Stingless bees serve as crucial pollinators and are increasingly recognized as models for investigating behavioral and genomic evolution in insects. In the genus Melipona, a major difference in heterochromatin organization defines two groups: Group I species (e.g., M. quadrifasciata) with < 50% of pericentromeric heterochromatin and Group II species (e.g., M. scutellaris) containing > 50% heterochromatin across their chromosomes. These differences are believed 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 through long-read sequencing and 3D chromosome conformation scaffolding. Comparative analyses reveal conserved synteny but marked divergence in structural variants and TE types. M. scutellaris exhibits 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 patterns across the genome and an expansion of histone deacetylase orthologs. The increased proportion of retrotransposons in M. scutellaris is counterbalanced by more DNA transposons in M. quadrifasciata, resulting in genomes of similar overall sizes but with distinct heterochromatin distributions. 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 compelling evidence that TE dynamics underlie the pronounced heterochromatic differences observed in Melipona.

Bumblebee populations are declining at an alarming rate due to anthropic-driven global changes. Increasing temperatures and more frequent extreme heat events impose substantial physiological stress on organisms, reducing fitness and contributing to population declines and biodiversity loss. While the immediate impact of heat stress (HS) on bumblebee behaviour and physiology has been largely documented, the delayed effect on individual fertility remains more elusive. We quantified the effect of HS on the viability of sperm stored in the male accessory testes of buff-tailed bumblebees (Bombus terrestris) following exposure to 25°C (control), 38°C (mild HS) or 42°C (severe HS) for 60 min, 1 day or 15 days after exposure. Sperm quality was assessed by the percentage of viable sperm (sperm viability) and the relative changes in total number of viable sperm cells (Δ viable sperm count) between controls and treatments. Sperm quality was significantly impaired following exposure to 38°C and 42°C compared to control males. However, reproductive impairment was driven by HS intensity, with severe stress causing an acute and immediate loss of the percentage of viable sperm, and mild stress eliciting delayed effects on the number of viable sperm cells. By inducing both immediate and delayed damages to male reproductive function, HS may have potential consequences for fertility under warming conditions.

Global warming and the increasing frequency of extreme weather events threaten organisms worldwide, with cold-adapted species like bumblebees being especially vulnerable. Although heat stress has been shown to affect bumblebee physiology and behaviour, whether it also impacts their epigenome remains unknown. In this study, we addressed this gap by investigating the short- and long-term effects of acute heat stress experienced early in adult life on DNA methylation profiles in the buff-tailed bumblebee (Bombus terrestris). The DNA methylomes of males exposed either to control conditions (25°C) or to acute heat stress (42 °C for 60 min) were sequenced 1 day and 15 days after exposure to disentangle the short- and long-term effects of thermal stress. Heat stress induced differential methylation at hundreds to thousands of sites in both the short- and long-term, yet these signatures were largely distinct across time points. Accordingly, random forest classification failed to identify a persistent long-term DNA methylation signature of heat stress, indicating that heat-induced DNA methylation patterns change over time. Similarly, epigenetic clock analyses revealed that epigenetic age was transiently increased in the short-term, but not in the long-term. By uncovering the temporal dynamics of DNA methylation profiles following heat stress inB. terrestrismales, our findings suggest that DNA methylation is actively regulated rather than passively maintaining environmentally induced alterations. These results shed new light on how environmental signals may dynamically shape the insect epigenome.

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.

Eusocial insect queens exhibit some of the most extreme durations of sperm storage in the animal kingdom. This extended lifespan of sperm within the queen’s storage organ (the spermatheca) after mating is largely sustained by the spermathecal fluid matrix—a rich and proteinaceous secretion that fills the void volume within the spermatheca. Here, we conducted a comparative proteomics study on mating-induced changes in spermathecal fluid of two long-lived hymenopteran species, Lasius niger and Apis mellifera. We found some similarities between species; for example, enolase and other enzymes responsible for carbohydrate metabolism were among the top differentially expressed proteins in both A. mellifera and L. niger. Additionally, both species exhibited post-mating upregulation of catalase, glutathione peroxidase, and Mn-conjugated superoxide dismutase (SOD), all of which are important antioxidant enzymes. However, we also identified notable differences, with Cu/Zn-conjugated SODs being consistently downregulated after mating in L. niger but upregulated in A. mellifera. Likewise, canonical immune effectors (phenoloxidase and lysozyme) showed similar patterns of expression in both species, (with phenoloxidase remaining unchanged and lysozyme increasing after mating), but ferritins, which are multifunctional antioxidant proteins that are also induced by immune challenges, differed, increasing in L. niger but decreasing in A. mellifera. Herein, we discuss expression patterns of these proteins and additional immune proteins, hexamerins, odorant binding proteins, and a key carbohydrate metabolism enzyme (glyceraldehyde-3-phosphate dehydrogenase) in the context of the differential life histories of these two eusocial insect species.

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. ### Competing Interest Statement The authors have declared no competing interest. Université Libre de Bruxelles, https://ror.org/01r9htc13, ARC 2025-2028, PDR T.0010.24 Fund for Scientific Research, FC 57457, RFE24/0071

Little is known about the influence of mating strategies that could potentially facilitate the colonization of new hosts in outbreeding species of the weevil subfamily Scolytinae. Individuals typically emerge from their host tree, disperse, and then mate with unrelated conspecifics in a new host where the females establish maternal galleries. Yet, in several spe-cies commonly classified as outbreeding, females have been found already mated before host colonization. Precolonization mating provides female with a sperm supply before they find a new host and allows them to establish a maternal gallery on their own. We compared the proportion of females mated before host colonization across 18 European and four American outbreeding Scolytinae species using a phylogenetically controlled analysis. To this end, we determined whether females caught in the spring had sperm in their spermathecae. We found that a proportion of females (range: 16-100%) mated before host colonization in all 22 species. Moreover, this trait was biased, although not significantly, toward invasiveness. Species known to have established outside their native range (Scolytinae with an Invasion History-SIH) displayed a higher proportion of females mated before host colonization than did species restricted to their native range (non-SIH). In Hylurgus ligniperda (Fabricius), a Palearctic species currently present across the globe, the proportions of females mated before host colonization reach 90% in the species' native range and up to 99% in its nonnative range (Argentina and New Zealand). Overall, these results show that precolonization mating is widespread among the Scolytinae. This trait could enhance the invasive capacities of outbreeding species by allowing females to establish a maternal gallery independently of any male during colonization, thus facilitating the establishment and spread of species introduced in new geographical areas.

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.

Hymenopteran queens are collectively highly fecund, often long-lived individuals that undergo dramatic physiological changes after they mate and establish a nest. However, the degree to which these changes are conserved among species with different life histories is not well-defined. We conducted a comparative proteomic study investigating differences between reproductive stages (virgin, mated and established queens) of Apis mellifera , Bombus impatiens , B. terrestris and Lasius niger . We analysed haemolymph for all species except L. niger , for which a whole-body analysis was performed due to the small size of these queens. We identified conserved upregulation of proteins involved in anatomical and system development as queens transition to establishing a nest in all species except B. terrestris . We also identified conserved patterns of vitellogenin, vitellogenin receptor and immune-responsive protein (IRP)30, all of which are proteins typically associated with oviposition. However, expression patterns of other immune proteins, heat-shock proteins (HSPs), detoxification enzymes and antioxidant enzymes were more dissimilar, with some species exhibiting similar trends and co-occurrence through reproductive stages, while others exhibited variable or opposite patterns. These conserved and unique profiles likely in part reflect similarities and differences in selective pressure on reproductive stages of each species and may indicate differing abilities to respond to emergent pathogens or environmental change.

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.

In many species, females have multiple mates, whose sperm compete for paternity. Males may subsequently invest in the increased production of sperm and/or seminal fluid. The latter is a complex mixture of proteins, peptides, and other compounds generated by the accessory glands (AGs) and is transferred to females along with a male’s sperm. Seminal fluid is known to be a key determinant of competitive outcomes among sperm, and its production may trade off with that of sperm. We show that AG size—a proxy for seminal fluid production—has a positive and phylogenetically robust correlation with both sperm competition intensity and sperm production in nine species of Cataglyphis desert ants. These results indicate a lack of trade-off between sperm production and seminal fluid production. They underscore that sperm competition may strongly shape sperm traits and could drive reproductive performance in eusocial hymenopterans.

Biological diversity often arises as organisms adapt to new ecological conditions (i.e., ecological opportunities) or colonize suitable areas (i.e., spatial opportunities). Cases of geographical expansion followed by local ecological divergence are well described; they result in clades comprising ecologically heterogeneous subclades. Here, we show that the desert ant genus Cataglyphis likely originated in open grassland habitats in the Middle East ∼18 million years ago and became a taxon of diverse species specializing in prey of different masses. The genus then colonized the Mediterranean Basin around 9 million years ago. The result was the rapid accumulation of species, and the appearance of local assemblages containing species from different lineages that still displayed ancestral foraging specialties. These findings highlight that, in Cataglyphis, ecological diversification preceded geographical expansion, resulting in a clade composed of ecologically homogeneous subclades.

Some ant species live in hot and arid environments, such as deserts and savannas. Worker polymorphism-variation in worker size and/or morphology within colonies-is adaptive in such ecosystems because it enhances resistance to heat stress and increases the efficiency of resource exploitation. However, species with small, monomorphic workers are also frequently found in these environments. How species with distinct worker size and degrees of polymorphism deal with such stressful environments remains poorly studied. We investigated the behavioral, physiological, and molecular adaptations that may enhance heat and desiccation tolerance in two sympatric species of Cataglyphis desert ants that differ dramatically in worker size and polymorphism: C. viatica is polymorphic, while C. cubica is small and monomorphic. We found that worker size, water content, water loss, and protein regulation play a key role in thermal resistance. (i) Large C. viatica workers better tolerated heat and desiccation stress than did small C. viatica or C. cubica workers. The former had greater water content and lost proportionally less water to evaporation under thermal stress. (ii) Despite their similar size distribution, workers of C. cubica are more heat tolerant than small C. viatica. This higher degree of tolerance likely stemmed from C. cubica workers having greater relative water content. (iii) Under thermal stress, small C. viatica workers metabolized larger quantities of fat and differentially expressed proteins involved in cellular homeostasis. In contrast, C. cubica downregulated the expression of numerous proteins involved in mitochondrial respiration likely reducing ROS accumulation. (iv) Consistent with these results, large C. viatica workers remained active throughout the day; C. cubica workers displayed a bimodal activity pattern, and small C. viatica remained poorly active outside the nest. Our study shows that ecologically similar ant species with different degrees of worker size polymorphism evolved distinct strategies for coping with extreme heat conditions.

Global climate changes may cause profound effects on species adaptation, particularly in ectotherms for whom even moderate warmer temperatures can lead to disproportionate heat failure. Still, several organisms evolved to endure high desert temperatures. Here, we describe the thermal tolerance survival and the transcriptomic heat stress response of three genera of desert (Cataglyphis, Melophorus, and Ocymyrmex) and two of temperate ants (Formica and Myrmica) and explore convergent and specific adaptations. We found heat stress led to either a reactive or a constitutive response in desert ants: Cataglyphis holgerseni and Melophorus bagoti differentially regulated very few transcripts in response to heat (0.12% and 0.14%, respectively), while Cataglyphis bombycina and Ocymyrmex robustior responded with greater expression alterations (respectively affecting 0.6% and 1.53% of their transcriptomes). These two responsive mechanisms-reactive and constitutive-were related to individual thermal tolerance survival and convergently evolved in distinct desert ant genera. Moreover, in comparison with desert species, the two temperate ants differentially expressed thousands of transcripts more in response to heat stress (affecting 8% and 12.71% of F. fusca and Myr. sabuleti transcriptomes). In summary, we show that heat adaptation in thermophilic ants involved changes in the expression response. Overall, desert ants show reduced transcriptional alterations even when under high thermal stress, and their expression response may be either constitutive or reactive to temperature increase.

Over recent decades, increasing attention has been paid to how low-molecular-weight molecules affect thermal tolerance in animals. Although the disaccharide sugar trehalose is known to serve as a thermal protectant in unicellular organisms, nothing is known about its potential role in insects. In this study, we investigated the effect of trehalose on heat tolerance in the Namib desert ant, Ocymyrmex robustior, one of the most thermotolerant animals found in terrestrial ecosystems. First, we tested whether a trehalose-supplemented diet increased worker survival following exposure to heat stress. Second, we assessed the degree of protein damage by comparing protein aggregation levels for trehalose-supplemented workers and control workers. Third, we compared the expression levels of three genes involved in trehalose metabolism. We found that trehalose supplementation significantly enhanced worker heat tolerance, increased metabolic levels of trehalose and reduced protein aggregation under conditions of heat stress. Expression levels of the three genes varied in a manner that was consistent with the maintenance of trehalose in the hemolymph and tissues under conditions of heat stress. Altogether, these results suggest that increased trehalose concentration may help protect Namib desert ant individuals against heat stress. More generally, they highlight the role played by sugar metabolites in boosting tolerance in extremophiles.

Group genetic diversity is usually associated with a reduced risk of disease outbreak and a slower rate of pathogen transmission. In social insects, multiple mating by queens (polyandry) evolved several times albeit reducing worker’s inclusive fitness. One major hypothesis suggests that polyandry has been selected for to mitigate the risk of outbreak thanks to increased genetic diversity within colonies. We investigated this hypothesis in the ant Cataglyphis mauritanica, in which nestmate workers are produced by several clonal, single-mated queens. Using natural colonies, we correlated genetic diversity with worker survival to a fungal entomopathogen. We further tested whether workers from different paternal lineages (but a common maternal genome) show differential resistance in experimentally single- or multiple-patriline groups, and whether an increased number of patrilines in a group improved disease resistance. We show that workers from distinct patrilines vary in their resistance to a pathogen in single-patriline colonies, but the difference among patrilines disappears when they are mixed in multiple-patriline colonies. Furthermore, pathogen resistance was affected by the number of patrilines in a group, with two- and three-patriline groups being more resistant than single-patriline groups. However, resistance did not differ between groups made of two and three patrilines; similarly, it was not associated with genetic diversity in natural colonies. Overall, our results suggest that collective disease defenses might homogenize workers’ resistance from different patrilines and, thereby, stabilize colony resistance. The occurrence of multiple breeders in insect societies has been hypothesized to be selected for because increased within-colony genetic diversity reduces the risk of severe outbreaks. We show that nestmate workers from distinct paternal lineages vary in their resistance to pathogens when reared in single-patriline groups. However, this difference disappears when workers are mixed in multiple-patriline groups. These results suggest that multiple mating by queens dilutes the deleterious consequences of a single patriline producing only susceptible offspring, rather than directly enhancing pathogen resistance.

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.

In the coming years, climate change is likely to increase the frequency and intensity of heatwaves. In many organisms, heat stress provokes physiological perturbations and can lead to decreased male fertility. Bumblebees are endo-heterothermic but display interspecific differences in thermotolerance that could have conservation implications. For the species of concern Bombus magnus, exposure to high temperatures can severely reduce sperm quality and, consequently, reproductive success. Such is not the case for B. terrestris, a ubiquitous species. To decipher the mechanisms at play, we characterized the seminal fluid proteomes of the two species. We quantified 1121 proteins, of which 522 were differentially expressed between B. terrestris and B. magnus. Several proteins with protective functions, such as proteases, antioxidant proteins and various heat-shock proteins, were present at higher levels in B. terrestris than in B. magnus under both control and heat-stress conditions. The same was true for proteins involved in cellular homeostasis, immunity, lipid/sugar metabolism and thermotolerance. Furthermore, proteins involved in the capture and elimination of reactive oxygen species also occurred at much high levels in B. terrestris. Overall, these results clearly indicate differences in the seminal proteome of the more thermotolerant B. terrestris versus B. magnus. The differences may contribute to explaining interspecific differences in sperm survival.

Scolytinae is a subfamily of weevils that contains many major pest species. Most scolytines employ an outbreeding mating system: individuals emerge from their natal host trees and fly to new hosts, where they mate with unrelated conspecifics. However, in several outbreeding species, some individuals mate prior to emergence, either with a sibling or an unrelated neighbor from another gallery. Preemergence mating allows females to start adult life with a supply of sperm; they can then dig a gallery on their own in a new host tree. In this study, we examined preemergence mating in Ips typographus, a supposedly outbred bark beetle that causes considerable damage to European spruce forests. Our field and laboratory studies have shown that 15–94