Sex- and caste-specific developmental responses to juvenile hormone in an ant with maternal caste determination.

Juvenile hormone is considered to be a master regulator of polyphenism in social insects. In the ant Cardiocondyla obscurior, whether a female egg develops into a queen or a worker is determined maternally and caste-specific differentiation occurs in embryos, so that queens and workers can be distinguished in a non-invasive manner from late embryogenesis onwards. This ant also exhibits two male morphs - winged and wingless males. Here, we used topical treatment with juvenile hormone III and its synthetic analogue methoprene, a method that influences caste determination and differentiation in some ant species, to investigate whether hormone manipulation affects the development and growth of male, queen- and worker-destined embryos and larvae. We found no effect of hormone treatment on female caste ratios or body sizes in any of the treated stages, even though individuals reacted to heightened hormone availability with increased expression of krüppel-homolog 1, a conserved JH first-response gene. In contrast, hormone treatment resulted in the emergence of significantly larger males, although male morph fate was not affected. These results show that in C. obscurior, maternal caste determination leads to irreversible and highly canalized caste-specific development and growth.

Infection with a non-lethal fungal parasite is associated with increased immune investment in the ant Myrmica scabrinodis.

Social insects, such as ants, are preferred host organisms of pathogens and parasites because colonies are densely populated, and the number of potential hosts is high in the same place and time. Within a colony, individuals are exposed differentially to risks according to their function and age. Thus, older individuals forage and are therefore the most exposed to infection, predation, or physical stress, while young workers mostly stay inside the sheltered nest being less exposed. Immune investment is considered to be dependent on an individual's age and pathogen pressure. Long-term exposure to a parasite could affect the immune activity of individuals in an intriguing way that interferes with the age-dependent decline in immunocompetence. However, there are only few cases in which such interferences can be studied. The myrmecopathogenic fungus Rickia wasmannii, which infects entire colonies without killing the workers, is a suitable candidate for such studies. We investigated the general immunocompetence of Myrmica scabrinodis ant workers associated with non-lethal fungal infection by measuring the levels of active phenoloxidase (PO) and total PO (PPO) (reflecting the amount of both active and inactive forms of the enzyme) in two age classes. The level of PO proved to be higher in infected workers than in uninfected ones, while the level of PPO increased with age but was not affected by infection. Overall, these results indicate that a long-term infection could go hand in hand with increased immune activity of ant workers, conferring them higher level of protection.

Convergent Loss of Chemoreceptors across Independent Origins of Slave-Making in Ants.

The evolution of an obligate parasitic lifestyle often leads to the reduction of morphological and physiological traits, which may be accompanied by loss of genes and functions. Slave-making ants are social parasites that exploit the work force of closely related ant species for social behaviors such as brood care and foraging. Recent divergence between these social parasites and their hosts enables comparative studies of gene family evolution. We sequenced the genomes of eight ant species, representing three independent origins of ant slavery. During the evolution of eusociality, chemoreceptor genes multiplied due to the importance of chemical communication in insect societies. We investigated the evolutionary fate of these chemoreceptors and found that slave-making ant genomes harbored only half as many gustatory receptors as their hosts', potentially mirroring the outsourcing of foraging tasks to host workers. In addition, parasites had fewer odorant receptors and their loss shows striking patterns of convergence across independent origins of parasitism, in particular in orthologs often implicated in sociality like the 9-exon odorant receptors. These convergent losses represent a rare case of convergent molecular evolution at the level of individual genes. Thus, evolution can operate in a way that is both repeatable and reversible when independent ant lineages lose important social traits during the transition to a parasitic lifestyle.

Queens control caste allocation in the ant Cardiocondyla obscurior.

Social insect queens and workers can engage in conflict over reproductive allocation when they have different fitness optima. Here, we show that queens have control over queen-worker caste allocation in the ant Cardiocondyla obscurior, a species in which workers lack reproductive organs. We describe crystalline deposits that distinguish castes from the egg stage onwards, providing the first report of a discrete trait that can be used to identify ant caste throughout pre-imaginal development. The comparison of queen and worker-destined eggs and larvae revealed size and weight differences in late development, but no discernible differences in traits that may be used in social interactions, including hair morphology and cuticular odours. In line with a lack of caste-specific traits, adult workers treated developing queens and workers indiscriminately. Together with previous studies demonstrating queen control over sex allocation, these results show that queens control reproductive allocation in C. obscurior and suggest that the fitness interests of colony members are aligned to optimize resource allocation in this ant.

Evidence for a conserved queen-worker genetic toolkit across slave-making ants and their ant hosts.

The ecological success of social Hymenoptera (ants, bees, wasps) depends on the division of labour between the queen and workers. Each caste exhibits highly specialized morphology, behaviour, and life-history traits, such as lifespan and fecundity. Despite strong defences against alien intruders, insect societies are vulnerable to social parasites, such as workerless inquilines or slave-making ants. Here, we investigate whether gene expression varies in parallel ways between lifestyles (slave-making versus host ants) across five independent origins of ant slavery in the "Formicoxenus-group" of the ant tribe Crematogastrini. As caste differences are often less pronounced in slave-making ants than in nonparasitic ants, we also compare caste-specific gene expression patterns between lifestyles. We demonstrate a substantial overlap in expression differences between queens and workers across taxa, irrespective of lifestyle. Caste affects the transcriptomes much more profoundly than lifestyle, as indicated by 37 times more genes being linked to caste than to lifestyle and by multiple caste-associated modules of coexpressed genes with strong connectivity. However, several genes and one gene module are linked to slave-making across the independent origins of this parasitic lifestyle, pointing to some evolutionary convergence. Finally, we do not find evidence for an interaction between caste and lifestyle, indicating that caste differences in gene expression remain consistent even when species switch to a parasitic lifestyle. Our findings strongly support the existence of a core set of genes whose expression is linked to the queen and worker caste in this ant taxon, as proposed by the "genetic toolkit" hypothesis.

Substantial direct fitness gains of workers in a highly eusocial ant.

Hamilton's theory of inclusive fitness suggests that helpers in animal societies gain fitness indirectly by increasing the reproductive performance of a related beneficiary. Helpers in cooperatively breeding birds, mammals and primitively eusocial wasps may additionally obtain direct fitness through inheriting the nest or mating partner of the former reproductive. Here, we show that also workers of a highly eusocial ant may achieve considerable direct fitness by producing males in both queenless and queenright colonies. We investigated the reproductive success of workers of the ant Temnothorax crassispinus in nature and the laboratory by dissecting workers and determining the origin of males by microsatellite analysis. We show that workers are capable of activating their ovaries and successfully producing their sons independently of the presence of a queen. Genotypes revealed that at least one fifth of the males in natural queenright colonies were not offspring of the queen. Most worker-produced males could be assigned to workers that were unrelated to the queen, suggesting egg-laying by drifting workers.

Body size and sperm quality in queen- And worker-produced ant males.

Workers of many species of social Hymenoptera have functional ovaries and are capable of laying haploid, unfertilized eggs, at least in the absence of a queen. Except for honeybees, it remains largely unknown whether worker-produced males have the same quality as queen-produced males and whether workers benefit in direct fitness by producing their sons. Previous studies in the monogynous ant Temnothorax crassispinus revealed that a high proportion of males in natural and laboratory colonies are worker offspring. Here, we compare longevity, body size, sperm length and sperm viability between queen- and worker-produced males. We either split queenright colonies into queenright and queenless halves or removed the queen from a fraction of the queenright colonies and then examined the newly produced males. Male quality traits varied considerably among colonies but differed only slightly between queen- and worker-produced males. Worker-produced males outnumbered queen-produced males and also had a longer lifespan, but under certain rearing conditions sperm from queen-produced males had a higher viability.

Lipid content influences division of labour in a clonal ant.

The fat body, a major metabolic hub in insects, is involved in many functions, e.g. energy storage, nutrient sensing and immune response. In social insects, fat appears to play an additional role in division of labour between egg layers and workers, which specialize in non-reproductive tasks inside and outside their nest. For instance, reproductives are more resistant to starvation, and changes in fat content have been associated with the transition from inside to outside work or reproductive activities. However, most studies have been correlative and we still need to unravel the causal interrelationships between fat content and division of both reproductive and non-reproductive labour. Clonal ants, e.g. Platythyrea punctata, are ideal models for studying task partitioning without confounding variation in genotype and morphology. In this study, we examined the range of variation and flexibility of fat content throughout the lifespan of workers, the threshold of corpulence associated with foraging or reproduction and whether low fat content is a cause rather than a consequence of the transition to foraging. We found that lipid stores change with division of labour from corpulent to lean and, in reverted nurses, back to corpulent. In addition, our data show the presence of fat content thresholds that trigger the onset of foraging or egg-laying behaviour. Our study supports the view that mechanisms that regulate reproduction and foraging in solitary insects, in particular the nutritional status of individuals, have been co-opted to regulate division of labour in colonies of social insects.

Population and colony structure of an ant with territorial males, Cardiocondyla venustula.

BACKGROUND: Many species of social insects have large-scale mating and dispersal flights and their populations are therefore often relatively homogenous. In contrast, dispersal on the wing appears to be uncommon in most species of the ant genus Cardiocondyla, because its males are wingless and the winged queens mate in their natal nests before dispersing on foot. Here we examine the population structure of C. venustula from South Africa. This species is of particular interest for the analysis of life history evolution in Cardiocondyla, as it occupies a phylogenetic position between tropical species with multi-queen (polygynous) colonies and fighting males and a Palearctic clade with single-queen colonies and mutually peaceful males. Males of C. venustula exhibit an intermediate strategy between lethal fighting and complete tolerance - they mostly engage in non-lethal fights and defend small territories inside their natal nests. We investigated how this reproductive behavior influences colony and population structure by analyzing samples on two geographic scales in South Africa: a small 40 × 40m2 plot and a larger area with distances up to 5 km between sampling sites in Rietvlei Nature Reserve near Pretoria. RESULTS: Colonies were found to be facultatively polygynous and queens appear to mate only with a single male. The extraordinarily high inbreeding coefficient suggests regular sib-mating. Budding by workers and young queens is the predominant mode of colony-founding and leads to high population viscosity. In addition, some queens appear to found colonies independently or through adoption into foreign nests. CONCLUSION: While C. venustula resembles tropical Cardiocondyla in queen number and mating frequency, it differs by the absence of winged disperser males. Dispersal by solitary, mated queens on foot or by short flights and their adoption by alien colonies might promote gene flow between colonies and counteract prolonged inbreeding. The abundance of suitable habitat and the high density of nests facilitate the spread of this species by budding and together with the apparent resistance against inbreeding make it a highly successful pioneer species and invader of degraded and man-made habitats.

Interruption points in the wing gene regulatory network underlying wing polyphenism evolved independently in male and female morphs in Cardiocondyla ants.

Wing polyphenism in ants, which produces a winged female queen caste and a wingless female worker caste, evolved approximately 150 million years ago and has been key to the remarkable success of ants. Approximately 20 million years ago, the myrmicine ant genus Cardiocondyla evolved an additional wing polyphenism among males producing two male morphs: wingless males that fight to enhance mating success and winged males that disperse. Here we show that interruption of rudimentary wing-disc development in larvae of the ant Cardiocondyla obscurior occurs further downstream in the network in wingless males as compared with wingless female workers. This pattern is corroborated in C. kagutsuchi, a species from a different clade within the genus, indicating that late interruption of wing development in males is conserved across Cardiocondyla. Therefore, our results show that the novel male wing polyphenism was not developmentally constrained by the pre-existing female wing polyphenism and evolved through independent alteration of interruption points in the wing gene network. Furthermore, a comparison of adult morphological characters in C. obscurior reveals that developmental trajectories lead to similar morphological trait integration between winged and wingless females, but dramatically different integration between winged and wingless males. This suggests that the alternative sex-specific developmental routes to achieve winglessness in the genus Cardiocondyla may have evolved through different selection regimes acting on wingless males and females.

Evolution of Social Insect Polyphenism Facilitated by the Sex Differentiation Cascade.

The major transition to eusociality required the evolution of a switch to canalize development into either a reproductive or a helper, the nature of which is currently unknown. Following predictions from the 'theory of facilitated variation', we identify sex differentiation pathways as promising candidates because of their pre-adaptation to regulating development of complex phenotypes. We show that conserved core genes, including the juvenile hormone-sensitive master sex differentiation gene doublesex (dsx) and a krüppel homolog 2 (kr-h2) with putative regulatory function, exhibit both sex and morph-specific expression across life stages in the ant Cardiocondyla obscurior. We hypothesize that genes in the sex differentiation cascade evolved perception of alternative input signals for caste differentiation (i.e. environmental or genetic cues), and that their inherent switch-like and epistatic behavior facilitated signal transfer to downstream targets, thus allowing them to control differential development into morphological castes.

Queens stay, workers leave: caste-specific responses to fatal infections in an ant.

BACKGROUND: The intense interactions among closely related individuals in animal societies provide perfect conditions for the spread of pathogens. Social insects have therefore evolved counter-measures on the cellular, individual, and social level to reduce the infection risk. One striking example is altruistic self-removal, i.e., lethally infected workers leave the nest and die in isolation to prevent the spread of a contagious disease to their nestmates. Because reproductive queens and egg-laying workers behave less altruistically than non-laying workers, e.g., when it comes to colony defense, we wondered whether moribund egg-layers would show the same self-removal as non-reproductive workers. Furthermore, we investigated how a lethal infection affects reproduction and studied if queens and egg-laying workers intensify their reproductive efforts when their residual reproductive value decreases ("terminal investment"). RESULTS: We treated queens, egg-laying workers from queenless colonies, and non-laying workers from queenright colonies of the monogynous (single-queened) ant Temnothorax crassispinus either with a control solution or a solution containing spores of the entomopathogenic fungus Metarhizium brunneum. Lethally infected workers left the nest and died away from it, regardless of their reproductive status. In contrast, infected queens never left the nest and were removed by workers only after they had died. The reproductive investment of queens strongly decreased after the treatment with both, the control solution and the Metarhizium brunneum suspension. The egg laying rate in queenless colonies was initially reduced in infected colonies but not in control colonies. Egg number increased again with decreasing number of infected workers. CONCLUSIONS: Queens and workers of the ant Temnothorax crassispinus differ in their reaction to an infection risk and a reduced life expectancy. Workers isolate themselves to prevent contagion inside the colony, whereas queens stay in the nest. We did not find terminal investment; instead it appeared that egg-layers completely shut down egg production in response to the lethal infection. Workers in queenless colonies resumed reproduction only after all infected individuals had died, probably again to minimize the risk of infecting the offspring.

Chromosomal variation among populations of a fungus-farming ant: implications for karyotype evolution and potential restriction to gene flow.

BACKGROUND: Intraspecific variation in chromosome structure may cause genetic incompatibilities and thus provides the first step in the formation of species. In ants, chromosome number varies tremendously from 2n = 2 to 2n = 120, and several studies have revealed considerable variation in karyotype within species. However, most previous studies were limited to the description of chromosome number and morphology, and more detailed karyomorphometric analyses may reveal additional, substantial variation. Here, we studied karyotype length, genome size, and phylogeography of five populations of the fungus-farming ant Trachymyrmex holmgreni in order to detect potential barriers to gene flow. RESULTS: Chromosome number and morphology did not vary among the five populations, but karyotype length and genome size were significantly higher in the southernmost populations than in the northern populations of this ant. Individuals or colonies with different karyotype lengths were not observed. Karyotype length variation appears to result from variation in centromere length. CONCLUSION: T. holmgreni shows considerable variation in karyotype length and might provide a second example of centromere drive in ants, similar to what has previously been observed in Solenopsis fire ants. Whether this variation leads to genetic incompatibilities between the different populations remains to be studied.

Stress and early experience underlie dominance status and division of labour in a clonal insect.

Cooperation and division of labour are fundamental in the 'major transitions' in evolution. While the factors regulating cell differentiation in multi-cellular organisms are quite well understood, we are just beginning to unveil the mechanisms underlying individual specialization in cooperative groups of animals. Clonal ants allow the study of which factors influence task allocation without confounding variation in genotype and morphology. Here, we subjected larvae and freshly hatched workers of the clonal ant Platythyrea punctata to different rearing conditions and investigated how these manipulations affected division of labour among pairs of oppositely treated, same-aged clonemates. High rearing temperature, physical stress, injury and malnutrition increased the propensity of individuals to become subordinate foragers rather than dominant reproductives. This is reflected in changed gene regulation: early stages of division of labour were associated with different expression of genes involved in nutrient signalling pathways, metabolism and the phenotypic response to environmental stimuli. Many of these genes appear to be capable of responding to a broad range of stressors. They might link environmental stimuli to behavioural and phenotypic changes and could therefore be more broadly involved in caste differentiation in social insects. Our experiments also shed light on the causes of behavioural variation among genetically identical individuals.

Group demography affects ant colony performance and individual speed of queen and worker aging.

BACKGROUND: The performance and fitness of social societies mainly depends on the efficiency of interactions between reproductive individuals and helpers. Helpers need to react to the group's requirements and to adjust their tasks accordingly, while the reproductive individual has to adjust its reproductive rate. Social insects provide a good system to study the interrelations between individual and group characteristics. In general, sterile workers focus on brood care and foraging while the queen lays eggs. Reproductive division of labor is determined by caste and not interchangeable as, e.g., in social mammals or birds. Hence, changing social and environmental conditions require a flexible response by each caste. In the ant Cardiocondyla obscurior, worker task allocation is based on age polyethism, with young workers focusing on brood care and old workers on foraging. Here, we examine how group age demography affects colony performance and fitness in colonies consisting of only old or young workers and a single old or young queen. We hypothesized that both groups will be fully functional, but that the forced task shift affects the individuals' performance. Moreover, we expected reduced worker longevity in groups with only young workers due to precocious foraging but no effect on queen longevity depending on group composition. RESULTS: Neither the performance of queens nor that of workers declined strongly with time per se, but offspring number and weight were influenced by queen age and the interaction between queen and worker age. Individual residual life expectancy strongly depended on colony demography instead of physiological age. While worker age affected queen longevity only slightly, exposing old workers to the conditions of colony founding increased their life spans by up to 50% relative to workers that had emerged shortly before colony set-up. CONCLUSIONS: The social environment strongly affected the tempo of aging and senescence in C. obscurior, highlighting the plasticity of life expectancy in social insects. Furthermore, colonies obtained the highest reproductive output when consisting of same-aged queens and workers independent of their physiological age. However, workers appeared to be able to adjust their behavior to the colony's needs and not to suffer from age-dependent restrictions.

Royal Darwinian Demons: Enforced Changes in Reproductive Efforts Do Not Affect the Life Expectancy of Ant Queens.

One of the central tenets of life-history theory is that organisms cannot simultaneously maximize all fitness components. This results in the fundamental trade-off between reproduction and life span known from numerous animals, including humans. Social insects are a well-known exception to this rule: reproductive queens outlive nonreproductive workers. Here, we take a step forward and show that under identical social and environmental conditions the fecundity-longevity trade-off is absent also within the queen caste. A change in reproduction did not alter life expectancy, and even a strong enforced increase in reproductive efforts did not reduce residual life span. Generally, egg-laying rate and life span were positively correlated. Queens of perennial social insects thus seem to maximize at the same time two fitness parameters that are normally negatively correlated. Even though they are not immortal, they best approach a hypothetical "Darwinian demon" in the animal kingdom.

Life-history evolution in ants: the case of Cardiocondyla.

Ants are important components of most terrestrial habitats, and a better knowledge of the diversity of their life histories is essential to understand many aspects of ecosystem functioning. The myrmicine genus Cardiocondyla shows a wide range of colony structures, reproductive behaviours, queen and male lifespans, and habitat use. Reconstructing the evolutionary pathways of individual and social phenotypic traits suggests that the ancestral life history of Cardiocondyla was characterized by the presence of multiple, short-lived queens in small-sized colonies and a male polyphenism with winged dispersers and wingless fighters, which engage in lethal combat over female sexuals within their natal nests. Single queening, queen polyphenism, the loss of winged males and tolerance among wingless males appear to be derived traits that evolved with changes in nesting habits, colony size and the spread from tropical to seasonal environments. The aim of this review is to bring together the information on life-history evolution in Cardiocondyla and to highlight the suitability of this genus for functional genomic studies of adaptation, phenotypic plasticity, senescence, invasiveness and other key life-history traits of ants.

Major Hurdles for the Evolution of Sociality.

Why do most animals live solitarily, while complex social life is restricted to a few cooperatively breeding vertebrates and social insects? Here, we synthesize concepts and theories in social evolution and discuss its underlying ecological causes. Social evolution can be partitioned into (a) formation of stable social groups, (b) evolution of helping, and (c) transition to a new evolutionary level. Stable social groups rarely evolve due to competition over food and/or reproduction. Food competition is overcome in social insects with central-place foraging or bonanza-type food resources, whereas competition over reproduction commonly occurs because staying individuals are rarely sterile. Hence, the evolution of helping is shaped by direct and indirect fitness options and helping is only altruism if it reduces the helper's direct fitness. The helper's capability to gain direct fitness also creates within-colony conflict. This prevents transition to a new evolutionary level.

Phylogeography of social polymorphism in a boreo-montane ant.

BACKGROUND: The disjunct distribution of several Palearctic species has been widely shaped by the changes in climatic conditions during the Quaternary. The observed genetic differentiation or reproductive isolation between extant populations may be the outcome of their contemporary geographic separation or reproductive incompatibility due to differences in phenotypic traits which have evolved in isolated refugia. In the boreal ant Leptothorax acervorum, colonies from central and peripheral populations differ in social structure: colonies from Central and Northern Europe may contain several equally reproductive queens (facultative polygyny), while in colonies from peripheral populations in Spain only one the most dominant of several queens lays eggs (functional monogyny). By reconstructing the specie's evolutionary and demographic history in Southwestern Europe we examine whether variation in social organization is associated with restricted gene flow between the two social forms. RESULTS: We show that multi-queen colonies from all so far known inner Iberian populations of L. acervorum are functionally monogynous, whereas multi-queen colonies from all Pyrenean populations are polygynous, like those from other previously studied areas in Europe. Our analyses revealed complex spatial-genetic structure, but no association between spatial-genetic structure and social organization in SW-Europe. The population in the western Pyrenees diverged most strongly from other Iberian populations. Moreover, microsatellite data suggest the occurrence of recent bottlenecks in Pyrenean and inner Iberian populations. CONCLUSIONS: Our study shows a lack of reproductive isolation between the two social forms in SW-Europe. This in turn suggests that demographic and spatial patterns in genetic variation as well as the distribution of social phenotypes are better explained by co-variation with climatic, ecological, and historical factors. Moreover, we for the first time show the existence of substantial spatial-genetic structure in L. acervorum, suggesting the existence of multiple refugia in SW-Europe, including two extra-Mediterranean refugia in France. While gene flow among inner Iberian refugia may have been larger during the late glacial, extra-Mediterranean refugia in southern France may have contributed to the post-glacial recolonization of W-Europe.

Transcriptomic Signatures Mirror the Lack of the Fecundity/Longevity Trade-Off in Ant Queens.

Life-history theory predicts a trade-off between reproductive investment and self-maintenance. The negative association between fertility and longevity found throughout multicellular organisms supports this prediction. As an important exception, the reproductives of many eusocial insects (ants, bees, and termites) are simultaneously very long-lived and highly fertile. Here, we examine the proximate basis for this exceptional relationship by comparing whole-body transcriptomes of differently aged queens of the ant Cardiocondyla obscurior. We show that the sets of genes differentially expressed with age significantly overlap with age-related expression changes previously found in female Drosophila melanogaster. We identified several developmental processes, such as the generation of neurons, as common signatures of aging. More generally, however, gene expression in ant queens and flies changes with age mainly in opposite directions. In contrast to flies, reproduction-associated genes were upregulated and genes associated with metabolic processes and muscle contraction were downregulated in old relative to young ant queens. Furthermore, we searched for putative C. obscurior longevity candidates associated with the previously reported lifespan-prolonging effect of mating by comparing the transcriptomes of queens that differed in mating and reproductive status. We found 21 genes, including the putative aging candidate NLaz (an insect homolog of APOD), which were consistently more highly expressed in short-lived, unmated queens than in long-lived, mated queens. Our study provides clear evidence that the alternative regulation of conserved molecular pathways that mediate the interplay among mating, egg laying, and aging underlies the lack of the fecundity/longevity trade-off in ant queens.