Canalized gene expression during development mediates caste differentiation in ants.

Ant colonies are higher-level organisms consisting of specialized reproductive and non-reproductive individuals that differentiate early in development, similar to germ-soma segregation in bilateral Metazoa. Analogous to diverging cell lines, developmental differentiation of individual ants has often been considered in epigenetic terms but the sets of genes that determine caste phenotypes throughout larval and pupal development remain unknown. Here, we reconstruct the individual developmental trajectories of two ant species, Monomorium pharaonis and Acromyrmex echinatior, after obtaining >1,400 whole-genome transcriptomes. Using a new backward prediction algorithm, we show that caste phenotypes can be accurately predicted by genome-wide transcriptome profiling. We find that caste differentiation is increasingly canalized from early development onwards, particularly in germline individuals (gynes/queens) and that the juvenile hormone signalling pathway plays a key role in this process by regulating body mass divergence between castes. We quantified gene-specific canalization levels and found that canalized genes with gyne/queen-biased expression were enriched for ovary and wing functions while canalized genes with worker-biased expression were enriched in brain and behavioural functions. Suppression in gyne larvae of Freja, a highly canalized gyne-biased ovary gene, disturbed pupal development by inducing non-adaptive intermediate phenotypes between gynes and workers. Our results are consistent with natural selection actively maintaining canalized caste phenotypes while securing robustness in the life cycle ontogeny of ant colonies.

Phylogenomic analysis and metabolic role reconstruction of mutualistic Rhizobiales hindgut symbionts of Acromyrmex leaf-cutting ants.

Rhizobiales are well-known plant-root nitrogen-fixing symbionts, but the functions of insect-associated Rhizobiales are poorly understood. We obtained genomes of three strains associated with Acromyrmex leaf-cutting ants and show that, in spite of being extracellular gut symbionts, they lost all pathways for essential amino acid biosynthesis, making them fully dependent on their hosts. Comparison with 54 Rhizobiales genomes showed that all insect-associated Rhizobiales lost the ability to fix nitrogen and that the Acromyrmex symbionts had exceptionally also lost the urease genes. However, the Acromyrmex strains share biosynthesis pathways for riboflavin vitamin, queuosine and a wide range of antioxidant enzymes likely to be beneficial for the ant fungus-farming symbiosis. We infer that the Rhizobiales symbionts catabolize excess of fungus-garden-derived arginine to urea, supplementing complementary Mollicutes symbionts that turn arginine into ammonia and infer that these combined symbiont activities stabilize the fungus-farming mutualism. Similar to the Mollicutes symbionts, the Rhizobiales species have fully functional CRISPR/Cas and R-M phage defenses, suggesting that these symbionts are important enough for the ant hosts to have precluded the evolution of metabolically cheaper defenseless strains.

A single-cell transcriptomic atlas tracking the neural basis of division of labour in an ant superorganism.

Ant colonies with permanent division of labour between castes and highly distinct roles of the sexes have been conceptualized to be superorganisms, but the cellular and molecular mechanisms that mediate caste/sex-specific behavioural specialization have remained obscure. Here we characterized the brain cell repertoire of queens, gynes (virgin queens), workers and males of Monomorium pharaonis by obtaining 206,367 single-nucleus transcriptomes. In contrast to Drosophila, the mushroom body Kenyon cells are abundant in ants and display a high diversity with most subtypes being enriched in worker brains, the evolutionarily derived caste. Male brains are as specialized as worker brains but with opposite trends in cell composition with higher abundances of all optic lobe neuronal subtypes, while the composition of gyne and queen brains remained generalized, reminiscent of solitary ancestors. Role differentiation from virgin gynes to inseminated queens induces abundance changes in roughly 35% of cell types, indicating active neurogenesis and/or programmed cell death during this transition. We also identified insemination-induced cell changes probably associated with the longevity and fecundity of the reproductive caste, including increases of ensheathing glia and a population of dopamine-regulated Dh31-expressing neurons. We conclude that permanent caste differentiation and extreme sex-differentiation induced major changes in the neural circuitry of ants.

Incomplete lineage sorting and phenotypic evolution in marsupials.

Incomplete lineage sorting (ILS) makes ancestral genetic polymorphisms persist during rapid speciation events, inducing incongruences between gene trees and species trees. ILS has complicated phylogenetic inference in many lineages, including hominids. However, we lack empirical evidence that ILS leads to incongruent phenotypic variation. Here, we performed phylogenomic analyses to show that the South American monito del monte is the sister lineage of all Australian marsupials, although over 31% of its genome is closer to the Diprotodontia than to other Australian groups due to ILS during ancient radiation. Pervasive conflicting phylogenetic signals across the whole genome are consistent with some of the morphological variation among extant marsupials. We detected hundreds of genes that experienced stochastic fixation during ILS, encoding the same amino acids in non-sister species. Using functional experiments, we confirm how ILS may have directly contributed to hemiplasy in morphological traits that were established during rapid marsupial speciation ca. 60 mya.

Competition-based screening helps to secure the evolutionary stability of a defensive microbiome.

BACKGROUND: The cuticular microbiomes of Acromyrmex leaf-cutting ants pose a conundrum in microbiome biology because they are freely colonisable, and yet the prevalence of the vertically transmitted bacteria Pseudonocardia, which contributes to the control of Escovopsis fungus garden disease, is never compromised by the secondary acquisition of other bacterial strains. Game theory suggests that competition-based screening can allow the selective recruitment of antibiotic-producing bacteria from the environment, by providing abundant resources to foment interference competition between bacterial species and by using Pseudonocardia to bias the outcome of competition in favour of antibiotic producers. RESULTS: Here, we use RNA-stable isotope probing (RNA-SIP) to confirm that Acromyrmex ants can maintain a range of microbial symbionts on their cuticle by supplying public resources. We then used RNA sequencing, bioassays, and competition experiments to show that vertically transmitted Pseudonocardia strains produce antibacterials that differentially reduce the growth rates of other microbes, ultimately biassing the bacterial competition to allow the selective establishment of secondary antibiotic-producing strains while excluding non-antibiotic-producing strains that would parasitise the symbiosis. CONCLUSIONS: Our findings are consistent with the hypothesis that competition-based screening is a plausible mechanism for maintaining the integrity of the co-adapted mutualism between the leaf-cutting ant farming symbiosis and its defensive microbiome. Our results have broader implications for explaining the stability of other complex symbioses involving horizontal acquisition.

Relaxed selection underlies genome erosion in socially parasitic ant species.

Inquiline ants are highly specialized and obligate social parasites that infiltrate and exploit colonies of closely related species. They have evolved many times convergently, are often evolutionarily young lineages, and are almost invariably rare. Focusing on the leaf-cutting ant genus Acromyrmex, we compared genomes of three inquiline social parasites with their free-living, closely-related hosts. The social parasite genomes show distinct signatures of erosion compared to the host lineages, as a consequence of relaxed selective constraints on traits associated with cooperative ant colony life and of inquilines having very small effective population sizes. We find parallel gene losses, particularly in olfactory receptors, consistent with inquiline species having highly reduced social behavioral repertoires. Many of the genomic changes that we uncover resemble those observed in the genomes of obligate non-social parasites and intracellular endosymbionts that branched off into highly specialized, host-dependent niches.

Proteomics reveals synergy between biomass degrading enzymes and inorganic Fenton chemistry in leaf-cutting ant colonies.

The symbiotic partnership between leaf-cutting ants and fungal cultivars processes plant biomass via ant fecal fluid mixed with chewed plant substrate before fungal degradation. Here we present a full proteome of the fecal fluid of Acromyrmex leaf-cutting ants, showing that most proteins function as biomass degrading enzymes and that ca. 85% are produced by the fungus and ingested, but not digested, by the ants. Hydrogen peroxide producing oxidoreductases were remarkably common in the proteome, inspiring us to test a scenario in which hydrogen peroxide reacts with iron to form reactive oxygen radicals after which oxidized iron is reduced by other fecal-fluid enzymes. Our biochemical assays confirmed that these so-called Fenton reactions do indeed take place in special substrate pellets, presumably to degrade plant cell wall polymers. This implies that the symbiotic partnership manages a combination of oxidative and enzymatic biomass degradation, an achievement that surpasses current human bioconversion technology.

Colonies of tropical leaf-cutting ants live in underground nests where a fungus grows that feeds them. The ants, in turn, provide the fungus with the freshly-cut leaf fragments it needs for nutrition. The relationship between the ants and the fungus, in which they live close together and help one another survive, is known as symbiosis. It is an ancient, extremely well integrated relationship, in which neither species can survive without the other. However, the details of how the ants and the fungus work together to break down the leaf fragments so they can be used for nutrition are not well understood. When the ants eat the fungus, they do not digest its enzymes (the proteins that accelerate chemical reactions in a cell). Instead, the fungal enzymes travel through the ants' gut and into their fecal liquid, which gets deposited on the fresh-cut leaves when the ants collect them. The ants then make temporary pellets out of the new leaf fragments before providing them to the fungus. To better understand how each species contributes to the breakdown of the leaf fragments, Schiøtt and Boomsma identified all the proteins present in the fecal fluid of the ants. Once they had a complete list of about 100 proteins, they determined which of them were produced by the fungus and which by the ant. Schiøtt and Boomsma observed that certain combinations of fungal and ant enzymes could trigger a Fenton reaction ­ a chemical reaction that efficiently begins the breakdown of the tough walls around plant cells. This reaction is so aggressive that it is rarely found in nature, but it could help explain the high efficiency of the fungus and the ants symbiotically processing leaf fragments. But could a Fenton reaction actually proceed in the ants' nest without hurting the ants or affecting the rest of the fungal garden? The evidence obtained suggested that the temporary pellets made by the ants serve to isolate the reaction, so the aggressive chemistry takes place away from the ants and detached from the fungal gardens. Schiøtt and Boomsma showed that the symbiotic relationship between the ants and the fungus has led to a sustainable and efficient way of breaking down plant materials to use them for nutrition. The Fenton reaction is economically important in many industries, including bioethanol production, the detergent industry, and food production. Emulating the methods used by leaf-cutting ants, which have been fine-tuned by millions of years of natural selection, may allow humans to develop more efficient technologies for breaking down organic compounds.

Nutritional niches reveal fundamental domestication trade-offs in fungus-farming ants.

During crop domestication, human farmers traded greater productivity for higher crop vulnerability outside specialized cultivation conditions. We found a similar domestication trade-off across the major co-evolutionary transitions in the farming systems of attine ants. First, the fundamental nutritional niches of cultivars narrowed over ~60 million years of naturally selected domestication, and laboratory experiments showed that ant farmers representing subsequent domestication stages strictly regulate protein harvest relative to cultivar fundamental nutritional niches. Second, ants with different farming systems differed in their abilities to harvest the resources that best matched the nutritional needs of their fungal cultivars. This was assessed by quantifying realized nutritional niches from analyses of items collected from the mandibles of laden ant foragers in the field. Third, extensive field collections suggest that among-colony genetic diversity of cultivars in small-scale farms may offer population-wide resilience benefits that species with large-scale farming colonies achieve by more elaborate and demanding practices to cultivate less diverse crops. Our results underscore that naturally selected farming systems have the potential to shed light on nutritional trade-offs that shaped the course of culturally evolved human farming.

A novel method for using RNA-seq data to identify imprinted genes in social Hymenoptera with multiply mated queens.

Genomic imprinting results in parent-of-origin-dependent gene expression biased towards either the maternally or paternally derived allele at the imprinted locus. The kinship theory of genomic imprinting argues that this unusual expression pattern can be a manifestation of intra-genomic conflict between the maternally and paternally derived halves of the genome that arises because they are not equally related to the genomes of social partners. The theory thus predicts that imprinting may evolve wherever there are close interactions among asymmetrically related kin. The social Hymenoptera with permanent caste differentiation are suitable candidates for testing the kinship theory because haplodiploid sex determination creates strong relatedness asymmetries and nursing workers interact closely with kin. However, progress in the search for imprinted genes in the social Hymenoptera has been slow, in part because tests for imprinting rely on reciprocal crosses that are impossible in most species. Here, we develop a method to systematically search for imprinting in haplodiploid social insects without crosses, using instead samples of pooled individuals collected from natural colonies. We tested this protocol using data available for the leaf-cutting ant Acromyrmex echinatior, providing the first genome-wide search for imprinting in any ant. Although we identified several genes as potentially imprinted, none of the four genes tested could be verified as imprinted using digital droplet PCR, highlighting the need for higher quality genomic assemblies that accurately map duplicated genes.

Seminal fluid compromises visual perception in honeybee queens reducing their survival during additional mating flights.

Queens of social insects make all mate-choice decisions on a single day, except in honeybees whose queens can conduct mating flights for several days even when already inseminated by a number of drones. Honeybees therefore appear to have a unique, evolutionarily derived form of sexual conflict: a queen's decision to pursue risky additional mating flights is driven by later-life fitness gains from genetically more diverse worker-offspring but reduces paternity shares of the drones she already mated with. We used artificial insemination, RNA-sequencing and electroretinography to show that seminal fluid induces a decline in queen vision by perturbing the phototransduction pathway within 24-48 hr. Follow up field trials revealed that queens receiving seminal fluid flew two days earlier than sister queens inseminated with saline, and failed more often to return. These findings are consistent with seminal fluid components manipulating queen eyesight to reduce queen promiscuity across mating flights.

Protein-Level Interactions as Mediators of Sexual Conflict in Ants.

All social insects with obligate reproductive division of labor evolved from strictly monogamous ancestors, but multiple queen-mating (polyandry) arose de novo, in several evolutionarily derived lineages. Polyandrous ant queens are inseminated soon after hatching and store sperm mixtures for a potential reproductive life of decades. However, they cannot re-mate later in life and are thus expected to control the loss of viable sperm because their lifetime reproductive success is ultimately sperm limited. In the leaf-cutting ant Atta colombica,, the survival of newly inseminated sperm is known to be compromised by seminal fluid of rival males and to be protected by secretions of the queen sperm storage organ (spermatheca). Here we investigate the main protein-level interactions that appear to mediate sperm competition dynamics and sperm preservation. We conducted an artificial insemination experiment and DIGE-based proteomics to identify proteomic changes when seminal fluid is exposed to spermathecal fluid followed by a mass spectrometry analysis of both secretions that allowed us to identify the sex-specific origins of the proteins that had changed in abundance. We found that spermathecal fluid targets only seven (2%) of the identified seminal fluid proteins for degradation, including two proteolytic serine proteases, a SERPIN inhibitor, and a semen-liquefying acid phosphatase. In vitro, and in vivo, experiments provided further confirmation that these proteins are key molecules mediating sexual conflict over sperm competition and viability preservation during sperm storage. In vitro, exposure to spermathecal fluid reduced the capacity of seminal fluid to compromise survival of rival sperm in a matter of hours and biochemical inhibition of these seminal fluid proteins largely eliminated that adverse effect. Our findings indicate that A. colombica, queens are in control of sperm competition and sperm storage, a capacity that has not been documented in other animals but is predicted to have independently evolved in other polyandrous social insects.

The evolution of abdominal microbiomes in fungus-growing ants.

The attine ants are a monophyletic lineage that switched to fungus farming ca. 55-60 MYA. They have become a model for the study of complex symbioses after additional fungal and bacterial symbionts were discovered, but their abdominal endosymbiotic bacteria remain largely unknown. Here, we present a comparative microbiome analysis of endosymbiotic bacteria spanning the entire phylogenetic tree. We show that, across 17 representative sympatric species from eight genera sampled in Panama, abdominal microbiomes are dominated by Mollicutes, α- and γ-Proteobacteria, and Actinobacteria. Bacterial abundances increase from basal to crown branches in the phylogeny reflecting a shift towards putative specialized and abundant abdominal microbiota after the ants domesticated gongylidia-bearing cultivars, but before the origin of industrial-scale farming based on leaf-cutting herbivory. This transition coincided with the ancestral single colonization event of Central/North America ca. 20 MYA, documented in a recent phylogenomic study showing that almost the entire crown group of the higher attine ants, including the leaf-cutting ants, evolved there and not in South America. Several bacterial species are located in gut tissues or abdominal organs of the evolutionarily derived, but not the basal attine ants. The composition of abdominal microbiomes appears to be affected by the presence/absence of defensive antibiotic-producing actinobacterial biofilms on the worker ants' cuticle, but the significance of this association remains unclear. The patterns of diversity, abundance and sensitivity of the abdominal microbiomes that we obtained explore novel territory in the comparative analysis of attine fungus farming symbioses and raise new questions for further in-depth research.

Correction to: The introduction history of invasive garden ants in Europe: integrating genetic, chemical and behavioural approaches.

Reinvestigation of the raw data revealed an unfortunate error in Ugelvig et al. 2008 [1].

Reconstructing the functions of endosymbiotic Mollicutes in fungus-growing ants.

Mollicutes, a widespread class of bacteria associated with animals and plants, were recently identified as abundant abdominal endosymbionts in healthy workers of attine fungus-farming leaf-cutting ants. We obtained draft genomes of the two most common strains harbored by Panamanian fungus-growing ants. Reconstructions of their functional significance showed that they are independently acquired symbionts, most likely to decompose excess arginine consistent with the farmed fungal cultivars providing this nitrogen-rich amino-acid in variable quantities. Across the attine lineages, the relative abundances of the two Mollicutes strains are associated with the substrate types that foraging workers offer to fungus gardens. One of the symbionts is specific to the leaf-cutting ants and has special genomic machinery to catabolize citrate/glucose into acetate, which appears to deliver direct metabolic energy to the ant workers. Unlike other Mollicutes associated with insect hosts, both attine ant strains have complete phage-defense systems, underlining that they are actively maintained as mutualistic symbionts.

Towards reconstructing the ancestral brain gene-network regulating caste differentiation in ants.

Specialized queens and life-time unmated workers evolved once in the common ancestor of all ants, but whether caste development across ants continues to be at least partly regulated by a single core set of genes remains obscure. We analysed brain transcriptomes from five ant species (three subfamilies) and reconstructed the origins of genes with caste-biased expression. Ancient genes predating the Neoptera were more likely to regulate gyne (virgin queen) phenotypes, while the caste differentiation roles of younger, ant-lineage-specific genes varied. Transcriptome profiling showed that the ancestral network for caste-specific gene regulation has been maintained, but that signatures of common ancestry are obscured by later modifications. Adjusting for such differences, we identified a core gene-set that: (1) consistently displayed similar directions and degrees of caste-differentiated expression; and (2) have mostly not been reported as being involved in caste differentiation. These core regulatory genes exist in the genomes of ant species that secondarily lost the queen caste, but expression differences for reproductive and sterile workers are minor and similar to social paper wasps that lack differentiated castes. Many caste-biased ant genes have caste-differentiated expression in honeybees, but directions of caste bias were uncorrelated, as expected when permanent castes evolved independently in both lineages.

Chemical warfare between leafcutter ant symbionts and a co-evolved pathogen.

Acromyrmex leafcutter ants form a mutually beneficial symbiosis with the fungus Leucoagaricus gongylophorus and with Pseudonocardia bacteria. Both are vertically transmitted and actively maintained by the ants. The fungus garden is manured with freshly cut leaves and provides the sole food for the ant larvae, while Pseudonocardia cultures are reared on the ant-cuticle and make antifungal metabolites to help protect the cultivar against disease. If left unchecked, specialized parasitic Escovopsis fungi can overrun the fungus garden and lead to colony collapse. We report that Escovopsis upregulates the production of two specialized metabolites when it infects the cultivar. These compounds inhibit Pseudonocardia and one, shearinine D, also reduces worker behavioral defenses and is ultimately lethal when it accumulates in ant tissues. Our results are consistent with an active evolutionary arms race between Pseudonocardia and Escovopsis, which modifies both bacterial and behavioral defenses such that colony collapse is unavoidable once Escovopsis infections escalate.

Association of Long-Term Risk of Respiratory, Allergic, and Infectious Diseases With Removal of Adenoids and Tonsils in Childhood.

Importance: Surgical removal of adenoids and tonsils to treat obstructed breathing or recurrent middle-ear infections remain common pediatric procedures; however, little is known about their long-term health consequences despite the fact that these lymphatic organs play important roles in the development and function of the immune system. Objective: To estimate long-term disease risks associated with adenoidectomy, tonsillectomy, and adenotonsillectomy in childhood. Design, Setting, and Participants: A population-based cohort study of up to 1 189 061 children born in Denmark between 1979 and 1999 and evaluated in linked national registers up to 2009, covering at least the first 10 and up to 30 years of their life, was carried out. Participants in the case and control groups were selected such that their health did not differ significantly prior to surgery. Exposures: Participants were classified as exposed if adenoids or tonsils were removed within the first 9 years of life. Main Outcomes and Measures: The incidence of disease (defined by International Classification of Diseases, Eighth Revision [ICD-8] and Tenth Revision [ICD-10] diagnoses) up to age 30 years was examined using stratified Cox proportional hazard regressions that adjusted for 18 covariates, including parental disease history, pregnancy complications, birth weight, Apgar score, sex, socioeconomic markers, and region of Denmark born. Results: A total of up to 1 189 061 children were included in this study (48% female); 17 460 underwent adenoidectomy, 11 830 tonsillectomy, and 31 377 adenotonsillectomy; 1 157 684 were in the control group. Adenoidectomy and tonsillectomy were associated with a 2- to 3-fold increase in diseases of the upper respiratory tract (relative risk [RR], 1.99; 95% CI, 1.51-2.63 and RR, 2.72; 95% CI, 1.54-4.80; respectively). Smaller increases in risks for infectious and allergic diseases were also found: adenotonsillectomy was associated with a 17% increased risk of infectious diseases (RR, 1.17; 95% CI, 1.10-1.25) corresponding to an absolute risk increase of 2.14% because these diseases are relatively common (12%) in the population. In contrast, the long-term risks for conditions that these surgeries aim to treat often did not differ significantly and were sometimes lower or higher. Conclusions and Relevance: In this study of almost 1.2 million children, of whom 17 460 had adenoidectomy, 11 830 tonsillectomy, and 31 377 adenotonsillectomy, surgeries were associated with increased long-term risks of respiratory, infectious, and allergic diseases. Although rigorous controls for confounding were used where such data were available, it is possible these effects could not be fully accounted for. Our results suggest it is important to consider long-term risks when making decisions to perform tonsillectomy or adenoidectomy.

Monogamous sperm storage and permanent worker sterility in a long-lived ambrosia beetle.

The lifetime monogamy hypothesis claims that the evolution of permanently unmated worker castes always requires maximal full-sibling relatedness to be established first. The long-lived diploid ambrosia beetle Austroplatypus incompertus (Schedl) is known to be highly social, but whether it has lifetime sterile castes has remained unclear. Here we show that the gallery systems of this beetle inside the heartwood of live Eucalyptus trees are always inhabited by a single core family, consisting of a lifetime-inseminated mother, permanently unmated daughter workers, and immatures that are always full siblings to each other and their adult caretakers. Overall sex ratios are even. Males always disperse and only survive as stored sperm, but female offspring either disperse to mate and found their own colony or assume unmated worker roles, probably surviving for many years without any reproductive potential because tarsal loss precludes later dispersal. A well-supported Platypodinae phylogeny has allowed us to infer that parental monogamy evolved before a lifetime-unmated worker caste emerged, confirming the prediction that monogamy and full-sibling relatedness are necessary conditions for the evolution of such workers. The initially very challenging but ultimately long-term stable nesting habitat in live trees appears to have provided the crucial benefit/cost factor for maintaining selection for permanently sterile workers after strict monogamy and lifetime sperm storage had become established in this curculionid coleopteran lineage.