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A study of Danish Island populations of the red ant Myrmica sulcinodis is presented. The findings suggest that given the isolation of the islands and the low investment in reproduction, the population was most likely established by a single queen, even though all three extant populations have within-colony relatedness.

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.

The mutualistic interaction specificity between attine ants and antibiotic-producing Actinobacteria has been controversial because Pseudonocardia strains cannot always be isolated from worker cuticles across attine ant species, while other actinobacteria can apparently replace Pseudonocardia and also inhibit growth of Escovopsis mycopathogens. Here we report that across field samples of Panamanian species: (i) Cuticular Pseudonocardia were largely restricted to species in the crown of the attine phylogeny and their appearance likely coincided with the first attines colonizing Central/North America. (ii) The phylogenetically basal attines almost always had cuticular associations with other Actinobacteria than Pseudonocardia . (iii) The sub-cuticular glands nourishing cuticular bacteria appear to be homologous throughout the phylogeny, consistent with an ancient general attine-Actinobacteria association. (iv) The basal attine species investigated always had Pseudonocardia as gut symbionts while Pseudonocardia presence appeared mutually exclusive between cuticular and gut microbiomes. (v) Gut-associated Pseudonocardia were phylogenetically ancestral while cuticular symbionts formed a derived crown group within the Pseudonocardia phylogeny. We further show that laboratory colonies often secondarily acquire cuticular Actinobacteria that they do not associate with in the field, suggesting that many previous studies were uninformative for questions of co-adaptation in the wild. An exhaustive literature survey showed that published studies concur with our present results, provided that they analyzed field colonies and that Actinobacteria were specifically isolated from worker cuticles shortly after field collection. Our results offer several testable hypotheses for a better overall understanding of attine- Pseudonocardia interaction dynamics and putative coevolution throughout the Americas., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

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., (© 2022. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

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., (© 2022. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2021. The Author(s).)

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.

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., Competing Interests: MS, JB No competing interests declared, (© 2021, Schiøtt and Boomsma.)

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.

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., (2020 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2020 European Society For Evolutionary Biology.)

Caste differentiation happens early in development to produce gynes as future colony germlines and workers as present colony soma. However, gynes need insemination to become functional queens, a transition that initiates reproductive role differentiation relative to unmated gynes. Here, we analyze the anatomy and transcriptomes of brains during this differentiation process within the reproductive caste of Monomorium pharaonis Insemination terminated brain growth, whereas unmated control gynes continued to increase brain volume. Transcriptomes revealed a specific gene regulatory network (GRN) mediating both brain anatomy changes and behavioral modifications. This reproductive role differentiation GRN hardly overlapped with the gyne-worker caste differentiation GRN, but appears to be also used by distantly related ants where workers became germline individuals after the queen caste was entirely or partially lost. The genes corazonin and neuroparsin A in the anterior neurosecretory cells were overexpressed in individuals with reduced or nonreproductive roles across all four ant species investigated., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

A major challenge in evolutionary biology has been to explain the variation in multicellularity across the many independently evolved multicellular lineages, from slime moulds to vertebrates. Social evolution theory has highlighted the key role of relatedness in determining multicellular complexity and obligateness; however, there is a need to extend this to a broader perspective incorporating the role of the environment. In this paper, we formally test Bonner's 1998 hypothesis that the environment is crucial in determining the course of multicellular evolution, with aggregative multicellularity evolving more frequently on land and clonal multicellularity more frequently in water. Using a combination of scaling theory and phylogenetic comparative analyses, we describe multicellular organizational complexity across 139 species spanning 14 independent transitions to multicellularity and investigate the role of the environment in determining multicellular group formation and in imposing constraints on multicellular evolution. Our results, showing that the physical environment has impacted the way in which multicellular groups form, highlight that environmental conditions might have affected the major evolutionary transition to obligate multicellularity.

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., Competing Interests: JL, JG, MW, RD, MS, YO, IC, JH, BB, JB, BB No competing interests declared, (© 2019, Liberti et al.)

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., (© 2019 Dosselli et al.)

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., (© 2018 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.)

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., Competing Interests: PS, MZ, JS, MS, JB No competing interests declared, (© 2018, Sapountzis et al.)

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.

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

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.

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.

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.

Background: Promiscuous mating and sperm competition often induce arms races between the sexes with detrimental outcomes for females. However, ants with multiply-inseminated queens have only a single time-window for sperm competition and queens are predicted to gain control over the outcome of sperm storage quickly. The seminal fluid of Acromyrmex leaf-cutting ants reduces the viability of rival sperm, but how confrontations between unrelated ejaculates affect sperm storage remains unknown., Results: We investigated the effects of ejaculate admixture on sperm motility in A. echinatior and found that the proportion of motile spermatozoa, sperm swimming speed, and linearity of sperm movement increased when rival ejaculates were mixed in vitro. Major effects induced by the seminal fluid of rival males were of similar magnitude to those generated by queen reproductive tract secretions, whereas own seminal fluid induced lower sperm activation levels., Conclusions: Our results suggest that ant sperm respond via a self-non-self recognition mechanism to similar or shared molecules expressed in the reproductive secretions of both sexes. Lower sperm motility in the presence of own seminal fluid indicates that enhanced motility is costly and may trade-off with sperm viability during sperm storage, consistent with studies in vertebrates. Our results imply that ant spermatozoa have evolved to adjust their energetic expenditure during insemination depending on the perceived level of sperm competition.

Leaf-cutting ant queens mate with multiple males during a single nuptial flight and store sperm for up to two decades. During mating, males transfer sperm from their accessory testes to the queen bursa copulatrix from where it enters the spermatheca, an insect sperm storage organ that has become highly specialized in long-lived ant queens who never re-mate later in life. Long-term storage without the possibility to obtain new sperm creates an immune defence dilemma, because recognition of non-self cells eliminates infections but may also target irreplaceable sperm and reduce lifetime reproductive success. We therefore hypothesized that non-specific immune responses, like pathogen melanization, should be silenced in the spermatheca, because they rely on general non-self recognition, and that specific responses such as antimicrobial peptides are activated instead as they specifically target pathogenic bacteria and/or fungi. The maintenance of uninfected sperm cells by males before mating is not constrained by non-self recognition, meaning immune regulation might be more liberal in male reproductive organs. To test this hypothesis, we measured gene expression of two antimicrobial peptides, abaecin and defensin, and prophenoloxidase, an important enzyme of the melanization pathway, in male accessory glands and testes and in queen bursae copulatrix and spermathecae of Acromyrmex echinatior and Atta colombica leaf-cutting ants. As expected, prophenoloxidase expression was low in reproductive organs that sustain prolonged contact with sperm, whereas antimicrobial peptides showed average to high expression, indicating that leaf-cutting ants invest in specific rather than generalist immune defences for pathogen protection in organs that store sperm., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

More than a century ago, William Morton Wheeler proposed that social insect colonies can be regarded as superorganisms when they have morphologically differentiated reproductive and nursing castes that are analogous to the metazoan germ-line and soma. Following the rise of sociobiology in the 1970s, Wheeler's insights were largely neglected, and we were left with multiple new superorganism concepts that are mutually inconsistent and uninformative on how superorganismality originated. These difficulties can be traced to the broadened sociobiological concept of eusociality, which denies that physical queen-worker caste differentiation is a universal hallmark of superorganismal colonies. Unlike early evolutionary naturalists and geneticists such as Weismann, Huxley, Fisher and Haldane, who set out to explain the acquisition of an unmated worker caste, the goal of sociobiology was to understand the evolution of eusociality, a broad-brush convenience category that covers most forms of cooperative breeding. By lumping a diverse spectrum of social systems into a single category, and drawing attention away from the evolution of distinct quantifiable traits, the sociobiological tradition has impeded straightforward connections between inclusive fitness theory and the major evolutionary transitions paradigm for understanding irreversible shifts to higher organizational complexity. We evaluate the history by which these inconsistencies accumulated, develop a common-cause approach for understanding the origins of all major transitions in eukaryote hierarchical complexity, and use Hamilton's rule to argue that they are directly comparable. We show that only Wheeler's original definition of superorganismality can be unambiguously linked to irreversible evolutionary transitions from context-dependent reproductive altruism to unconditional differentiation of permanently unmated castes in the ants, corbiculate bees, vespine wasps and higher termites. We argue that strictly monogamous parents were a necessary, albeit not sufficient condition for all transitions to superorganismality, analogous to single-zygote bottlenecking being a necessary but not sufficient condition for the convergent origins of complex soma across multicellular eukaryotes. We infer that conflict reduction was not a necessary condition for the origin of any of these major transitions, and conclude that controversies over the status of inclusive fitness theory primarily emanate from the arbitrarily defined sociobiological concepts of superorganismality and eusociality, not from the theory itself., (© 2017 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

The social Hymenoptera have distinct larval and adult stages separated by metamorphosis, which implies striking remodeling of external and internal body structures during the pupal stage. This imposes challenges to gut symbionts as existing cultures are lost and may or may not need to be replaced. To elucidate the extent to which metamorphosis interrupts associations between bacteria and hosts, we analyzed changes in gut microbiota during development and traced the transmission routes of dominant symbionts from the egg to adult stage in the leaf-cutting ants Acromyrmex echinatior and Atta cephalotes , which are both important functional herbivores in the New World tropics. Bacterial density remained similar across the developmental stages of Acromyrmex , but Atta brood had very low bacterial prevalences suggesting that bacterial gut symbionts are not actively maintained. We found that Wolbachia was the absolute dominant bacterial species across developmental stages in Acromyrmex and we confirmed that Atta lacks Wolbachia also in the immature stages, and had mostly Mollicutes bacteria in the adult worker guts. Wolbachia in Acromyrmex appeared to be transovarially transmitted similar to transmission in solitary insects. In contrast, Mollicutes were socially transmitted from old workers to newly emerged callows. We found that larval and pupal guts of both ant species contained Pseudomonas and Enterobacter bacteria that are also found in fungus gardens, but hardly or not in adult workers, suggesting they are beneficial only for larval growth and development. Our results reveal that transmission pathways for bacterial symbionts may be very different both between developmental stages and between sister genera and that identifying the mechanisms of bacterial acquisition and loss will be important to clarify their putative mutualistic functions.

Background and objectives: Pre-eclampsia often has detrimental health effects for pregnant women and their fetuses, but whether exposure in the womb has long-term health-consequences for children as they grow up remains poorly understood. We assessed overall morbidity of children following exposure to either mild or severe pre-eclampsia up to 30 years after birth and related disease risks to duration of exposure, i.e. the time from diagnosis to delivery. Methodology: We did a registry-based retrospective cohort study in Denmark covering the years 1979-2009, using the separate diagnoses of mild and severe pre-eclampsia and the duration of exposure as predictor variables for specific and overall risks of later disease. We analysed 3 537 525 diagnoses for 14 disease groups, accumulated by 758 524 singleton children, after subdividing deliveries in six gestational age categories, partialing out effects of eight potentially confounding factors. Results: Exposure to mild pre-eclampsia appeared to have consistent negative effects on health later in life, although only a few specific disease cases remained significant after corrections for multiple testing. Morbidity risks associated with mild pre-eclampsia were of similar magnitude as those associated with severe pre-eclampsia. Apart from this overall trend in number of diagnoses incurred across disease groups, hazard ratios for several disorders also increased with the duration of exposure, including disorders related to the metabolic syndrome. Conclusions and implications: Maternal pre-eclampsia has lasting effects on offspring health and differences between exposure to severe and mild pre-eclampsia appear to be less than previously assumed. Our results suggest that it would be prudent to include the long-term health prospects of children in the complex clinical management of mild pre-eclampsia.

The attine ants of South and Central America are ancient farmers, having evolved a symbiosis with a fungal food crop >50 million years ago. The most evolutionarily derived attines are the Atta and Acromyrmex leafcutter ants, which harvest fresh leaves to feed their fungus. Acromyrmex and many other attines vertically transmit a mutualistic strain of Pseudonocardia and use antifungal compounds made by these bacteria to protect their fungal partner against co-evolved fungal pathogens of the genus Escovopsis . Pseudonocardia mutualists associated with the attines Apterostigma dentigerum and Trachymyrmex cornetzi make novel cyclic depsipeptide compounds called gerumycins, while a mutualist strain isolated from derived Acromyrmex octospinosus makes an unusual polyene antifungal called nystatin P1. The novelty of these antimicrobials suggests there is merit in exploring secondary metabolites of Pseudonocardia on a genome-wide scale. Here, we report a genomic analysis of the Pseudonocardia phylotypes Ps1 and Ps2 that are consistently associated with Acromyrmex ants collected in Gamboa, Panama. These were previously distinguished solely on the basis of 16S rRNA gene sequencing but genome sequencing of five Ps1 and five Ps2 strains revealed that the phylotypes are distinct species and each encodes between 11 and 15 secondary metabolite biosynthetic gene clusters (BGCs). There are signature BGCs for Ps1 and Ps2 strains and some that are conserved in both. Ps1 strains all contain BGCs encoding nystatin P1-like antifungals, while the Ps2 strains encode novel nystatin-like molecules. Strains show variations in the arrangement of these BGCs that resemble those seen in gerumycin gene clusters. Genome analyses and invasion assays support our hypothesis that vertically transmitted Ps1 and Ps2 strains have antibacterial activity that could help shape the cuticular microbiome. Thus, our work defines the Pseudonocardia species associated with Acromyrmex ants and supports the hypothesis that Pseudonocardia species could provide a valuable source of new antimicrobials.

A visionary Darwinian ahead of his time, George C. Williams developed in his 1966 book Adaptation and Natural Selection the essentials of a unifying theory of adaptation that remains robust today and has inspired immense progress in understanding how natural selection works., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

How differentiation between cell types evolved is a fundamental question in biology, but few studies have explored single-gene phenotypes that mediate first steps towards division of labour with selective advantage for groups of cells. Here, we show that differential expression of the FLO11 gene produces stable fractions of Flo11 + and Flo11 - cells in clonal Saccharomyces cerevisiae biofilm colonies on medium with intermediate viscosity. Differentiated Flo11 +/- colonies, consisting of adhesive and non-adhesive cells, obtain a fourfold growth advantage over undifferentiated colonies by overgrowing glucose resources before depleting them, rather than depleting them while they grow as undifferentiated Flo11 - colonies do. Flo11 +/- colonies maintain their structure and differentiated state by switching non-adhesive cells to adhesive cells with predictable probability. Mixtures of Flo11 + and Flo11 - cells from mutant strains that are unable to use this epigenetic switch mechanism produced neither integrated colonies nor growth advantages, so the condition-dependent selective advantages of differentiated FLO11 expression can only be reaped by clone-mate cells. Our results show that selection for cell differentiation in clonal eukaryotes can evolve before the establishment of obligate undifferentiated multicellularity, and without necessarily leading to more advanced organizational complexity., (© 2016 The Author(s).)

Queens of Acromyrmex leaf-cutting ants store sperm of multiple males after a single mating flight, and never remate even though they may live for decades and lay tens of thousands of eggs. Sperm of different males are initially transferred to the bursa copulatrix and compete for access to the long-term storage organ of queens, but the factors determining storage success or failure have never been studied. We used in vitro experiments to show that reproductive tract secretions of Acromyrmex echinatior queens increase sperm swimming performance by at least 50% without discriminating between sperm of brothers and unrelated males. Indiscriminate female-induced sperm chemokinesis makes the likelihood of storage directly dependent on initial sperm viability and thus provides a simple mechanism to secure maximal possible reproductive success of queens, provided that initial sperm motility is an accurate predictor of viability during later egg fertilization., (© 2016 The Authors.)

Background and Objectives: Effects of maternal and paternal age on offspring autism and schizophrenia risks have been studied for over three decades, but inconsistent risks have often been found, precluding well-informed speculation on why these age-related risks might exist., Methodology: To help clarify this situation we analysed a massive single population sample from Denmark including the full spectrum of autistic and schizophrenic disorders (eliminating between-study confounding), used up to 30 follow-up years, controlled for over 20 potentially confounding factors and interpret the ultimate causation of the observed risk patterns using generally accepted principles of parent-offspring conflict and life-history theory., Results: We evaluated the effects of paternal age, maternal age and parental age difference on offspring mental disorders and found consistently similar risk patterns for related disorders and markedly different patterns between autistic and schizophrenic disorders. Older fathers and mothers both conferred increased risk for autistic but not schizophrenic disorders, but autism risk was reduced in younger parents and offspring of younger mothers had increased risk for many schizophrenic disorders. Risk for most disorders also increased when parents were more dissimilarly aged. Monotonically increasing autism risk is consistent with mutation accumulation as fathers' age, but this explanation is invalid for schizophrenic disorders, which were not related to paternal age and were negatively correlated with maternal age., Conclusions and Implications: We propose that the observed maternally induced risk patterns ultimately reflect a shifting ancestral life-history trade-off between current and future reproduction, mediated by an initially high but subsequently decreasing tendency to constrain foetal provisioning as women proceed from first to final pregnancy., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health.)

Attine ants evolved farming 55-60 My before humans. Although evolutionarily derived leafcutter ants achieved industrial-scale farming, extant species from basal attine genera continue to farm loosely domesticated fungal cultivars capable of pursuing independent reproductive interests. We used feeding experiments with the basal attine Mycocepurus smithii to test whether reproductive allocation conflicts between farmers and cultivars constrain crop yield, possibly explaining why their mutualism has remained limited in scale and productivity. Stoichiometric and geometric framework approaches showed that carbohydrate-rich substrates maximize growth of both edible hyphae and inedible mushrooms, but that modest protein provisioning can suppress mushroom formation. Worker foraging was consistent with maximizing long-term cultivar performance: ant farmers could neither increase carbohydrate provisioning without cultivars allocating the excess toward mushroom production, nor increase protein provisioning without compromising somatic cultivar growth. Our results confirm that phylogenetically basal attine farming has been very successful over evolutionary time, but that unresolved host-symbiont conflict may have precluded these wild-type symbioses from rising to ecological dominance. That status was achieved by the evolutionarily derived leafcutter ants following full domestication of a coevolving cultivar 30-35 Mya after the first attine ants committed to farming., Competing Interests: The authors declare no conflict of interest.

Fungus-growing ants (Attini) have evolved an obligate dependency upon a basidiomycete fungus that they cultivate as their food. Less well known is that the crop fungus is also used by many attine species to cover their eggs, larvae and pupae. The adaptive functional significance of this brood covering is poorly understood. One hypothesis to account for this behaviour is that it is part of the pathogen protection portfolio when many thousands of sister workers live in close proximity and larvae and pupae are not protected by cells, as in bees and wasps, and are immobile. We performed behavioural observations on brood covering in the leaf-cutting ant Acromyrmex echinatior, and we experimentally manipulated mycelial cover on pupae and exposed them to the entomopathogenic fungus Metarhizium brunneum to test for a role in pathogen resistance. Our results show that active mycelial brood covering by workers is a behaviourally plastic trait that varies temporally, and across life stages and castes. The presence of a fungal cover on the pupae reduced the rate at which conidia appeared and the percentage of pupal surface that produced pathogen spores, compared to pupae that had fungal cover experimentally removed or naturally had no mycelial cover. Infected pupae with mycelium had higher survival rates than infected pupae without the cover, although this depended upon the time at which adult sister workers were allowed to interact with pupae. Finally, workers employed higher rates of metapleural gland grooming to infected pupae without mycelium than to infected pupae with mycelium. Our results imply that mycelial brood covering may play a significant role in suppressing the growth and subsequent spread of disease, thus adding a novel layer of protection to their defence portfolio., (© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society.)