Serge Aron

Global warming and the increasing frequency of extreme weather events threaten organisms worldwide, with cold-adapted species like bumblebees being especia

Stingless bees serve as crucial pollinators and are increasingly recognized as models for investigating behavioral and genomic evolution in insects. In the

Epigenetic clocks are machine learning models that predict an organism's chronological age (the time elapsed since birth) or biological age (a proxy for physiological integrity) based on methylation levels from multiple genomic sites. To date, all epigenetic clocks rely exclusively on C5‐methylcytosine (5 mC), the predominant DNA methylation mark in vertebrates. However, not all species possess detectable 5 mC levels. Here, we used N6‐methyladenine (6 mA), a less‐characterized DNA modification type, to develop a series of epigenetic clocks in the buff‐tailed bumblebee ( Bombus terrestris ). Using long‐read Nanopore sequencing, we generated genome‐wide, base‐resolution profiles of 6 mA and 5 mC in males of different ages ( n = 15), and developed multiple epigenetic clocks based on distinct features of the aging DNA methylome. All clocks showed strong correlations between predicted epigenetic and chronological age. Moreover, they also detected pharmacologically induced lifespan extension, reflected by a reduction in predicted epigenetic age relative to chronological age, indicating that these clocks capture biological aging. These findings demonstrate that 6 mA can be used to build accurate epigenetic clocks and establish 6 mA as a promising biomarker of aging in animals.

Background/Objectives: The DNA methylome allows environmental signals to be converted into stable and adaptive changes in gene expression. While 5-methylcytosine (5mC) has been extensively studied, alternative epigenetic marks such as N6-methyladenine (6mA) and 5-hydroxymethylcytosine (5hmC) remain poorly understood. Comparative studies of these marks are rare, and their results are often confounded by phylogeny, tissue type, developmental stage, or methodology. Here, we aimed to disentangle the constitutive, somatic- and germline-specific, and/or age-related patterns displayed by 6mA, 5mC, and 5hmC within a single species. Methods: We generated long-read nanopore sequencing data for somatic tissues of buff-tailed bumblebee (Bombus terrestris) males and their sperm, enabling simultaneous detection of 6mA, 5mC, and 5hmC. We used a stepwise approach to successively identify (i) constitutive patterns conserved between somatic tissues and sperm, (ii) differences between the soma and the germline, and (iii) age-related changes between young and old males. Results: We found distinct constitutive, somatic and sperm, and age-related specific signatures in the genomic contexts, maintenance fidelity, and biological functions associated with 6mA, 5mC, and 5hmC. Sperm cells consistently displayed lower methylation entropy than did somatic tissues, indicating more stable methylation patterns in the germline. 5mC exhibited the greatest variation across all genomic contexts; 6mA and 5hmC displayed less dramatic differences. The influence of age was subtler but revealed context-dependent remodeling of methylation, particularly for 5hmC. Conclusions: We observed that 6mA, 5mC, and 5hmC displayed constitutive, somatic- and sperm-specific, and age-related differences that were associated with distinct genomic contexts and biological functions, supporting the complementarity of these methylation marks and their diverging epigenetic roles.

Epigenetic alterations are a hallmark of aging. Age-specific DNA methylation patterns can be used to create ‘epigenetic clocks’—machine-learning algorithms that use methylation data from multiple genomic sites to predict an organism’s chronological age ( i . e ., the number of years or time passed since birth) or biological age ( i . e ., a measure of an organism’s health and functional status). Epigenetic clocks have been developed for mammals and, to a lesser extent, for birds, fish, amphibians, crustaceans, and insects. At present, all epigenetic clocks utilise C5-methylcytosine (5mC), a prevalent DNA methylation mark in vertebrates. However, in some species, 5mC marks are rare or even undetectable. Here, we describe epigenetic clocks based on N6-methyladenine (6mA), a DNA methylation mark whose role in aging has remained unexplored. Using Oxford Nanopore Technology (ONT) sequencing, we measured genome-wide base-resolution levels of 6mA and 5mC in males of the buff-tailed bumblebee Bombus terrestris ( n = 24). We constructed a series of epigenetic clocks using age-specific patterns in 6mA or 5mC. For each clock, predicted epigenetic age and chronological age were highly correlated. Furthermore, we pharmacologically increased individual lifespan with pharmacological agents and showed that, for individuals whose lifespan had been pharmacologically increased, each clock predicted younger epigenetic age than chronological age, indicating that the clocks captured signals of biological aging. Our results demonstrate that 6mA patterns can be used to build epigenetic clocks that accurately predict both chronological and biological age in animals, paving the way toward the use of 6mA as a reliable biomarker of aging.

Stingless bees are essential pollinators and emerging models for studying behavioral and genomic evolution. In the genus Melipona , a major difference in heterochromatin organization defines two groups: Group I species (e.g., M. quadrifasciata ) with 50% heterochromatin across their chromosomes. These differences are thought to correlate with genome size and transposable element (TE) content, offering a unique opportunity to explore how heterochromatin variation, TE dynamics, and chromosomal evolution interact in a phylogenetic context. We present pseudo-chromosome-level genome assemblies for M. quadrifasciata and M. scutellaris obtained by long-read sequencing and Hi-C scaffolding. Comparative analyses reveal conserved synteny but marked divergence in structural variants and TEs. M. scutellaris shows an expansion of retrotransposons, particularly Gypsy/DIRS1 elements, concentrated in TE hotspots linked to chromosomal rearrangements and structural variants. This coincides with distinct methylation entropy and an expansion of histone deacetylase orthologs, potentially affecting heterochromatin organization. The increased ratio of retrotransposons in M. scutellaris is counterbalanced by more DNA transposons in M. quadrifasciata , resulting in genomes of similar overall sizes but of distinct heterochromatin distribution. Advancing our understanding of genome evolution in eusocial insects, we provide high-resolution genomic resources for two Melipona species that differ in heterochromatin content. Our results highlight the complex role of TEs in shaping genomes and underscore their influence on chromosomal and epigenetic innovation, providing strong evidence that TE dynamics underly the striking heterochromatic differences observed in Melipona .

Social organization, dispersal and fecundity coevolve, but whether they are genetically linked remains little known. Supergenes are prime candidates for coupling adaptive traits and mediating sex-specific trade-offs. Here, we test whether a supergene that controls social structure in Formica selysi also influences dispersal-related traits and fecundity within each sex. In this ant species, single-queen colonies contain only the ancestral supergene haplotype M and produce MM queens and M males, while multi-queen colonies contain the derived haplotype P and produce MP queens, PP queens and P males. By combining multiple experiments, we show that the M haplotype induces phenotypes with higher dispersal potential and higher fecundity in both sexes. Specifically, MM queens, MP queens and M males are more aerodynamic and more fecund than PP queens and P males, respectively. Differences between MP and PP queens from the same colonies reveal a direct genetic effect of the supergene on dispersal-related traits and fecundity. The derived haplotype P , associated with multi-queen colonies, produces queens and males with reduced dispersal abilities and lower fecundity. More broadly, similarities between the Formica and Solenopsis systems reveal that supergenes play a major role in linking behavioural, morphological and physiological traits associated with intraspecific social polymorphisms.

Epigenetic alterations are a primary hallmark of ageing. In mammals, age-related epigenetic changes alter gene expression profiles, disrupt cellular homeostasis and physiological functions and, therefore, promote ageing. It remains unclear whether ageing is also driven by epigenetic mechanisms in invertebrates. Here, we used a pharmacological hypomethylating agent (RG108) to evaluate the effects of DNA methylation (DNAme) on lifespan in an insect—the bumblebee Bombus terrestris . RG108 extended mean lifespan by 43% and induced the differential methylation of genes involved in hallmarks of ageing, including DNA damage repair and chromatin organization. Furthermore, the longevity gene sirt1 was overexpressed following the treatment. Functional experiments demonstrated that SIRT1 protein activity was positively associated with lifespan. Overall, our study indicates that epigenetic mechanisms are conserved regulators of lifespan in both vertebrates and invertebrates and provides new insights into how DNAme is involved in the ageing process in insects.