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.

Phylogenomics of palearctic Formica species suggests a single origin of temporary parasitism and gives insights to the evolutionary pathway toward slave-making behaviour.

BACKGROUND: The ants of the Formica genus are classical model species in evolutionary biology. In particular, Darwin used Formica as model species to better understand the evolution of slave-making, a parasitic behaviour where workers of another species are stolen to exploit their workforce. In his book "On the Origin of Species" (1859), Darwin first hypothesized that slave-making behaviour in Formica evolved in incremental steps from a free-living ancestor. METHODS: The absence of a well-resolved phylogenetic tree of the genus prevent an assessment of whether relationships among Formica subgenera are compatible with this scenario. In this study, we resolve the relationships among the 4 palearctic Formica subgenera (Formica str. s., Coptoformica, Raptiformica and Serviformica) using a phylogenomic dataset of 945 genes for 16 species. RESULTS: We provide a reference tree resolving the relationships among the main Formica subgenera with high bootstrap supports. DISCUSSION: The branching order of our tree suggests that the free-living lifestyle is ancestral in the Formica genus and that parasitic colony founding could have evolved a single time, probably acting as a pre-adaptation to slave-making behaviour. CONCLUSION: This phylogenetic tree provides a solid backbone for future evolutionary studies in the Formica genus and slave-making behaviour.

Not only for egg yolk--functional and evolutionary insights from expression, selection, and structural analyses of Formica ant vitellogenins.

Vitellogenin (Vg), a storage protein, has been extensively studied for its egg-yolk precursor role, and it has been suggested to be fundamentally involved in caste differences in social insects. More than one Vg copy has been reported in several oviparous species, including ants. However, the number and function of different Vgs, their phylogenetic relatedness, and their role in reproductive queens and nonreproductive workers have been studied in few species only. We studied caste-biased expression of Vgs in seven Formica ant species. Only one copy of conventional Vg was identified in Formica species, and three Vg homologs, derived from ancient duplications, which represent yet undiscovered Vg-like genes. We show that each of these Vg-like genes is present in all studied Hymenoptera and some of them in other insects as well. We show that after each major duplication event, at least one of the Vg-like genes has experienced a period of positive selection. This, combined with the observation that the Vg-like genes have acquired or lost specific protein domains suggests sub- or neofunctionalization between Vg and the duplicated genes. In contrast to earlier studies, Vg was not consistently queen biased in its expression, and the caste bias of the three Vg-like genes was highly variable among species. Furthermore, a truncated and Hymenoptera-specific Vg-like gene, Vg-like-C, was consistently worker biased. Multispecies comparisons are essential for Vg expression studies, and for gene expression studies in general, as we show that expression and also, putative functions cannot be generalized even among closely related species.

Tools and applications for integrative analysis of DNA methylation in social insects.

DNA methylation is a common epigenetic signalling tool and an important biological process which is widely studied in a large array of species. The presence, level and function of DNA methylation vary greatly across species. In some insects, DNA methylation systems are minimal, and overall methylation rates tend to be low in all studied insect species. Low methylation levels probed by whole-genome bisulphite sequencing require great care with respect to data quality control and interpretation. Here, we introduce BWASP/R, a complete workflow that allows efficient, scalable and entirely reproducible analyses of raw DNA methylation sequencing data. Consistent application of quality control filters and analysis parameters provides fair comparisons among different studies and an integrated view of all experiments on one species. We describe the capabilities of the BWASP/R workflow by re-analysing several publicly available social insect WGBS data sets, comprising 70 samples and cumulatively 147 replicates from four different species. We show that the CpG methylome comprises only about 1.5% of CpG sites in the honeybee genome and that the cumulative data are consistent with genetic signatures of site accessibility and physiological control of methylation levels.

Molecular evolution of bumble bee vitellogenin and vitellogenin-like genes.

Vitellogenin (Vg), a storage protein, has been significantly studied for its egg yolk precursor role in oviparous animals. Recent studies found that vitellogenin and its Vg-like homologs were fundamentally involved in many other biological processes in social insects such as female caste differences and oxidative stress resilience. In this study, we conducted the first large-scale molecular evolutionary analyses of vitellogenin coding genes (Vg) and Vg-like genes of bumble bees, a primitively eusocial insect belonging to the genus Bombus. We obtained sequences for each of the four genes (Vg, Vg-like-A, Vg-like-B, and Vg-like-C) from 27 bumble bee genomes (nine were newly sequenced in this study), and sequences from the two closest clades of Bombus, including five Apis species and five Tetragonula species. Our molecular evolutionary analyses show that in bumble bee, the conventional Vg experienced strong positive selection, while the Vg-like genes showed overall relaxation of purifying selection. In Apis and Tetragonula; however, all four genes were found under purifying selection. Furthermore, the conventional Vg showed signs of strong positive selection in most subgenera in Bombus, apart from the obligate parasitic subgenus Psithyrus which has no caste differentiation. Together, these results indicate that the conventional Vg, a key pleiotropic gene in social insects, is the most rapidly evolving copy, potentially due to its multiple known social functions for both worker and queen castes. This study shows that concerted evolution and purifying selection shaped the evolution of the Vg gene family following their ancient gene duplication and may be the leading forces behind the evolution of new potential protein function enabling functional social pleiotropy.

Researchers collaborate with same-gendered colleagues more often than expected across the life sciences.

Evidence suggests that women in academia are hindered by conscious and unconscious biases, and often feel excluded from formal and informal opportunities for research collaboration. In addition to ensuring fairness and helping to redress gender imbalance in the academic workforce, increasing women's access to collaboration could help scientific progress by drawing on more of the available human capital. Here, we test whether researchers tend to collaborate with same-gendered colleagues, using more stringent methods and a larger dataset than in past work. Our results reaffirm that researchers co-publish with colleagues of the same gender more often than expected by chance, and show that this 'gender homophily' is slightly stronger today than it was 10 years ago. Contrary to our expectations, we found no evidence that homophily is driven mostly by senior academics, and no evidence that homophily is stronger in fields where women are in the minority. Interestingly, journals with a high impact factor for their discipline tended to have comparatively low homophily, as predicted if mixed-gender teams produce better research. We discuss some potential causes of gender homophily in science.

De novo transcriptome assembly and its annotation for the black ant Formica fusca at the larval stage.

Communication and nutrition are major drivers of fitness in ants. While communication is paramount to colony cohesion, nutrition is decisive in regulating reproductive division of labor among colony members. However, neither of these has been studied from a molecular perspective in developing individuals. Here, we report the availability of the first transcriptome resources for larvae of the ant Formica fusca, a species with excellent discrimination abilities and thus the potential to become a model system for studying molecular mechanisms of communication. We generated a comprehensive, high-coverage RNA-seq data set using Illumina RNA-seq technology by sequencing 24 individual 1st - 2nd instar larvae collected from four experimental groups (6 samples per treatment, 49 million mean reads per sample, coverage between 194-253×). A total of 24,765 unigenes were generated using a combination of genome-guided and de novo transcriptome assembly. A comprehensive assembly pipeline and annotation lists are provided. This dataset adds valuable transcriptomic resources for further study of developmental gene expression, transcriptional regulation and functional gene activity in ant larvae.

Evolutionary constraints shape caste-specific gene expression across 15 ant species.

Development of polymorphic phenotypes from similar genomes requires gene expression differences. However, little is known about how morph-specific gene expression patterns vary on a broad phylogenetic scale. We hypothesize that evolution of morph-specific gene expression, and consequently morph-specific phenotypic evolution, may be constrained by gene essentiality and the amount of pleiotropic constraints. Here, we use comparative transcriptomics of queen and worker morphs, that is, castes, from 15 ant species to understand the constraints of morph-biased gene expression. In particular, we investigate how measures of evolutionary constraints at the sequence level (expression level, connectivity, and number of gene ontology [GO] terms) correlate with morph-biased expression. Our results show that genes indeed vary in their potential to become morph-biased. The existence of genes that are constrained in becoming caste-biased potentially limits the evolutionary decoupling of the caste phenotypes, that is, it might result in "caste load" occasioning from antagonistic fitness variation, similarly to sexually antagonistic fitness variation between males and females. On the other hand, we suggest that genes under low constraints are released from antagonistic variation and thus more likely to be co-opted for morph specific use. Overall, our results suggest that the factors that affect sequence evolutionary rates and evolution of plastic expression may largely overlap.

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.

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.