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Caste differentiation involves many functional traits that diverge during larval growth and metamorphosis to produce adults irreversibly adapted to reproductive division of labor. Investigating developmental differentiation is important for general biological understanding and has increasingly been explored for social phenotypes that diverge in parallel from similar genotypes. Here, we use Monomorium pharaonis ants to investigate the extent to which canalized worker development can be shifted toward gyne (virgin-queen) phenotypes by juvenile hormone (JH) treatment. We show that excess JH can activate gyne-biased development in workers so that wing-buds, ocelli, antennal and genital imaginal discs, flight muscles, and gyne-like fat bodies and brains emerge after pupation. However, ovary development remained unresponsive to JH treatment, indicating that JH-sensitive germline sequestration happens well before somatic differentiation. Our findings reveal important qualitative restrictions in the extent to which JH treatment can redirect larval development and that these constraints are independent of body size. Our findings corroborate that JH is a key hormone for inducing caste differentiation but show that this process can be asymmetric for higher colony-level germline versus somatic caste differentiation in superorganisms as defined a century ago by Wheeler. We quantified gene expression changes in response to JH treatment throughout development and identified a set of JH-sensitive genes responsible for the emergence of gyne-like somatic traits. Our study suggests that the gonadotropic role of JH in ovary maturation has shifted from the individual level in solitary insects to the colony level in an evolutionary-derived and highly polygynous superorganism like the pharaoh ant., Competing Interests: Competing interests statement:The authors declare no competing interest.

Meiosis is essential for eukaryotic reproduction and provides the basis for Mendel's segregation laws. A recent study by Lacy et al. identified a significant deviation from these laws in a clonal ant, hinting at a potentially overlooked meiotic feature. This discovery may have broader implications for recombination in nonclonal eukaryotes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Fungus-farming ants cultivate multiple lineages of fungi for food, but, because fungal cultivar relationships are largely unresolved, the history of fungus-ant coevolution remains poorly known. We designed probes targeting >2000 gene regions to generate a dated evolutionary tree for 475 fungi and combined it with a similarly generated tree for 276 ants. We found that fungus-ant agriculture originated ~66 million years ago when the end-of-Cretaceous asteroid impact temporarily interrupted photosynthesis, causing global mass extinctions but favoring the proliferation of fungi. Subsequently, ~27 million years ago, one ancestral fungal cultivar population became domesticated, i.e., obligately mutualistic, when seasonally dry habitats expanded in South America, likely isolating the cultivar population from its free-living, wet forest-dwelling conspecifics. By revealing these and other major transitions in fungus-ant coevolution, our results clarify the historical processes that shaped a model system for nonhuman agriculture.

The naturally selected fungal crop (Leucoagaricus gongylophorus) farmed by leafcutter ants shows striking parallels with artificially selected plant crops domesticated by humans (e.g. polyploidy, engorged nutritional rewards, and dependence on cultivation). To date, poorly resolved L. gongylophorus genome assemblies based on short-read sequencing have constrained hypotheses about how millions of years under cultivation by ants shaped the fungal crop genome and potentially drove domestication. We use PacBio HiFi sequencing of L. gongylophorus from the leafcutter ant Atta colombica to identify 18 putatively novel biosynthetic gene clusters that likely cemented life as a cultivar (e.g. plant fragment degradation, ant-farmer communication, and antimicrobial defense). Comparative analyses with cultivated and free-living fungi showed genomic signatures of stepwise domestication transitions: (i) free-living to ant-cultivated: loss of genes conferring stress response and detoxification; (ii) hyphal food to engorged nutritional rewards: expansions of genes governing cellular homeostasis, carbohydrate metabolism, and siderophore biosynthesis; and (iii) detrital provisioning to freshly cut plant fragments: gene expansions promoting cell wall biosynthesis, fatty acid metabolism, and DNA repair. Comparisons across L. gongylophorus fungi farmed by 3 leafcutter ant species highlight genomic signatures of exclusively vertical clonal propagation and widespread transposable element activity. These results show how natural selection can shape domesticated cultivar genomes toward long-term ecological resilience of farming systems that have thrived across millennia., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Knowledge of viral biodiversity within insects, particularly within ants, is extremely limited with only a few environmental viruses from invasive ant species identified to date. This study documents and explores the viral communities in ants. We comprehensively profile the metagenomes of a phylogenetically broad group of 35 ant species with varied ecological traits and report the discovery of 3710 novel and unique ant-associated viral genomes. These previously unknown viruses discovered within this study constitute over 95% of all currently described ant viruses, significantly increasing our knowledge of the ant virosphere. The identified RNA and DNA viruses fill gaps in insect-associated viral phylogenies and uncover evolutionary histories characterized by both frequent host switching and co-divergence. Many ants also host diverse bacterial communities, and we discovered that approximately one-third of these new ant-associated viruses are bacteriophages. Two ecological categories, bacterial abundance in the host and habitat degradation are both correlated with ant viral diversity and help to structure viral communities within ants. These data demonstrate that the ant virosphere is remarkably diverse phylogenetically and genomically and provide a substantial foundation for studies in virus ecology and evolution within eukaryotes. We highlight the importance of studying insect-associated viruses in natural ecosystems in order to more thoroughly and effectively understand host-microbe evolutionary dynamics., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Island biotas provide unparalleled opportunities to examine evolutionary processes. Founder effects and bottlenecks, e.g., typically decrease genetic diversity in island populations, while selection for reduced dispersal can increase population structure. Given that support for these generalities mostly comes from single-species analyses, assemblage-level comparisons are needed to clarify how (i) colonization affects the gene pools of interacting insular organisms, and (ii) patterns of genetic differentiation vary within assemblages of organisms. Here, we use genome-wide sequence data from ultraconserved elements (UCEs) to compare the genetic diversity and population structure of mainland and island populations of nine ant species in coastal southern California. As expected, island populations (from Santa Cruz Island) had lower expected heterozygosity and Watterson's theta compared to mainland populations (from the Lompoc Valley). Island populations, however, exhibited smaller genetic distances among samples, indicating less population subdivision. Within the focal assemblage, pairwise Fst values revealed pronounced interspecific variation in mainland-island differentiation, which increases with gyne body size. Our results reveal population differences across an assemblage of interacting species and illuminate general patterns of insularization in ants. Compared to single-species studies, our analysis of nine conspecific population pairs from the same island-mainland system offers a powerful approach to studying fundamental evolutionary processes., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE). All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The basidiomycete fungus Leucoagaricus gongylophorus is able to grow in the fungus garden of leaf-cutter ants. This mutualistic interaction has driven the evolutionary adaptation of L. gongylophorus , shaping its metabolism to produce enzymes adept at lignocellulosic biomass degradation. In this study, we undertook the comprehensive sequencing, assembly, and functional annotation of the genome of L. gongylophorus strain LEU18496, mutualistic fungus of the Atta mexicana . Our genomic analyses revealed a distinctive bimodal nature to the genome: a predominant region characterized by AT enrichment and low genetic density, alongside a smaller region exhibiting higher GC content and higher genetic density. The presence of transposable elements (TEs) within the AT-enriched region suggests genomic compartmentalization, facilitating differential evolutionary rates. With a gene count of 6748, the assembled genome of L. gongylophorus LEU18496 surpasses previous reports for this fungal species. Inspection of genes associated with central metabolism unveiled a remarkable abundance of carbohydrate-active enzymes (CAZymes) and fungal oxidative lignin enzymes (FOLymes), underscoring their pivotal roles in the life cycle of this fungus.

Nestmate recognition in ants is regulated through the detection of cuticular hydrocarbons by odorant receptors (ORs) in the antennae. These ORs are crucial for maintaining colony cohesion that allows invasive ant species to dominate colonized environments. In the invasive Argentine ant, Linepithema humile, ORs regulating nestmate recognition are thought to be present in a clade of nine-exon odorant receptors, but the identity of the specific genes remains unknown. We sought to narrow down the list of candidate genes using transcriptomics and phylostratigraphy. Comparative transcriptomic analyses were conducted on the antennae, head, thorax, and legs of Argentine ant workers. We have identified a set of twenty-one nine-exon odorant receptors enriched in the antennae compared to the other tissues, allowing for downstream verification of whether they can detect Argentine ant cuticular hydrocarbons. Further investigation of these ORs could allow us to further understand the mechanisms underlying nestmate recognition and colony cohesion in ants., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dittmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Communication is essential for social organisms. In eusocial insects, olfaction facilitates communication and recognition between nestmates. The study of certain model organisms has led to the hypothesis that odorant receptors are expanded in eusocial Hymenoptera. This has become a widely mentioned idea in the literature, albeit with conflicting reports, and has not been tested with a broad comparative analysis. Here we combined existing genomic and new neuroanatomical data, including from an approximately 100 Myr old fossil ant, across a phylogenetically broad sample of hymenopteran lineages. We find no evidence that variation in the size and evolutionary tempo of odorant receptor repertoires is related to eusociality. Post hoc exploration of our data hinted at loss of flight as a possible factor shaping some of the variation in OR repertoires in Hymenoptera. Nevertheless, our analyses revealed a complex pattern of evolutionary variation, and raise new questions about the ecological, behavioural and social factors that shape olfactory abilities.

Assigning a query individual animal or plant to its derived population is a prime task in diverse applications related to organismal genealogy. Such endeavors have conventionally relied on short DNA sequences under a phylogenetic framework. These methods naturally show constraints when the inferred population sources are ambiguously phylogenetically structured, a scenario demanding substantially more informative genetic signals. Recent advances in cost-effective production of whole-genome sequences and artificial intelligence have created an unprecedented opportunity to trace the population origin for essentially any given individual, as long as the genome reference data are comprehensive and standardized. Here, we developed a convolutional neural network method to identify population origins using genomic SNPs. Three empirical datasets (an Asian honeybee, a red fire ant, and a chicken datasets) and two simulated populations are used for the proof of concepts. The performance tests indicate that our method can accurately identify the genealogy origin of query individuals, with success rates ranging from  93 % to 100 %. We further showed that the accuracy of the model can be significantly increased by refining the informative sites through F ST filtering. Our method is robust to configurations related to batch sizes and epochs, whereas model learning benefits from the setting of a proper preset learning rate. Moreover, we explained the importance score of key sites for algorithm interpretability and credibility, which has been largely ignored. We anticipate that by coupling genomics and deep learning, our method will see broad potential in conservation and management applications that involve natural resources, invasive pests and weeds, and illegal trades of wildlife products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Southeast Asia is a biodiversity hotspot characterized by a complex paleogeography, and its Polypodiopsida flora is particularly diverse. While hybridization is recognized as common in ferns, further research is needed to investigate the relationship between hybridization events and fern diversity. Lecanopteris s.s., an ant-associated fern, has been subject to debate regarding species delimitations primarily due to limited DNA markers and species sampling. Our study integrates 22 newly generated plastomes, 22 transcriptomes, and flow cytometry of all native species along with two cultivated hybrids. Our objective is to elucidate the reticulate evolutionary history within Lecanopteris s.s. through the integration of phylobiogeographic reconstruction, gene flow inference, and genome size estimation. Key findings of our study include: (1) An enlarged plastome size (178-187 Kb) in Lecanopteris s.s., attributed to extreme expansion of the Inverted Repeat (IR) regions; (2) The traditional 'pumila' and 'crustacea' groups are paraphyletic; (3) Significant cytonuclear discordance attributed to gene flow; (4) Natural hybridization and introgression in the 'pumila' and 'darnaedii' groups; (5) L. luzonensis is the maternal parent of L. 'Yellow Tip', with L. pumila suggested as a possible paternal parent; (6) L. 'Tatsuta' is a hybrid between L. luzonensis and L. crustacea; (7) Lecanopteris s.s. first diverged during the Neogene and then during the middle Miocene climatic optimum in the Indochina and Sundaic regions. In conclusion, the biogeographic history and speciation of Lecanopteris have been profoundly shaped by past climate changes and geodynamics of Southeast Asia. Dispersals, hybridization and introgression between species act as pivotal factors in the evolutionary trajectory of Lecanopteris s.s.. This research provides a robust framework for further exploration and understanding of the complex dynamics driving the diversification and distribution patterns within Polypodiaceae subfamily Microsoroideae., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Phenotypic plasticity displayed by an animal in response to different environmental conditions is supposedly crucial for its survival and reproduction. The female adults of some ant lineages display phenotypic plasticity related to reproductive role. In pharaoh ant queens, insemination induces substantial physiological/behavioral changes and implicates remarkable gene regulatory network (GRN) shift in the brain. Here, we report a neuropeptide neuroparsin A (NPA) showing a conserved expression pattern associated with reproductive activity across ant species. Knock-down of NPA in unmated queen enhances ovary activity, whereas injection of NPA peptide in fertilized queen suppresses ovary activity. We found that NPA mainly affected the downstream gene JHBP in the ovary, which is positively regulated by NPA and suppression of which induces elevated ovary activity, and shadow which is negatively regulated by NPA. Furthermore, we show that NPA was also employed into the brain-ovary axis in regulating the worker reproductive changes in other distantly related species, such as Harpegnathos venator ants., Competing Interests: The authors have declared that no competing interests., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: High-throughput experimental technologies can provide deeper insights into pathway perturbations in biomedical studies. Accordingly, their usage is central to the identification of molecular targets and the subsequent development of suitable treatments for various diseases. Classical interpretations of generated data, such as differential gene expression and pathway analyses, disregard interconnections between studied genes when looking for gene-disease associations. Given that these interconnections are central to cellular processes, there has been a recent interest in incorporating them in such studies. The latter allows the detection of gene modules that underlie complex phenotypes in gene interaction networks. Existing methods either impose radius-based restrictions or freely grow modules at the expense of a statistical bias towards large modules. We propose a heuristic method, inspired by Ant Colony Optimization, to apply gene-level scoring and module identification with distance-based search constraints and penalties, rather than radius-based constraints., Results: We test and compare our results to other approaches using three datasets of different neurodegenerative diseases, namely Alzheimer's, Parkinson's, and Huntington's, over three independent experiments. We report the outcomes of enrichment analyses and concordance of gene-level scores for each disease. Results indicate that the proposed approach generally shows superior stability in comparison to existing methods. It produces stable and meaningful enrichment results in all three datasets which have different case to control proportions and sample sizes., Conclusion: The presented network-based gene expression analysis approach successfully identifies dysregulated gene modules associated with a certain disease. Using a heuristic based on Ant Colony Optimization, we perform a distance-based search with no radius constraints. Experimental results support the effectiveness and stability of our method in prioritizing modules of high relevance. Our tool is publicly available at github.com/GhadiElHasbani/ACOxGS.git., (© 2024. The Author(s).)

According to Mendel's second law, chromosomes segregate randomly in meiosis. Non-random segregation is primarily known for cases of selfish meiotic drive in females, in which particular alleles bias their own transmission into the oocyte. Here we report a rare example of unselfish meiotic drive for crossover inheritance in the clonal raider ant, Ooceraea biroi, in which both alleles are co-inherited at all loci across the entire genome. This species produces diploid offspring parthenogenetically via fusion of two haploid nuclei from the same meiosis. This process should cause rapid genotypic degeneration due to loss of heterozygosity, which results if crossover recombination is followed by random (Mendelian) segregation of chromosomes. However, by comparing whole genomes of mothers and daughters, we show that loss of heterozygosity is exceedingly rare, raising the possibility that crossovers are infrequent or absent in O. biroi meiosis. Using a combination of cytology and whole-genome sequencing, we show that crossover recombination is, in fact, common but that loss of heterozygosity is avoided because crossover products are faithfully co-inherited. This results from a programmed violation of Mendel's law of segregation, such that crossover products segregate together rather than randomly. This discovery highlights an extreme example of cellular 'memory' of crossovers, which could be a common yet cryptic feature of chromosomal segregation., (© 2024. The Author(s).)

The evolution of animals and their gut symbionts is a complex phenomenon, obscured by lability and diversity. In social organisms, transmission of symbionts among relatives may yield systems with more stable associations. Here, we study the history of a social insect symbiosis involving cephalotine ants and their extracellular gut bacteria, which come predominantly from host-specialized lineages. We perform multi-locus phylogenetics for symbionts from nine bacterial orders, and map prior amplicon sequence data to lineage-assigned symbiont genomes, studying distributions of rigorously defined symbionts across 20 host species. Based on monophyly and additional hypothesis testing, we estimate that these specialized gut bacteria belong to 18 distinct lineages, of which 15 have been successfully isolated and cultured. Several symbiont lineages showed evidence for domestication events that occurred later in cephalotine evolutionary history, and only one lineage was ubiquitously detected in all 20 host species and 48 colonies sampled with amplicon 16S rRNA sequencing. We found evidence for phylogenetically constrained distributions in four symbionts, suggesting historical or genetic impacts on community composition. Two lineages showed evidence for frequent intra-lineage co-infections, highlighting the potential for niche divergence after initial domestication. Nearly all symbionts showed evidence for occasional host switching, but four may, more often, co-diversify with their hosts. Through our further assessment of symbiont localization and genomic functional profiles, we demonstrate distinct niches for symbionts with shared evolutionary histories, prompting further questions on the forces underlying the evolution of hosts and their gut microbiomes., (© 2024 John Wiley & Sons Ltd.)

The fitness effects of overt parasites, and host resistance to them, are well documented. Most symbionts, however, are more covert and their interactions with their hosts are less well understood. Wolbachia , an intracellular symbiont of insects, is particularly interesting because it is thought to be unaffected by the host immune response and to have fitness effects mostly focussed on sex ratio manipulation. Here, we use quantitative PCR to investigate whether host genotype affects Wolbachia infection density in the leaf-cutting ant Acromyrmex echinatior , and whether Wolbachia infection density may affect host morphology or caste determination. We found significant differences between host colonies in the density of Wolbachia infections, and also smaller intracolonial differences in infection density between host patrilines. However, the density of Wolbachia infections did not appear to affect the morphology of adult queens or likelihood of ants developing as queens. The results suggest that both host genotype and environment influence the host- Wolbachia relationship, but that Wolbachia infections carry little or no physiological effect on the development of larvae in this system., Competing Interests: The authors declare there are no competing interests., (©2024 Frost et al.)

Eusocial Hymenoptera have the highest recombination rates among all multicellular animals studied so far, but it is unclear why this is and how this affects the biology of individual species. A high-resolution linkage map for the ant Cardiocondyla obscurior corroborates genome-wide high recombination rates reported for ants (8.1 cM/Mb). However, recombination is locally suppressed in regions that are enriched with TEs, that have strong haplotype divergence, or that show signatures of epistatic selection in C. obscurior The results do not support the hypotheses that high recombination rates are linked to phenotypic plasticity or to modulating selection efficiency. Instead, genetic diversity and the frequency of structural variants correlate positively with local recombination rates, potentially compensating for the low levels of genetic variation expected in haplodiploid social Hymenoptera with low effective population size. Ultimately, the data show that recombination contributes to within-population polymorphism and to the divergence of the lineages within C. obscurior ., (© 2024 Errbii et al.; Published by Cold Spring Harbor Laboratory Press.)

The number of chromosomes varies tremendously across species. It is not clear whether having more or fewer chromosomes could be advantageous. The probability of non-disjunction should theoretically decrease with smaller karyotypes, but too long chromosomes should enforce spatial constraint for their segregation during the mitotic anaphase. Here, we propose a new experimental cell system to acquire novel insights into the mechanisms underlying chromosome segregation. We collected the endemic Australian ant Myrmecia croslandi, the only known species with the simplest possible karyotype of a single chromosome in the haploid males (and one pair of chromosomes in the diploid females), since males are typically haploid in hymenopteran insects. Five colonies, each with a queen and a few hundreds of workers, were collected in the Canberra district (Australia), underwent karyotype analysis to confirm the presence of a single pair of chromosomes in worker pupae, and were subsequently maintained in the laboratory in Paris (France). Starting from dissociated male embryos, we successfully conducted primary cell cultures comprised of single-chromosome cells. This could be developed into a unique model that will be of great interest for future genomic and cell biology studies related to mitosis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Coastal dunes are unique habitats, threatened by human activities. In biogeographical terms, coastal dunes are habitat islands, being discrete and distinct patches of similar habitat among themselves, separated from each other by a different type of habitat. Furthermore, coastal dunes harbor endemic species, adapted to living solely in the habitats found on specific dune systems. For example, the honeypot ant Myrmecocystus baja is endemic and restricted to coastal dunes of Mexico's Baja California Pacific coast. This ecological and biogeographical scenario led to the questions whether their geographical isolation is reflected in their genetic diversity and structuring, and how their demographic history is related with the formation of the dune system habitats. To answer these questions, population genetic, isolation-with-migration, and phylogeographical analyses were carried out, based on mitochondrial and five nuclear intronic markers. Minimal gene flow was detected only between two of the dune systems sampled; otherwise, the M. baja populations were found to be isolated and genetically structured, and their divergence generally pre-dated the modern-day dune systems. It is therefore highly likely that these ants were already present in paleodunes and that each of the populations was established from founder populations as the dunes formed. These findings highlight the importance of coastal dunes for species such as the honeypot ant from Baja California, in promoting genetic differentiation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: Ants are ecologically dominant insects in most terrestrial ecosystems, with more than 14,000 extant species in about 340 genera recorded to date. However, genomic resources are still scarce for most species, especially for species endemic in East or Southeast Asia, limiting the study of phylogeny, speciation and adaptation of this evolutionarily successful animal lineage. Here, we assemble and annotate the genomes of Odontoponera transversa and Camponotus friedae, two ant species with a natural distribution in China, to facilitate future study of ant evolution., Data Description: We obtained a total of 16 Gb and 51 Gb PacBio HiFi data for O. transversa and C. friedae, respectively, which were assembled into the draft genomes of 339 Mb for O. transversa and 233 Mb for C. friedae. Genome assessments by multiple metrics showed good completeness and high accuracy of the two assemblies. Gene annotations assisted by RNA-seq data yielded a comparable number of protein-coding genes in the two genomes (10,892 for O. transversa and 11,296 for C. friedae), while repeat annotations revealed a remarkable difference of repeat content between these two ant species (149.4 Mb for O. transversa versus 49.7 Mb for C. friedae). Besides, complete mitochondrial genomes for the two species were assembled and annotated., (© 2024. The Author(s).)

A co-evolutionary arms race ensues when parasites exhibit exploitative behaviour, which prompts adaptations in their hosts, in turn triggering counter-adaptations by the parasites. To unravel the genomic basis of this coevolution from the host's perspective, we collected ants of the host species Temnothorax longispinosus, parasitized by the social parasite Temnothorax americanus, from 10 populations in the northeastern United States exhibiting varying levels of parasite prevalence and living under different climatic conditions. We conducted a genome-wide association study (GWAS) to identify single nucleotide polymorphisms (SNPs) associated with both prevalence and climate. Our investigation highlighted a multitude of candidate SNPs associated with parasite prevalence, particularly in genes responsible for sensory perception of smell including odorant receptor genes. We further focused on population-specific compositions of cuticular hydrocarbons, a complex trait important for signalling, communication and protection against desiccation. The relative abundances of n-alkanes were correlated with climate, while there was only a trend between parasite prevalence and the relative abundances of known recognition cues. Furthermore, we identified candidate genes likely involved in the synthesis and recognition of specific hydrocarbons. In addition, we analysed the population-level gene expression in the antennae, the primary organ for odorant reception, and established a strong correlation with parasite prevalence. Our comprehensive study highlights the intricate genomic patterns forged by the interplay of diverse selection factors and how these are manifested in the expression of various phenotypes., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Obligatory ant-plant symbioses often appear to be single evolutionary shifts within particular ant lineages; however, convergence can be revealed once natural history observations are complemented with molecular phylogenetics. Here, we describe a remarkable example of convergent evolution in an ant-plant symbiotic system. Exclusively arboreal, Myrmelachista species can be generalized opportunists nesting in several plant species or obligately symbiotic, live-stem nesters of a narrow set of plant species. Instances of specialization within Myrmelachista are known from northern South America and throughout Middle America. In Middle America, a diverse radiation of specialists occupies understory treelets of lowland rainforests. The morphological and behavioural uniformity of specialists suggests that they form a monophyletic assemblage, diversifying after a single origin of specialization. Using ultraconserved element phylogenomics and ancestral state reconstructions, we show that shifts from opportunistic to obligately symbiotic evolved independently in South and Middle America. Furthermore, our analyses support a remarkable case of convergence within the Middle American radiation, with two independently evolved specialist clades, arising nearly simultaneously from putative opportunistic ancestors during the late Pliocene. This repeated evolution of a complex phenotype suggests similar mechanisms behind trait shifts from opportunists to specialists, generating further questions about the selective forces driving specialization.

Formica is a large genus in the family Formicidae with high diversity in its distribution, morphology, and physiology. To better understand evolutionary characteristics of Formica, the complete mitochondrial genomes (mitogenomes) of two Formica species were determined and a comparative mitogenomic analysis for this genus was performed. The two newly sequenced Formica mitogenomes each included 37 typical mitochondrial genes and a large non-coding region (putative control region), as observed in other Formica mitogenomes. Base composition, gene order, codon usage, and tRNA secondary structure were well conserved among Formica species, whereas diversity in sequence size and structural characteristics was observed in control regions. We also observed several conserved motifs in the intergenic spacer regions. These conserved genomic features may be related to mitochondrial function and their highly conserved physiological constraints, while the diversity of the control regions may be associated with adaptive evolution among heterogenous habitats. A negative AT-skew value on the majority chain was presented in each of Formica mitogenomes, indicating a reversal of strand asymmetry in base composition. Strong codon usage bias was observed in Formica mitogenomes, which was predominantly determined by nucleotide composition. All 13 mitochondrial protein-coding genes of Formica species exhibited molecular signatures of purifying selection, as indicated by the ratio of non-synonymous substitutions to synonymous substitutions being less than 1 for each protein-coding gene. Phylogenetic analyses based on mitogenomic data obtained fairly consistent phylogenetic relationships, except for two Formica species that had unstable phylogenetic positions, indicating mitogenomic data are useful for constructing phylogenies of ants. Beyond characterizing two additional Formica mitogenomes, this study also provided some key evolutionary insights into Formica., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Olfaction is essential for complex social behavior in insects. To discriminate complex social cues, ants evolved an expanded number of odorant receptor ( Or ) genes. Mutations in the obligate odorant co-receptor gene orco lead to the loss of ~80% of the antennal lobe glomeruli in the jumping ant Harpegnathos saltator . However, the cellular mechanism remains unclear. Here, we demonstrate massive apoptosis of odorant receptor neurons (ORNs) in the mid to late stages of pupal development, possibly due to ER stress in the absence of Orco. Further bulk and single-nucleus transcriptome analysis shows that, although most orco -expressing ORNs die in orco mutants, a small proportion of them survive: They express ionotropic receptor ( Ir ) genes that form IR complexes. In addition, we found that some Or genes are expressed in mechanosensory neurons and nonneuronal cells, possibly due to leaky regulation from nearby non- Or genes. Our findings provide a comprehensive overview of ORN development and Or expression in H. saltator .

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.

Animals have evolved various sex determination systems. Here, we describe a newly found mechanism. A long noncoding RNA (lncRNA) transduces complementary sex determination (CSD) signal in the invasive Argentine ant. In this haplodiploid species, we identified a 5-kilobase hyper-polymorphic region underlying CSD: Heterozygous embryos become females, while homozygous and hemizygous embryos become males. Heterozygosity at the CSD locus correlates with higher expression of ANTSR , a gene that overlaps with the CSD locus and specifies an lncRNA transcript. ANTSR knockdown in CSD heterozygotes leads to male development. Comparative analyses indicated that, in Hymenoptera, ANTSR is an ancient yet rapidly evolving gene. This study reveals an lncRNA involved in genetic sex determination, alongside a previously unknown regulatory mechanism underlying sex determination based on complementarity among noncoding alleles.

RNAi plays a crucial role in insect gene function research and pest control field. Nonetheless, the variable efficiency of RNAi across diverse insects and off-target effects also limited its further application. In this study, we cloned six essential housekeeping genes from Solenopsis invicta and conducted RNAi experiments by orally administering dsRNA. Then, we found that mixing with liposomes significantly enhanced the RNAi efficiency by targeting for SiV-ATPaseE . Additionally, we observed a certain lethal effect of this dsRNA on queens by our established RNAi system. Furthermore, no strict sequence-related off-target effects were detected. Finally, the RNAi effect of large-scale bacteria expressing dsRNA was successfully confirmed for controlling S. invicta . In summary, this study established an RNAi system for S. invicta and provided a research template for the future development of nucleic acid drugs based on RNAi.

Background: Social insects vary considerably in their social organization both between and within species. In the California harvester ant, Pogonomyrmex californicus (Buckley 1867), colonies are commonly founded and headed by a single queen (haplometrosis, primary monogyny). However, in some populations in California (USA), unrelated queens cooperate not only during founding (pleometrosis) but also throughout the life of the colony (primary polygyny). The genetic architecture and evolutionary dynamics of this complex social niche polymorphism (haplometrosis vs pleometrosis) have remained unknown., Results: We provide a first analysis of its genomic basis and evolutionary history using population genomics comparing individuals from a haplometrotic population to those from a pleometrotic population. We discovered a recently evolved (< 200 k years), 8-Mb non-recombining region segregating with the observed social niche polymorphism. This region shares several characteristics with supergenes underlying social polymorphisms in other socially polymorphic ant species. However, we also find remarkable differences from previously described social supergenes. Particularly, four additional genomic regions not in linkage with the supergene show signatures of a selective sweep in the pleometrotic population. Within these regions, we find for example genes crucial for epigenetic regulation via histone modification (chameau) and DNA methylation (Dnmt1)., Conclusions: Altogether, our results suggest that social morph in this species is a polygenic trait involving a potential young supergene. Further studies targeting haplo- and pleometrotic individuals from a single population are however required to conclusively resolve whether these genetic differences underlie the alternative social phenotypes or have emerged through genetic drift., (© 2024. The Author(s).)

Myrmecophytic plants utilise defensive services offered by obligate ant partners nesting in their domatia in a novel means of survival in tropical habitats. Although much is known about the ecology of myrmecophytism, there aren't enough empirical examples to demonstrate whether it substantially influences evolutionary patterns in host plant lineages. In this study, we make use of the species-rich Macaranga (Euphorbiaceae) ant-plant symbiosis distributed in the Southeast Asian Sundaland to delve into the evolutionary dynamics of myrmecophytism in host plants. We generated the most comprehensive dated phylogeny of myrmecophytic Macaranga till date using genotyping-by-sequencing (GBS). With this in hand, we traced the evolutionary history of myrmecophytism in Macaranga using parametric biogeography and ancestral state reconstruction. Diversification rate analysis methods were employed to determine if myrmecophytism enhanced diversification rates in the genus. Our results demonstrate that myrmecophytism is labile and easily lost. Ancestral state reconstruction supported a single origin of myrmecophytism in Macaranga ∼18 mya on Borneo followed by multiple losses. Diversification rate analysis methods did not yield sufficient evidence to support the hypothesis that myrmecophytism enhanced diversification rates in Macaranga; we found that topographical features on Borneo may have played a more direct role in the divergence of clades instead. Our study provides evidence that while the acquisition of domatia clearly functions as a key innovation that has enabled host plants to exploit the environment in novel ways, it may not necessarily enhance diversification rates. In fact, we hypothesise that overly specialised cases of myrmecophytism may even be an evolutionary dead end., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The N 6 -methyladenosine (m 6 A) modification of RNA has been reported to remodel gene expression in response to environmental conditions; however, the biological role of m 6 A in social insects remains largely unknown. In this study, we explored the role of m 6 A in the division of labour by worker ants (Solenopsis invicta). We first determined the presence of m 6 A in RNAs from the brains of worker ants and found that m 6 A methylation dynamics differed between foragers and nurses. Depletion of m 6 A methyltransferase or chemical suppression of m 6 A methylation in foragers resulted in a shift to 'nurse-like' behaviours. Specifically, mRNAs of dopamine receptor 1 (Dop1) and dopamine transporter (DAT) were modified by m 6 A, and their expression increased dopamine levels to promote the behavioural transition from foragers to nurses. The abundance of Dop1 and DAT mRNAs and their stability were reduced by the inhibition of m 6 A modification caused by the silencing of Mettl3, suggesting that m 6 A modification in worker ants modulates dopamine synthesis, which regulates labour division. Collectively, our results provide the first example of the epitranscriptomic regulation of labour division in social insects and implicate m 6 A regulatory mechanism as a potential novel target for controlling red imported fire ants., (© 2024 John Wiley & Sons Ltd.)

Aphids and ants are mutualistic species with a close space-time relationship, which may facilitate the occurrence of horizontal transfer events between these insect groups. Myrmar-like mariner elements were previously isolated from two ant (Myrmica ruginodis and Tapinoma ibericum) and two aphid species (Aphis fabae and Aphis hederae). The aim of this work is to determine the presence of Myrmar-like mariner elements in new ant and aphid species, as well as to analyze the likelihood of horizontal transfer events between these taxa. To accomplish this, the Myrmar-like element has been isolated from five aphid species and six ant species. Among these new analyzed species, full-length Myrmar-like mariner elements with very high sequence similarity have been isolated from the aphids Aphis nerii, Aphis spiraecola, Brachycaudus cardui, and Rhopalosiphum maidis as well as from the ants Lasius grandis and Lasius niger, even though aphids and ants belong to two insect orders (Hemiptera and Hymenoptera) that have evolved independently for at least 300 million-years. Both Lasius species establish frequent mutualistic relationships with multiple aphid species, including A. nerii, A. spiraecola, and B. cardui. The study of the putative protein encoded by them and the phylogenetic analysis suggests that they could be active transposons shared by aphids and ants through horizontal transfer events. Additionally, mariner elements with internal deletion were found in several aphids and one ant species, showing a high degree of sequence similarity among them. The characteristics of these elements with internal deletion suggest a complex origin involving various evolutionary processes, possibly including also horizontal transfer events. Myrmar-like elements have also been isolated from the other ant species, although without similarity with the aphid mariner sequences. Myrmar-like elements are also present in phylogenetically distant insect species, as well as in one crustacean species. The phylogenetic study carried out with all Myrmar-like elements suggests the probable occurrence of horizontal transfer events., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mitochondria play a key role in cell biology and have their own genome, residing in a highly oxidative environment that induces faster changes than the nuclear genome. Because of this, mitochondrial markers have been exploited to reconstruct phylogenetic and phylogeographic relationships in studies of adaptation and molecular evolution. In this study, we determined the complete mitogenome of the fungus-farming ant Mycetophylax simplex (Hymenoptera, Formicidae) and conducted a comparative analysis among 29 myrmicine ant mitogenomes. Mycetophylax simplex is an endemic ant that inhabits sand dunes along the southern Atlantic coast. Specifically, the species occur in the ecosystem known as "restinga", within the Atlantic Forest biome. Due to habitat degradation, land use and decline of restinga habitats, the species is considered locally extinct in extremely urban beaches and is listed as vulnerable on the Brazilian Red List (ICMBio). We employed a mitochondrion-targeting approach to obtain the complete mitogenome through high-throughput DNA sequencing technology. This method allowed us to determine the mitogenome with high performance, coverage and low cost. The circular mitogenome has a length of 16,367 base pairs enclosing 37 genes (13 protein-coding genes, 22 tRNAs and 2 rRNAs) along with one control region (CR). All the protein-coding genes begin with a typical ATN codon and end with the canonical stop codons. All tRNAs formed the fully paired acceptor stems and fold into the typical cloverleaf-shaped secondary structures. The gene order is consistent with the shared Myrmicinae structure, and the A + T content of the majority strand is 81.51%. Long intergenic spacers were not found but some gene are slightly shorter. The phylogenetic relationships based on concatenated nucleotide and amino acid sequences of the 13 protein-coding genes, using Maximum Likelihood and Bayesian Inference methods, indicated that mitogenome sequences were useful in resolving higher-level relationship within Formicidae., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The highly invasive Argentine ant (Linepithema humile) started its colonisation from the species' native range in South America approximately 150 years ago and has since become one of the major pests in the world. We investigated how the shifts into new ranges have affected the evolution of Argentine ants' immune genes. To the best of our knowledge, this is the first broadscale population genetic study focusing on ants' immune genes. We analysed comprehensive targeted-seq data of immune and non-immune genes containing 174 genes from 18 Argentine ant supercolonies covering the species' native and introduced ranges. We predicted that the immune gene evolution of introduced supercolonies differs from that of the native supercolonies and proposed two different, non-mutually exclusive hypotheses for this: 1) the enemy release hypothesis and 2) the higher pathogen pressure hypothesis - both of which seem to explain the observed evolutionary patterns on their behalf. Our results show that the introduced supercolonies were targeted by weaker selection than natives, but positive selection was evident among supercolonies of both ranges. Moreover, in some cases, such as the antiviral RNAi genes, introduced range supercolonies harboured a higher proportion of positively selected genes than natives. This observation was striking, knowing the recent demographic history and the detected generally lower selection efficacy of introduced supercolonies. In conclusion, it is evident that pathogen pressure is ubiquitous and strongly affects the immune gene evolution in Argentine ants., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Evolutionary Biology.)

Carpenter ants in the genus Camponotus are large, conspicuous ants that are abundant and ecologically influential in many terrestrial ecosystems. The bicolored carpenter ant, Camponotus vicinus Mayr, is distributed across a wide range of elevations and latitudes in western North America, where it is a prominent scavenger and predator. Here, we present a high-quality genome assembly of C. vicinus from a sample collected in Sonoma County, California, near the type locality of the species. This genome assembly consists of 38 scaffolds spanning 302.74 Mb, with contig N50 of 15.9 Mb, scaffold N50 of 19.9 Mb, and BUSCO completeness of 99.2%. This genome sequence will be a valuable resource for exploring the evolutionary ecology of C. vicinus and carpenter ants generally. It also provides an important tool for clarifying cryptic diversity within the C. vicinus species complex, a genetically diverse set of populations, some of which are quite localized and of conservation interest., (© The American Genetic Association. 2023.)

A highly endemic ant fauna is found in the arid regions of southern Africa, including species in the genus Ocymyrmex. This genus of ants has higher species richness in the western arid regions of southern Africa compared to tropical and subtropical parts of the continent. The processes that have produced these patterns of diversity and distribution of arid adapted ants in southern Africa have never been investigated. The diversification of many other taxa in the region has been associated with past climate fluctuations that occurred during the Miocene epoch. In this study, the nature and timing of historical processes that may have led to the diversification within Ocymyrmex were assessed. We hypothesized that past climate oscillations, characterized by long periods of aridification, have driven the current distribution of Ocymyrmex species that resulted in the highest species richness of the genus in the Deserts & xeric shrublands biome in southern Africa. Ninety-four Ocymyrmex worker specimens from Botswana, Kenya, Namibia, South Africa, Tanzania and Zimbabwe, representing 21 currently described species and six morphospecies, were included in a phylogenomic analysis. Phylogenies for the genus, based on next generation sequencing data from ultraconserved elements, were inferred using Maximum Likelihood, and a dating analysis was performed using secondary age estimates as calibration points. A distribution database of Ocymyrmex records was used to assign species ranges, which were then coded according to major biomes in southern Africa and used as input for biogeographical analysis. We explored the phylogenomic relationships of Ocymyrmex and analysed these within a biogeographical and paleoclimatic framework to disentangle the potential processes responsible for diversification in this group. Dating analyses estimated that the crown age of Ocymyrmex dates to the Oligocene, around 32 Ma. Diversification within this group occurred between the mid-Miocene (∼12.5 Ma) and Pleistocene (∼2 Ma). Our biogeographic analyses suggest that Ocymyrmex species originated in the south-western region of southern Africa, which is now part of the Deserts & xeric shrublands biome and diversified into eastern subtropical areas during the Pliocene. Paleoclimatic changes resulting in increased aridity during the Miocene likely drove the diversification of the genus Ocymyrmex. It is most likely that the diversification of grasslands, because of historical climate change, facilitated the diversification of these ants to the eastern parts of southern Africa when open grasslands replaced forests during the early Miocene., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Venoms have evolved independently over a hundred times in the animal kingdom to deter predators and/or subdue prey. Venoms are cocktails of various secreted toxins, whose origin and diversification provide an appealing system for evolutionary researchers. Previous studies of the ant venom of Tetramorium bicarinatum revealed several Myrmicitoxin (MYRTX) peptides that gathered into seven precursor families suggesting different evolutionary origins. Analysis of the T. bicarinatum genome enabling further genomic approaches was necessary to understand the processes underlying the evolution of these myrmicitoxins., Results: Here, we sequenced the genome of Tetramorium bicarinatum and reported the organisation of 44 venom peptide genes (vpg). Of the eleven chromosomes that make up the genome of T. bicarinatum, four carry the vpg which are organized in tandem repeats. This organisation together with the ML evolutionary analysis of vpg sequences, is consistent with evolution by local duplication of ancestral genes for each precursor family. The structure of the vpg into two or three exons is conserved after duplication events while the promoter regions are the least conserved parts of the vpg even for genes with highly identical sequences. This suggests that enhancer sequences were not involved in duplication events, but were recruited from surrounding regions. Expression level analysis revealed that most vpg are highly expressed in venom glands, although one gene or group of genes is much more highly expressed in each family. Finally, the examination of the genomic data revealed that several genes encoding transcription factors (TFs) are highly expressed in the venom glands. The search for binding sites (BS) of these TFs in the vpg promoters revealed hot spots of GATA sites in several vpg families., Conclusion: In this pioneering investigation on ant venom genes, we provide a high-quality assembly genome and the annotation of venom peptide genes that we think can fosters further genomic research to understand the evolutionary history of ant venom biochemistry., (© 2024. The Author(s).)

Ants are the most ubiquitous and ecologically dominant arthropods on Earth, and understanding their phylogeny is crucial for deciphering their character evolution, species diversification, and biogeography. Although recent genomic data have shown promise in clarifying intrafamilial relationships across the tree of ants, inconsistencies between molecular datasets have also emerged. Here I re-examine the most comprehensive published Sanger-sequencing and genome-scale datasets of ants using model comparison methods that model among-site compositional heterogeneity to understand the sources of conflict in phylogenetic studies. My results under the best-fitting model, selected on the basis of Bayesian cross-validation and posterior predictive model checking, identify contentious nodes in ant phylogeny whose resolution is modelling-dependent. I show that the Bayesian infinite mixture CAT model outperforms empirical finite mixture models (C20, C40 and C60) and that, under the best-fitting CAT-GTR + G4 model, the enigmatic Martialis heureka is sister to all ants except Leptanillinae, rejecting the more popular hypothesis supported under worse-fitting models, that place it as sister to Leptanillinae. These analyses resolve a lasting controversy in ant phylogeny and highlight the significance of model comparison and adequate modelling of among-site compositional heterogeneity in reconstructing the deep phylogeny of insects., (© 2024. The Author(s).)

Insects and mammals have independently evolved odorant receptor genes that are arranged in large genomic tandem arrays. In mammals, each olfactory sensory neuron chooses to express a single receptor in a stochastic process that includes substantial chromatin rearrangements. Here, we show that ants, which have the largest odorant receptor repertoires among insects, employ a different mechanism to regulate gene expression from tandem arrays. Using single-nucleus RNA sequencing, we found that ant olfactory sensory neurons choose different transcription start sites along an array but then produce mRNA from many downstream genes. This can result in transcripts from dozens of receptors being present in a single nucleus. Such rampant receptor co-expression at first seems difficult to reconcile with the narrow tuning of the ant olfactory system. However, RNA fluorescence in situ hybridization showed that only mRNA from the most upstream transcribed odorant receptor seems to reach the cytoplasm where it can be translated into protein, whereas mRNA from downstream receptors gets sequestered in the nucleus. This implies that, despite the extensive co-expression of odorant receptor genes, each olfactory sensory neuron ultimately only produces one or very few functional receptors. Evolution has thus found different molecular solutions in insects and mammals to the convergent challenge of selecting small subsets of receptors from large odorant receptor repertoires., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The red imported fire ant ( Solenopsis invicta Buren) is a social pest species with a robust reproductive ability that causes extensive damage. Identification of the genes involved in queen fertility is critical in order to better understand the reproductive biology and screening for the potential molecular targets in S. invicta . Here, we used the mRNA deep sequencing (RNA-seq) approach to identify differentially expressed genes (DEGs) in the transcriptomes of three reproductive caste types of S. invicta , including queen (QA) and winged female (FA) and male (MA) ants. The genes that were specific to and highly expressed in the queens were then screened, and the Vg2 and Vg3 genes were chosen as targets to explore their functions in oogenesis and fertility. A minimum of 6.08 giga bases (Gb) of clean reads was obtained from all samples, with a mapping rate > 89.78%. There were 7524, 7133, and 977 DEGs identified in the MA vs. QA, MA vs. FA, and FA vs. QA comparisons, respectively. qRT-PCR was used to validate 10 randomly selected DEGs, including vitellogenin 2 ( Vg2 ) and 3 ( Vg3 ), and their expression patterns were mostly consistent with the RNA-seq data. The S. invicta Vgs included conserved domains and motifs that are commonly found in most insect Vgs. SiVg2 and SiVg3 were highly expressed in queens and winged females and were most highly expressed in the thorax, followed by the fat body, head, and epidermis. Evaluation based on a loss-of-function-based knockdown analysis showed that the downregulation of either or both of these genes resulted in smaller ovaries, less oogenesis, and less egg production. The results of transcriptional sequencing provide a foundation for clarifying the regulators of queen fertility in S. invicta . The functions of SiVg2 and SiVg3 as regulators of oogenesis highlight their importance in queen fecundity and their potential as targets of reproductive disruption in S. invicta control.

Antagonistic selection has long been considered a major driver of the formation and expansion of sex chromosomes. For example, sexually antagonistic variation on an autosome can select for suppressed recombination between that autosome and the sex chromosome, leading to a neo-sex chromosome. Autosomal supergenes, chromosomal regions containing tightly linked variants affecting the same complex trait, share similarities with sex chromosomes, raising the possibility that sex chromosome evolution models can explain the evolution of genome structure and recombination in other contexts. We tested this premise in a Formica ant species, wherein we identified four supergene haplotypes on chromosome 3 underlying colony social organization and sex ratio. We discovered a novel rearranged supergene variant (9r) on chromosome 9 underlying queen miniaturization. The 9r is in strong linkage disequilibrium with one chromosome 3 haplotype (P 2 ) found in multi-queen (polygyne) colonies. We suggest that queen miniaturization is strongly disfavored in the single-queen (monogyne) background and is thus socially antagonistic. As such, divergent selection experienced by ants living in alternative social "environments" (monogyne and polygyne) may have contributed to the emergence of a genetic polymorphism on chromosome 9 and associated queen-size dimorphism. Consequently, an ancestral polygyne-associated haplotype may have expanded to include the polymorphism on chromosome 9, resulting in a larger region of suppressed recombination spanning two chromosomes. This process is analogous to the formation of neo-sex chromosomes and consistent with models of expanding regions of suppressed recombination. We propose that miniaturized queens, 16%-20% smaller than queens without 9r, could be incipient intraspecific social parasites., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Estrogen-related receptor (ERR) is a key regulator of insect growth, development, and metabolic processes in insects; however, the molecular mechanisms underlying its effects are not fully understood. We investigated roles of 20-hydroxyecdysone (20E) and insulin/insulin-like signaling/target of rapamycin (IIS/TOR) signaling pathways in the effects of PvERR on larval development, metamorphosis, and adult growth in ant Polyrhachis vicina Roger. PvFOXO expression levels depended on caste and developmental stage. PvERR RNAi significantly reduced the expression levels of IIS/TOR signaling pathway genes and 20E signaling pathway genes in fourth-instar larvae, pupae, females, and workers and significantly increased the expression levels of IIS/TOR signaling pathway genes PvFOXO and PvAkt in males. PvFOXO RNAi resulted in developmental defects and increased mortality. After PvFOXO RNAi, the expression of PvERR, 20E signaling pathway genes, and IIS/TOR signaling pathway genes decreased significantly in pupae, females, and workers and increased significantly in fourth-instar larvae. Exogenous 20E attenuated expression changes induced by PvFOXO RNAi in a sex- and stage-specific manner. These results indicate that ERR interacts with 20E and IIS/TOR signaling pathways to regulate caste determination, metamorphosis, and male fertility in P. vicina and that correlations between PvERR and PvFOXO are caste- and stage-specific., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The transmission of microbial symbionts across animal species could strongly affect their biology and evolution, but our understanding of transmission patterns and dynamics is limited. Army ants (Formicidae: Dorylinae) and their hundreds of closely associated insect guest species (myrmecophiles) can provide unique insights into interspecific microbial symbiont sharing. Here, we compared the microbiota of workers and larvae of the army ant Eciton burchellii with those of 13 myrmecophile beetle species using 16S rRNA amplicon sequencing. We found that the previously characterized specialized bacterial symbionts of army ant workers were largely absent from ant larvae and myrmecophiles, whose microbial communities were usually dominated by Rickettsia, Wolbachia, Rickettsiella and/or Weissella. Strikingly, different species of myrmecophiles and ant larvae often shared identical 16S rRNA genotypes of these common bacteria. Protein-coding gene sequences confirmed the close relationship of Weissella strains colonizing army ant larvae, some workers and several myrmecophile species. Unexpectedly, these strains were also similar to strains infecting dissimilar animals inhabiting very different habitats: trout and whales. Together, our data show that closely interacting species can share much of their microbiota, and some versatile microbial species can inhabit and possibly transmit across a diverse range of hosts and environments., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Overlooking cryptic species diversity has grave implications on assessments of climate change impacts on biodiversity, ecosystems and organismal populations. Discriminating between cryptic species has long been challenging even for seasoned taxonomists, as interspecies morphological differences are often indiscernible by visual observation. Multi-disciplinary methods involving genetic analyses in conjunction with quantitative morphological data, should therefore be used to investigate boundaries between cryptic species. We adopted an integrated approach combining analyses of mitochondrial COI barcodes, a genome-wide dataset obtained via multiplexed inter-simple sequence repeats (ISSRs) genotyping by sequencing (MIG-seq), and geometric morphometrics to investigate species divergences in the inscrutable Rhopalomastix javana species complex. Objective clustering of COI suggested five putative molecular species units divergent from each other by thresholds within 4.2-10.6% uncorrected pairwise distance. Phylogenetic analyses based on concatenated MIG-seq data also recovered and strongly supported the monophyly of five major lineages in agreement with COI clusters. Co-ancestry analyses based on MIG-seq data using fineRADstructure resolved variable patterns of admixture linked to geography, and potential genetic drift within some putative species. Geometric morphometric analyses of specimen images further detected statistically significant differences in at least one of three anatomical aspects (Head, Meso, Profile) between all pairs of putative species. Head shape (full-face view) was determined to be the most informative character for species diagnosis, with relatively high classification accuracy. Thin-plate spline deformation grids highlighted areas of high variation between species in each shape for deeper taxonomic scrutiny. The presence of species from multiple distinct lineages existing in near-sympatry firmly demonstrates that R. javana comprises more than one closely-related species, but exact species boundaries are difficult to ascertain. Differences in elevation and its associated abiotic effects on ant adaptations and reproductive phenology may contribute to restricting gene flow and maintaining species boundaries between sympatric populations of the R. javana complex. We further assess the advantages and limitations of geometric morphometrics as a taxonomic tool. Despite its drawbacks, our combined approach has helped draw important insights on cryptic diversity in R. javana , and also identified gaps of knowledge that await address. Results from this study will inform and prime future in-depth taxonomic investigation on the R. javana complex, including formal descriptions and establishment of the five putative species., Competing Interests: The authors declare that they have no competing interests., (© 2023 Wang and Yamada.)

Parasites with complex life cycles often manipulate the phenotype of their intermediate hosts to increase the probability of transmission to their definitive hosts. Infection with Anomotaenia brevis, a cestode that uses Temnothorax nylanderi ants as intermediate hosts, leads to a multiple-fold extension of host lifespan and to changes in behaviour, morphology and colouration. The mechanisms behind these changes are unknown, as is whether the increased longevity is achieved through parasite manipulation. Here, we demonstrate that the parasite releases proteins into its host with functions that might explain the observed changes. These parasitic proteins make up a substantial portion of the proteome of the hosts' haemolymph, and thioredoxin peroxidase and superoxide dismutase, two antioxidants, exhibited the highest abundances among them. The largest part of the secreted proteins could not be annotated, indicating they are either novel or severely altered during recent coevolution to function in host manipulation. We also detected shifts in the hosts' proteome with infection, in particular an overabundance of vitellogenin-like A in infected ants, a protein that regulates division of labour in Temnothorax ants, which could explain the observed behavioural changes. Our results thus suggest two different strategies that might be employed by this parasite to manipulate its host: secreting proteins with immediate influence on the host's phenotype and altering the host's translational activity. Our findings highlight the intricate molecular interplay required to influence the phenotype of a host and point to potential signalling pathways and genes involved in parasite-host communication., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Social insects are models for studies of phenotypic plasticity. Ant queens and workers vary in fecundity and lifespan, which are enhanced and extended in queens. Yet, the regulatory mechanisms underlying this variation are not well understood. Ant queens live and reproduce for years, so that they need to protect their germline from transposable element (TE) activity, which may be redundant in short-lived, often sterile workers. We analysed the expression of two protective classes of small RNAs, microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), in various tissues, castes and age classes of the ant Temnothorax rugatulus. In queens, piRNAs were highly abundant in ovaries with TEs being their clear targets, with reduced but still detectable piRNA-specific ping-pong signatures in thorax and brains. piRNA pathway activity varied little with age in queens. Moreover, the reduced ovaries of workers also exhibited similar piRNA activity and this not only in young, fertile workers, but also in older foragers with regressed ovaries. Therefore, these ants protect their germline through piRNA activity, regardless of ovarian development, age or caste, even in sterile workers often considered the soma of the superorganism. Our tissue-specific miRNA analysis detected the expression of 304 miRNAs, of which 105 were expressed in all tissues, 10 enriched in the brain, three in the thorax, whereas 83 were ovarian-specific. We identified ovarian miRNAs whose expression was related to caste, fecundity and age, and which likely regulate group-specific gene expression. sRNA shifts in young- to middle-aged queens were minor, suggesting delayed senescence in this reproductive caste., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Division of labor (DOL) is a characteristic trait of insect societies, where tasks are generally performed by specialized individuals. Inside workers focus on brood or nest care, while others take risks by foraging outside. Theory proposes that workers have different thresholds to perform certain tasks when confronted with task-related stimuli, leading to specialization and consequently DOL. Workers are presumed to vary in their response to task-related cues rather than in how they perceive such information. Here, we test the hypothesis that DOL instead stems from workers varying in their efficiency to detect stimuli of specific tasks. We use transcriptomics to measure mRNA expression levels in the antennae and brain of nurses and foragers of the ant Temnothorax longispinosus. We find seven times as many genes to be differentially expressed between behavioral phenotypes in the antennae compared to the brain. Moreover, half of all odorant receptors are differentially expressed, with an overrepresentation of the 9-exon gene family upregulated in the antennae of nurses. Nurses and foragers thus apparently differ in the perception of their olfactory environment and task-related signals. Our study supports the hypothesis that antennal sensory filters predispose workers to specialize in specific tasks., (© 2023. Springer Nature Limited.)

Eusocial insects exemplify a remarkable system of division of labor within the same colony. This behavioral range, which is sometimes accompanied by morphological or physiological differences, provides an opportunity to study the relationship between complex behaviors and their underlying molecular mechanisms. This is especially true in ants because certain genera have an elaborate caste system and can dramatically change their stereotypical behavior over their lifetime. Recent studies experimentally alter ant behavior over short times, thus opening the study of underlying plasticity pathways. The molecular underpinnings of these behaviors are neuromodulators as well as the regulation of chromatin. Here, we concisely review the current understanding of the relationship between neuromodulators, epigenetics, and social behavior in ants. We discuss future directions in light of experimental limitations of the ant system., (© 2023 New York Academy of Sciences.)

Ecitonides constanceae sp. nov., a new species of the myrmecophilous genus Ecitonides Wasmann, 1894, which is associated with army ants (Hymenoptera: Formicidae: Ecitoninae), is described from Peru. The holotype, which is the only known specimen, was collected at light at Los Amigos Biological Station, Madre de Dios department, Peru. The army ant host species is unknown. The new species is distinguished from congeners and an updated key of the species, as well as a checklist of Ecitonides and their associated host ants are provided. Based on the holotype we non-destructively extracted DNA and sequenced two genes which were incorportated into an existing molecular phylogenetic dataset and analysed using Bayesian Inference. Ecitonides is recovered as sister to recently described thermitophilous genus Ruptor ya et al. 2022 and placed in the so-called Medonina and allied taxa clade. Beyond Bayesian Inference phylogenetic analysis and their associated nodal support methods, we also explored the position of the Ecitonides within the posterior sample of trees produced in the Bayesian analyses, which further supported its placement.

The red imported fire ant (Solenopsis invicta) is classified as one of the worst invasive alien species 1 and as the fifth costliest worldwide 2 , impacting ecosystems, agriculture and human health 3 . We report the establishment of S. invicta in Europe for the first time, documenting a mature population in Sicily. We use genetic analyses to assess its putative origin, as well as wind tracking and species distribution modelling to predict its potential range on the continent. We show that half of the urban areas in Europe are already suitable and that climate warming expected under current trends will favor the expansion of this invasive ant., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Insect social parasites are characterized by exploiting the hosts' social behaviour. Why exactly hosts direct their caring behaviour towards these parasites and their offspring remains largely unstudied. One hypothesis is that hosts do not perceive their social environment as altered and accept the parasitic colony as their own. We used the ant Leptothorax acervorum, host of the dulotic, obligate social parasite Harpagoxenus sublaevis, to shed light on molecular mechanisms underlying behavioural exploitation by contrasting tissue-specific transcriptomes in young host workers. Host pupae were experimentally (re-)introduced into fragments of their original, another conspecific, heterospecific or parasitic colony. Brain and antennal mRNA was extracted and sequenced from adult ants after they had lived in the experimental colony for at least 50 days after eclosion. The resulting transcriptomes of L. acervorum revealed that ants were indeed affected by their social environment. Host brain transcriptomes were altered by the presence of social parasites, suggesting that the parasitic environment influences brain activity, which may be linked to behavioural changes. Transcriptional activity in the antennae changed most with the presence of unrelated individuals, regardless of whether they were conspecifics or parasites. This suggests early priming of odour perception, which was further supported by sensory perception of odour as an enriched function of differentially expressed genes. Furthermore, gene expression in the antennae, but not in the brain corresponded to ant worker behaviour before sampling. Our study demonstrated that the exploitation of social behaviours by brood parasites correlates with transcriptomic alterations in the central and peripheral nervous systems., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)