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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., In this study, we conducted the first large‐scale molecular evolutionary analyses of vitellogenin‐coding genes (Vg) and Vg‐like genes (Vg‐like‐A, Vg‐like‐B, and Vg‐like‐C) of bumble bees, a primitively eusocial insects belonging to the genus Bombus. The overall dN/dS ratio of the conventional Vg was 1.311, and this gene was under strong positive selection in most (9/10) subgenera in Bombus, apart from the obligate parasitic subgenus Psithyrus (dN/dS = 0.713) which has no caste differentiation.

Social insects depend on communication to regulate social behaviour. This also applies to their larvae, which are commonly exposed to social interactions and can react to social stimulation. However, how social insect larvae sense their environment is not known. Using RNAseq, we characterized expression of sensory-related genes in larvae of the ant Formica fusca, upon exposure to two social environments: isolation without contact to other individuals, and stimulation via the presence of other developing individuals. Expression of key sensory-related genes was higher following social stimulation, and larvae expressed many of the same sensory-related genes as adult ants and larvae of other insects, including genes belonging to the major insect chemosensory gene families. Our study provides first insights into the molecular changes associated with social information perception in social insect larvae.

Social insect colonies are characterized by division of labour, and extensive morphological, physiological and behavioural differences between queens and workers. The storage protein vitellogenin (Vg) affects multiple aspects of social insect life histories, and has been suggested as a key player for caste differentiation and maintenance. Recently, three genes homologous to Vg have been described in the antFormica exsecta. Their role is currently unclear but their structural variation suggests variable functions. We examined the expression patterns of the conventional Vg and the three Vg-like genes using qRT-PCR in the common black antFormica fuscabetween queens and workers, between nurse and foragers workers, and across social contexts (queenless vs. queenright nests), and sampling time. As expected, we found a significant queen caste and nurse task-related increase for the conventional Vg, while Vg-like-C displayed a consistent forager-biased expression pattern. Task (forager vs. nurse) was the only factor that explained expression variation among workers in any of the studied genes. The removal of the queen did not affect expression, although the proportion of fertile nurses increased in queenless nests. The observed expression biases suggest that inFormica fusca,the ancestral duplication has led to alternative social functions for Vg-like genes across castes and tasks. To get a broader picture of the role of gene duplications in social evolution and the roles of Vg-like genes in caste differentiation and maintenance, how these genes achieve these roles at a molecular level need to be investigated further.

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. Machine-accessible metadata file describing the reported data (ISA-Tab format)

Understanding how a single genome creates and maintains distinct phenotypes is a central goal in evolutionary biology. Social insects are a striking example of co-opted genetic backgrounds giving rise to dramatically different phenotypes, such as queen and worker castes. A conserved set of molecular pathways, previously envisioned as a set of 'toolkit' genes, has been hypothesized to underlie queen and worker phenotypes in independently evolved social insect lineages. Here, we investigated the toolkit from a developmental point of view, using RNA-Seq to compare caste-biased gene expression patterns across three life stages (pupae, emerging adult and old adult) and two female castes (queens and workers) in the ant Formica exsecta. We found that the number of genes with caste-biased expression increases dramatically from pupal to old adult stages. This result suggests that phenotypic differences between queens and workers at the pupal stage may derive from a relatively low number of caste-biased genes, compared to higher number of genes required to maintain caste differences at the adult stage. Gene expression patterns were more similar among castes within developmental stages than within castes despite the extensive phenotypic differences between queens and workers. Caste-biased expression was highly variable among life stages at the level of single genes, but more consistent when gene functions (gene ontology terms) were investigated. Finally, we found that a large part of putative toolkit genes were caste-biased at least in some life stages in F. exsecta, and the caste-biases, but not their direction, were more often shared between F. exsecta and other ant species than between F. exsecta and bees. Our results indicate that gene expression should be examined across several developmental stages to fully reveal the genetic basis of polyphenisms.

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.

Much of the variation among insects is derived from the different ways that chitin has been moulded to form rigid structures, both internal and external. In this study, we identify a highly conserved expression pattern in an insect-only gene family, the Osiris genes, that is essential for development, but also plays a significant role in phenotypic plasticity and in immunity/toxicity responses. The majority of Osiris genes exist in a highly syntenic cluster, and the cluster itself appears to have arisen very early in the evolution of insects. We used developmental gene expression in the fruit fly, Drosophila melanogaster, the bumble bee, Bombus terrestris, the harvester ant, Pogonomyrmex barbatus, and the wood ant, Formica exsecta, to compare patterns of Osiris gene expression both during development and between alternate caste phenotypes in the polymorphic social insects. Developmental gene expression of Osiris genes is highly conserved across species and correlated with gene location and evolutionary history. The social insect castes are highly divergent in pupal Osiris gene expression. Sets of co-expressed genes that include Osiris genes are enriched in gene ontology terms related to chitin/cuticle and peptidase activity. Osiris genes are essential for cuticle formation in both embryos and pupae, and genes co-expressed with Osiris genes affect wing development. Additionally, Osiris genes and those co-expressed seem to play a conserved role in insect toxicology defences and digestion. Given their role in development, plasticity, and protection, we propose that the Osiris genes play a central role in insect adaptive evolution.

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.

Background Understanding how dissimilar phenotypes are produced from a similar set of genes is a crucial topic in evolutionary biology. In social insects, both workers and queens develop from diploid eggs, and the extent of phenotypic dimorphism between castes varies between species. Gene expression differences underlying the two female castes, queens and workers, are crucial for social insect evolution. However, it is unknown how much the caste biased proportion of the genome varies in quantity or quality and how this relates to the level of caste polymorphism. This project investigates the differences in gene expression between female castes in Wasmannia auropunctata using deep transcriptional sequencing to understand the role of coding, and potentially non-coding sequences on caste differentiation.