit from diapause I is synchronous and cells enter directly into the S phase without transiting through G1. The developmental trajectories of embryos entering diapause and of those that continue to develop are different. In particular, embryos entering diapause have reduced growth along the medio-lateral axis. Finally, exit from diapause II is synchronous for all cells and is characterized by a burst of mitotic activity and growth along the medio-lateral axis such that, by the end of this phase, the morphology of the embryos is identical to that of direct-developing embryos. CONCLUSIONS: Our study reveals surprising levels of coordination of cellular dynamics during diapause and provides a reference framework for further developmental analyses of this remarkable developmental quiescent state.

Relaxed Selection Limits Lifespan by Increasing Mutation Load.

To uncover the selective forces shaping life-history trait evolution across species, we investigate the genomic basis underlying adaptations to seasonal habitat desiccation in African killifishes, identifying the genetic variants associated with positive and relaxed purifying selection in 45 killifish species and 231 wild individuals distributed throughout sub-Saharan Africa. In annual species, genetic drift led to the expansion of nuclear and mitochondrial genomes and caused the accumulation of deleterious genetic variants in key life-history modulating genes such as mtor, insr, ampk, foxo3, and polg. Relaxation of purifying selection is also significantly associated with mitochondrial function and aging in human populations. We find that relaxation of purifying selection prominently shapes genomes and is a prime candidate force molding the evolution of lifespan and the distribution of genetic variants associated with late-onset diseases in different species. VIDEO ABSTRACT.

Loss of genomic integrity induced by lysosphingolipid imbalance drives ageing in the heart.

Cardiac dysfunctions dramatically increase with age. Revealing a currently unknown contributor to cardiac ageing, we report the age-dependent, cardiac-specific accumulation of the lysosphingolipid sphinganine (dihydrosphingosine, DHS) as an evolutionarily conserved hallmark of the aged vertebrate heart. Mechanistically, the DHS-derivative sphinganine-1-phosphate (DHS1P) directly inhibits HDAC1, causing an aberrant elevation in histone acetylation and transcription levels, leading to DNA damage. Accordingly, the pharmacological interventions, preventing (i) the accumulation of DHS1P using SPHK2 inhibitors, (ii) the aberrant increase in histone acetylation using histone acetyltransferase (HAT) inhibitors, (iii) the DHS1P-dependent increase in transcription using an RNA polymerase II inhibitor, block DHS-induced DNA damage in human cardiomyocytes. Importantly, an increase in DHS levels in the hearts of healthy young adult mice leads to an impairment in cardiac functionality indicated by a significant reduction in left ventricular fractional shortening and ejection fraction, mimicking the functional deterioration of aged hearts. These molecular and functional defects can be partially prevented in vivo using HAT inhibitors. Together, we report an evolutionarily conserved mechanism by which increased DHS levels drive the decline in cardiac health.

Inverse resource allocation between vision and olfaction across the genus Drosophila.

Divergent populations across different environments are exposed to critical sensory information related to locating a host or mate, as well as avoiding predators and pathogens. These sensory signals generate evolutionary changes in neuroanatomy and behavior; however, few studies have investigated patterns of neural architecture that occur between sensory systems, or that occur within large groups of closely-related organisms. Here we examine 62 species within the genus Drosophila and describe an inverse resource allocation between vision and olfaction, which we consistently observe at the periphery, within the brain, as well as during larval development. This sensory variation was noted across the entire genus and appears to represent repeated, independent evolutionary events, where one sensory modality is consistently selected for at the expense of the other. Moreover, we provide evidence of a developmental genetic constraint through the sharing of a single larval structure, the eye-antennal imaginal disc. In addition, we examine the ecological implications of visual or olfactory bias, including the potential impact on host-navigation and courtship.