Queen fecundity, worker entourage and cuticular chemistry in the ant Formica fusca.

Cooperative breeding entails conflicts over reproductive shares that may be settled in different ways. In ants, where several queens simultaneously reproduce in a colony, both queens and workers may influence the reproductive apportionment and offspring quality. Queens may vary in their intrinsic fecundity, which may influence the size of the worker entourage attending individual queens, and this may eventually dictate the reproductive output of a queen. We tested whether the reproductive success of queens is affected by the size of their worker entourage, their fecundity at the onset of the reproductive season, and whether the queen cuticular hydrocarbon profile carries information on fecundity. We show that in the ant Formica fusca both queen fecundity and egg hatching success increase with the size of their entourage, and that newly hatched larvae produced by initially highly fecund queens are smaller. Furthermore, higher relatedness among workers increased queen fecundity. Finally, the queens that received a large worker entourage differed in the cuticular chemistry from those that received a small worker entourage. Our results thus show that workers play a pivotal role in determining queen fitness, that high intracolony relatedness among workers enhances the overall reproductive output in the colony, and that queen fecundity is reflected in their cuticular hydrocarbon profile.

Relatedness modulates reproductive competition among queens in ant societies with multiple queens.

Reproductive sharing in animal groups with multiple breeders, insects and vertebrates alike, contains elements of both conflict and cooperation, and depends on both relatedness between co-breeders, as well as their internal and external conditions. We studied how queens of the ant Formica fusca adjust their reproductive efforts in response to experimental manipulations of the kin competition regime in their nest. Queens respond to the presence of competitors by increasing their egg laying efforts, but only if the competitors are highly fecund and distantly related. Such a mechanism is likely to decrease harmful competition among close relatives. We demonstrate that queens of Formica fusca fine-tune their cooperative breeding behaviors in response to kinship and fecundity of others in a remarkably precise and flexible manner.

The bacterial and fungal community composition in time and space in the nest mounds of the ant Formica exsecta (Hymenoptera: Formicidae).

In a subarctic climate, the seasonal shifts in temperature, precipitation, and plant cover drive the temporal changes in the microbial communities in the topsoil, forcing soil microbes to adapt or decline. Many organisms, such as mound-building ants, survive the cold winter owing to the favorable microclimate in their nest mounds. We have previously shown that the microbial communities in the nest of the ant Formica exsecta are significantly different from those in the surrounding bulk soil. In the current study, we identified taxa, which were consistently present in the nests over a study period of three years. Some taxa were also significantly enriched in the nest samples compared with spatially corresponding reference soils. We show that the bacterial communities in ant nests are temporally stable across years, whereas the fungal communities show greater variation. It seems that the activities of the ants contribute to unique biochemical processes in the secluded nest environment, and create opportunities for symbiotic interactions between the ants and the microbes. Over time, the microbial communities may come to diverge, due to drift and selection, especially given the long lifespan (up to 30 years) of the ant colonies.

The Scent of Ant Brood: Caste Differences in Surface Hydrocarbons of Formica exsecta Pupae.

Chemical communication is common across all organisms. Insects in particular use predominantly chemical stimuli in assessing their environment and recognizing their social counterparts. One of the chemical stimuli used for recognition in social insects, such as ants, is the suite of long-chain, cuticular hydrocarbons. In addition to providing waterproofing, these surface hydrocarbons serve as a signature mixture, which ants can perceive, and use to distinguish between strangers and colony mates, and to determine caste, sex, and reproductive status of another individual. They can be both environmentally and endogenously acquired. The surface chemistry of adult workers has been studied extensively in ants, yet the pupal stage has rarely been considered. Here we characterized the surface chemistry of pupae of Formica exsecta, and examine differences among sexes, castes (reproductive vs. worker), and types of sample (developing individual vs. cocoon envelope). We found quantitative and qualitative differences among both castes and types of sample, but male and female reproductives did not differ in their surface chemistry. We also found that the pupal surface chemistry was more complex than that of adult workers in this species. These results improve our understanding of the information on which ants base recognition, and highlights the diversity of surface chemistry in social insects across developmental stages.

An Ancient and Eroded Social Supergene Is Widespread across Formica Ants.

Supergenes, clusters of tightly linked genes, play a key role in the evolution of complex adaptive variation [1, 2]. Although supergenes have been identified in many species, we lack an understanding of their origin, evolution, and persistence [3]. Here, we uncover 20-40 Ma of evolutionary history of a supergene associated with polymorphic social organization in Formica ants [4]. We show that five Formica species exhibit homologous divergent haplotypes spanning 11 Mbp on chromosome 3. Despite the supergene's size, only 142 single nucleotide polymorphisms (SNPs) consistently distinguish alternative supergene haplotypes across all five species. These conserved trans-species SNPs are localized in a small number of disjunct clusters distributed across the supergene. This unexpected pattern of divergence indicates that the Formica supergene does not follow standard models of sex chromosome evolution, in which distinct evolutionary strata reflect an expanding region of suppressed recombination [5]. We propose an alternative "eroded strata model" in which clusters of conserved trans-species SNPs represent functionally important areas maintained by selection in the face of rare recombination between ancestral haplotypes. The comparison of whole-genome sequences across 10 additional Formica species reveals that the most conserved region of the supergene contains a transcription factor essential for motor neuron development in Drosophila [6]. The discovery that a very small portion of this large and ancient supergene harbors conserved trans-species SNPs linked to colony social organization suggests that the ancestral haplotypes have been eroded by recombination, with selection preserving differentiation at one or a few genes generating alternative social organization.

Comparison of Twelve Ant Species and Their Susceptibility to Fungal Infection.

Eusocial insects, such as ants, have access to complex disease defenses both at the individual, and at the colony level. However, different species may be exposed to different diseases, and/or deploy different methods of coping with disease. Here, we studied and compared survival after fungal exposure in 12 species of ants, all of which inhabit similar habitats. We exposed the ants to two entomopathogenic fungi (Beauveria bassiana and Metarhizium brunneum), and measured how exposure to these fungi influenced survival. We furthermore recorded hygienic behaviors, such as autogrooming, allogrooming and trophallaxis, during the days after exposure. We found strong differences in autogrooming behavior between the species, but none of the study species performed extensive allogrooming or trophallaxis under the experimental conditions. Furthermore, we discuss the possible importance of the metapleural gland, and how the secondary loss of this gland in the genus Camponotus could favor a stronger behavioral response against pathogen threats.

The first draft genomes of the ant Formica exsecta, and its Wolbachia endosymbiont reveal extensive gene transfer from endosymbiont to host.

BACKGROUND: Adapting to changes in the environment is the foundation of species survival, and is usually thought to be a gradual process. However, transposable elements (TEs), epigenetic modifications, and/or genetic material acquired from other organisms by means of horizontal gene transfer (HGTs), can also lead to novel adaptive traits. Social insects form dense societies, which attract and maintain extra- and intracellular accessory inhabitants, which may facilitate gene transfer between species. The wood ant Formica exsecta (Formicidae; Hymenoptera), is a common ant species throughout the Palearctic region. The species is a well-established model for studies of ecological characteristics and evolutionary conflict. RESULTS: In this study, we sequenced and assembled draft genomes for F. exsecta and its endosymbiont Wolbachia. The F. exsecta draft genome is 277.7 Mb long; we identify 13,767 protein coding genes, for which we provide gene ontology and protein domain annotations. This is also the first report of a Wolbachia genome from ants, and provides insights into the phylogenetic position of this endosymbiont. We also identified multiple horizontal gene transfer events (HGTs) from Wolbachia to F. exsecta. Some of these HGTs have also occurred in parallel in multiple other insect genomes, highlighting the extent of HGTs in eukaryotes. CONCLUSION: We present the first draft genome of ant F. exsecta, and its endosymbiont Wolbachia (wFex), and show considerable rates of gene transfer from the symbiont to the host. We expect that especially the F. exsecta genome will be valuable resource in further exploration of the molecular basis of the evolution of social organization.

The possible role of ant larvae in the defence against social parasites.

Temporary social parasite ant queens initiate new colonies by entering colonies of host species, where they begin laying eggs. As the resident queen can be killed during this process, host colonies may lose their entire future reproductive output. Selection thus favours the evolution of defence mechanisms, before and after parasite intrusion. Most studies on social parasites focus on host worker discrimination of parasite queens and their offspring. However, ant larvae can also influence brood composition by consuming eggs. This raises the question whether host larvae can aid in preventing colony takeover by consuming eggs laid by parasite queens. To test whether larvae could play a role in anti-parasite defence, we compared the rates at which larvae of a common host species, Formica fusca, consumed eggs laid by social parasite, non-parasite, nest-mate, or conspecific non-nest-mate queens. Larvae consumed social parasite eggs more than eggs laid by a heterospecific non-parasite queen, irrespective of the chemical distance between the egg cuticular profiles. Also, larvae consumed eggs laid by conspecific non-nest-mate queens more than those laid by nest-mate queens. Our study suggests that larvae may act as players in colony defence against social parasitism, and that social parasitism is a key factor shaping discrimination behaviour in ants.

Stress responses upon starvation and exposure to bacteria in the ant Formica exsecta.

Organisms are simultaneously exposed to multiple stresses, which requires regulation of the resistance to each stress. Starvation is one of the most severe stresses organisms encounter, yet nutritional state is also one of the most crucial conditions on which other stress resistances depend. Concomitantly, organisms often deploy lower immune defenses when deprived of resources. This indicates that the investment into starvation resistance and immune defenses is likely to be subject to trade-offs. Here, we investigated the impact of starvation and oral exposure to bacteria on survival and gene expression in the ant Formica exsecta. Of the three bacteria used in this study, only Serratia marcescens increased the mortality of the ants, whereas exposure to Escherichia coli and Pseudomonas entomophila alleviated the effects of starvation. Both exposure to bacteria and starvation induced changes in gene expression, but in different directions depending on the species of bacteria used, as well as on the nutritional state of the ants.

Genome organization and molecular characterization of the three Formica exsecta viruses-FeV1, FeV2 and FeV4.

We present the genome organization and molecular characterization of the three Formica exsecta viruses, along with ORF predictions, and functional annotation of genes. The Formica exsecta virus-4 (FeV4; GenBank ID: MF287670) is a newly discovered negative-sense single-stranded RNA virus representing the first identified member of order Mononegavirales in ants, whereas the Formica exsecta virus-1 (FeV1; GenBank ID: KF500001), and the Formica exsecta virus-2 (FeV2; GenBank ID: KF500002) are positive single-stranded RNA viruses initially identified (but not characterized) in our earlier study. The new virus FeV4 was found by re-analyzing data from a study published earlier. The Formica exsecta virus-4 genome is 9,866 bp in size, with an overall G + C content of 44.92%, and containing five predicted open reading frames (ORFs). Our bioinformatics analysis indicates that gaps are absent and the ORFs are complete, which based on our comparative genomics analysis suggests that the genomes are complete. Following the characterization, we validate virus infection for FeV1, FeV2 and FeV4 for the first time in field-collected worker ants. Some colonies were infected by multiple viruses, and the viruses were observed to infect all castes, and multiple life stages of workers and queens. Finally, highly similar viruses were expressed in adult workers and queens of six other Formica species: F. fusca, F. pressilabris, F. pratensis, F. aquilonia, F. truncorum and F. cinerea. This research indicates that viruses can be shared between ant species, but further studies on viral transmission are needed to understand viral infection pathways.

Changes in gene DNA methylation and expression networks accompany caste specialization and age-related physiological changes in a social insect.

Social insects provide systems for studying epigenetic regulation of phenotypes, particularly with respect to differentiation of reproductive and worker castes, which typically arise from a common genetic background. The role of gene expression in caste specialization has been extensively studied, but the role of DNA methylation remains controversial. Here, we perform well replicated, integrated analyses of DNA methylation and gene expression in brains of an ant (Formica exsecta) with distinct female castes using traditional approaches (tests of differential methylation) combined with a novel approach (analysis of co-expression and co-methylation networks). We found differences in expression and methylation profiles between workers and queens at different life stages, as well as some overlap between DNA methylation and expression at the functional level. Large portions of the transcriptome and methylome are organized into "modules" of genes, some significantly associated with phenotypic traits of castes and developmental stages. Several gene co-expression modules are preserved in co-methylation networks, consistent with possible regulation of caste-specific gene expression by DNA methylation. Surprisingly, brain co-expression modules were highly preserved when compared with a previous study that examined whole-body co-expression patterns in 16 ant species, suggesting that these modules are evolutionarily conserved and for specific functions in various tissues. Altogether, these results suggest that DNA methylation participates in regulation of caste specialization and age-related physiological changes in social insects.

Trends in bacterial and fungal communities in ant nests observed with Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and Next Generation Sequencing (NGS) techniques-validity and compatibility in ecological studies.

Microbes are ubiquitous and often occur in functionally and taxonomically complex communities. Unveiling these community dynamics is one of the main challenges of microbial research. Combining a robust, cost effective and widely used method such as Terminal Restriction Fragment Length Polymorphism (T-RFLP) with a Next Generation Sequencing (NGS) method (Illumina MiSeq), offers a solid alternative for comprehensive assessment of microbial communities. Here, these two methods were combined in a study of complex bacterial and fungal communities in the nest mounds of the ant Formica exsecta, with the aim to assess the degree to which these methods can be used to complement each other. The results show that these methodologies capture similar spatiotemporal variations, as well as corresponding functional and taxonomical detail, of the microbial communities in a challenging medium consisting of soil, decomposing plant litter and an insect inhabitant. Both methods are suitable for the analysis of complex environmental microbial communities, but when combined, they complement each other well and can provide even more robust results. T-RFLP can be trusted to show similar general community patterns as Illumina MiSeq and remains a good option if resources for NGS methods are lacking.

Weak population structure in the ant Formica fusca.

Dispersal is a fundamental trait of a species' biology. High dispersal results in weakly structured or even panmictic populations over large areas, whereas weak dispersal enables population differentiation and strong spatial structuring. We report on the genetic population structure in the polygyne ant Formica fusca and the relative contribution of the dispersing males and females to this. We sampled 12 localities across a ∼35 km2 study area in Finland and generated mitochondrial DNA (mtDNA) haplotype data and microsatellite data. First, we assessed queen dispersal by estimating population differentiation from mtDNA haplotype data. Second, we analysed nuclear DNA microsatellite data to determine overall population genetic substructure in the study area with principal components analysis, Bayesian clustering, hierarchical F statistics and testing for evidence of isolation-by-distance. Third, we directly compared genetic differentiation estimates from maternally inherited mtDNA and bi-parentally inherited DNA microsatellites to test for sex-bias in dispersal. Our results showed no significant spatial structure or isolation by distance in neither mtDNA nor DNA microsatellite data, suggesting high dispersal of both sexes across the study area. However, mitochondrial differentiation was weaker (Fst-mt = 0.0047) than nuclear differentiation (Fst-nuc = 0.027), which translates into a sixfold larger female migration rate compared to that of males. We conclude that the weak population substructure reflects high dispersal in both sexes, and it is consistent with F. fusca as a pioneer species exploiting unstable habitats in successional boreal forests.

Transcriptome sequencing reveals high isoform diversity in the ant Formica exsecta.

Transcriptome resources for social insects have the potential to provide new insight into polyphenism, i.e., how divergent phenotypes arise from the same genome. Here we present a transcriptome based on paired-end RNA sequencing data for the ant Formica exsecta (Formicidae, Hymenoptera). The RNA sequencing libraries were constructed from samples of several life stages of both sexes and female castes of queens and workers, in order to maximize representation of expressed genes. We first compare the performance of common assembly and scaffolding software (Trinity, Velvet-Oases, and SOAPdenovo-trans), in producing de novo assemblies. Second, we annotate the resulting expressed contigs to the currently published genomes of ants, and other insects, including the honeybee, to filter genes that have annotation evidence of being true genes. Our pipeline resulted in a final assembly of altogether 39,262 mRNA transcripts, with an average coverage of >300X, belonging to 17,496 unique genes with annotation in the related ant species. From these genes, 536 genes were unique to one caste or sex only, highlighting the importance of comprehensive sampling. Our final assembly also showed expression of several splice variants in 6,975 genes, and we show that accounting for splice variants affects the outcome of downstream analyses such as gene ontologies. Our transcriptome provides an outstanding resource for future genetic studies on F. exsecta and other ant species, and the presented transcriptome assembly can be adapted to any non-model species that has genomic resources available from a related taxon.

Survival and gene expression under different temperature and humidity regimes in ants.

Short term variation in environmental conditions requires individuals to adapt via changes in behavior and/or physiology. In particular variation in temperature and humidity are common, and the physiological adaptation to changes in temperature and humidity often involves alterations in gene expression, in particular that of heat-shock proteins. However, not only traits involved in the resistance to environmental stresses, but also other traits, such as immune defenses, may be influenced indirectly by changes in temperature and humidity. Here we investigated the response of the ant F. exsecta to two temperature regimes (20°C & 25°C), and two humidity regimes (50% & 75%), for two populations. We measured the survival and the expression of six metabolism- and immunity-related genes, and furthermore compared the expression levels in each condition with the pre-experiment expression levels. Both populations survived equally well at the two humidities, but one population showed higher mortality at 25°C than 20°, at 50% humidity. Similarly, the two populations showed striking differences in their gene expression before the experiment, and in their responses to the environmental conditions. Surprisingly, instead of converging to similar expression levels in the same environmental conditions, gene expression diverged further apart. This indicates different reaction norms to both temperature and humidity for the two populations. Furthermore, our results suggest that also immune defenses are indirectly affected by environmental conditions.

Starvation resistance and tissue-specific gene expression of stress-related genes in a naturally inbred ant population.

Starvation is one of the most common and severe stressors in nature. Not only does it lead to death if not alleviated, it also forces the starved individual to allocate resources only to the most essential processes. This creates energetic trade-offs which can lead to many secondary challenges for the individual. These energetic trade-offs could be exacerbated in inbred individuals, which have been suggested to have a less efficient metabolism. Here, we studied the effect of inbreeding on starvation resistance in a natural population of Formica exsecta ants, with a focus on survival and tissue-specific expression of stress, metabolism and immunity-related genes. Starvation led to large tissue-specific changes in gene expression, but inbreeding had little effect on most of the genes studied. Our results illustrate the importance of studying stress responses in different tissues instead of entire organisms.

Comparative transcriptomics reveals the conserved building blocks involved in parallel evolution of diverse phenotypic traits in ants.

BACKGROUND: Reproductive division of labor in eusocial insects is a striking example of a shared genetic background giving rise to alternative phenotypes, namely queen and worker castes. Queen and worker phenotypes play major roles in the evolution of eusocial insects. Their behavior, morphology and physiology underpin many ecologically relevant colony-level traits, which evolved in parallel in multiple species. RESULTS: Using queen and worker transcriptomic data from 16 ant species we tested the hypothesis that conserved sets of genes are involved in ant reproductive division of labor. We further hypothesized that such sets of genes should also be involved in the parallel evolution of other key traits. We applied weighted gene co-expression network analysis, which clusters co-expressed genes into modules, whose expression levels can be summarized by their 'eigengenes'. Eigengenes of most modules were correlated with phenotypic differentiation between queens and workers. Furthermore, eigengenes of some modules were correlated with repeated evolution of key phenotypes such as complete worker sterility, the number of queens per colony, and even invasiveness. Finally, connectivity and expression levels of genes within the co-expressed network were strongly associated with the strength of selection. Although caste-associated sets of genes evolve faster than non-caste-associated, we found no evidence for queen- or worker-associated co-expressed genes evolving faster than one another. CONCLUSIONS: These results identify conserved functionally important genomic units that likely serve as building blocks of phenotypic innovation, and allow the remarkable breadth of parallel evolution seen in ants, and possibly other eusocial insects as well.

Ancient Duplications Have Led to Functional Divergence of Vitellogenin-Like Genes Potentially Involved in Inflammation and Oxidative Stress in Honey Bees.

Protection against inflammation and oxidative stress is key in slowing down aging processes. The honey bee (Apis mellifera) shows flexible aging patterns linked to the social role of individual bees. One molecular factor associated with honey bee aging regulation is vitellogenin, a lipoglycophosphoprotein with anti-inflammatory and antioxidant properties. Recently, we identified three genes in Hymenopteran genomes arisen from ancient insect vitellogenin duplications, named vg-like-A, -B, and -C. The function of these vitellogenin homologs is unclear. We hypothesize that some of them might share gene- and protein-level similarities and a longevity-supporting role with vitellogenin. Here, we show how the structure and modifications of the vg-like genes and proteins have diverged from vitellogenin. Furthermore, all three vg-like genes show signs of positive selection, but the spatial location of the selected protein sites differ from those found in vitellogenin. We show that all these genes are expressed in both long-lived winter worker bees and in summer nurse bees with intermediate life expectancy, yet only vg-like-A shows elevated expression in winter bees as found in vitellogenin. Finally, we show that vg-like-A responds more strongly than vitellogenin to inflammatory and oxidative conditions in summer nurse bees, and that also vg-like-B responds to oxidative stress. We associate vg-like-A and, to lesser extent, vg-like-B to the antiaging roles of vitellogenin, but that vg-like-C probably is involved in some other function. Our analysis indicates that an ancient duplication event facilitated the adaptive and functional divergence of vitellogenin and its paralogs in the honey bee.

Natal Dispersal, Mating Patterns, and Inbreeding in the Ant Formica exsecta.

Sex-biased dispersal and multiple mating may prevent or alleviate inbreeding and its outcome, inbreeding depression, but studies demonstrating this in the wild are scarce. Perennial ant colonies offer a unique system to investigate the relationships between natal dispersal behavior and inbreeding. Due to the sedentary life of ant colonies and lifetime sperm storage by queens, measures of dispersal distance and mating strategy are easier to obtain than in most taxa. We used a suite of molecular markers to infer the natal colonies of queens and males in a wild population of the ant Formica exsecta. Dispersal was male biased, with median male dispersal distances (∼140 m) twice those of queens (∼60 m). The results also showed that the population was inbred and that inbreeding avoidance behaviors--sex-biased dispersal, queen dispersal distance, and multiple mating--were all ineffective in reducing homozygosity among colony workers. Queen homozygosity did not affect dispersal behavior, but more homozygous queens had lower colony-founding success and were more incestuously mated themselves, with potentially accumulating effects on colony fitness. We also provide independent evidence that dispersal is sex biased and show that our estimate corresponds well with dispersal estimates derived from population-genetic estimates.