Eusocial insects use cuticular hydrocarbons as components of pheromones that mediate social behaviours, such as caste and nestmate recognition, and regulation of reproduction. In ants such as Harpegnathos saltator, the queen produces a pheromone which suppresses the development of workers’ ovaries and if she is removed, workers can transition to a reproductive state known as gamergate. Here we functionally characterize a subfamily of odorant receptors (Ors) with a nine-exon gene structure that have undergone a massive expansion in ants and other eusocial insects. We deorphanize 22 representative members and find they can detect cuticular hydrocarbons from different ant castes, with one (HsOr263) that responds strongly to gamergate extract and a candidate queen pheromone component. After systematic testing with a diverse panel of hydrocarbons, we find that most Harpegnathos saltator Ors are narrowly tuned, suggesting that several receptors must contribute to detection and discrimination of different cuticular hydrocarbons important in mediating eusocial behaviour.

Ants are a highly successful family of insects that thrive in a variety of habitats across the world. Perhaps their best-known features are complex social organization and strict division of labor, separating reproduction from the day-to-day maintenance and care of the colony, as well as strict discrimination against foreign individuals. Since these social characteristics in ants are thought to be mediated by semiochemicals, a thorough analysis of these signals, and the receptors that detect them, is critical in revealing mechanisms that lead to stereotypic behaviors. To address these questions, we have defined and characterized the major chemoreceptor families in a pair of behaviorally and evolutionarily distinct ant species, Camponotus floridanus and Harpegnathos saltator. Through comprehensive re-annotation, we show that these ant species harbor some of the largest yet known repertoires of odorant receptors (Ors) among insects, as well as a more modest number of gustatory receptors (Grs) and variant ionotropic glutamate receptors (Irs).

Our phylogenetic analyses further demonstrate remarkably rapid gains and losses of ant Ors, while Grs and Irs have also experienced birth-and-death evolution to different degrees. In addition, comparisons of antennal transcriptomes between sexes identify many chemoreceptors that are differentially expressed between males and females and between species. We have also revealed an agonist for a worker-enriched OR from C. floridanus, representing the first case of a heterologously characterized ant tuning Or. Collectively, our analysis reveals a large number of ant chemoreceptors exhibiting patterns of differential expression and evolution consistent with sex/species-specific functions. These differentially expressed genes are likely associated with sex-based differences, as well as the radically different social lifestyles observed between C. floridanus and H. saltator, and thus are targets for further functional characterization. Our findings represent an important advance toward understanding the molecular basis of social interactions and the differential chemical ecologies among ant species.

Although eusociality evolved independently within several orders of insects, research into the molecular underpinnings of the transition towards social complexity has been confined primarily to Hymenoptera (for example, ants and bees). Here we sequence the genome and stage-specific transcriptomes of the dampwood termite Zootermopsis nevadensis (Blattodea) and compare them with similar data for eusocial Hymenoptera, to better identify commonalities and differences in achieving this significant transition. We show an expansion of genes related to male fertility, with upregulated gene expression in male reproductive individuals reflecting the profound differences in mating biology relative to the Hymenoptera. For several chemoreceptor families, we show divergent numbers of genes, which may correspond to the more claustral lifestyle of these termites. We also show similarities in the number and expression of genes related to caste determination mechanisms. Finally, patterns of DNA methylation and alternative splicing support a hypothesized epigenetic regulation of caste differentiation.

The termites evolved eusociality and complex societies before the ants, but have been studied much less. The recent publication of the first two termite genomes provides a unique comparative opportunity, particularly because the sequenced termites represent opposite ends of the social complexity spectrum. Zootermopsis nevadensis has simple colonies with totipotent workers that can develop into all castes (dispersing reproductives, nest-inheriting replacement reproductives, and soldiers). In contrast, the fungus-growing termite Macrotermes natalensis belongs to the higher termites and has very large and complex societies with morphologically distinct castes that are life-time sterile. Here we compare key characteristics of genomic architecture, focusing on genes involved in communication, immune defenses, mating biology and symbiosis that were likely important in termite social evolution. We discuss these in relation to what is known about these genes in the ants and outline hypothesis for further testing.

The sophisticated organization of eusocial insect societies is largely based on the regulation of complex behaviors by hydrocarbon pheromones present on the cuticle. We used electrophysiology to investigate the detection of cuticular hydrocarbons (CHCs) by female-specific olfactory sensilla basiconica on the antenna of Camponotus floridanus ants through the utilization of one of the largest family of odorant receptors characterized so far in insects. These sensilla, each of which contains multiple olfactory receptor neurons, are differentially sensitive to CHCs and allow them to be classified into three broad groups that collectively detect every hydrocarbon tested, including queen and worker-enriched CHCs. This broad-spectrum sensitivity is conserved in a related species, Camponotus laevigatus, allowing these ants to detect CHCs from both nestmates and non-nestmates. Behavioral assays demonstrate that these ants are excellent at discriminating CHCs detected by the antenna, including enantiomers of a candidate queen pheromone that regulates the reproductive division of labor.

Insects communicate with pheromones using sensitive antennal sensilla. Although trace amounts of pheromones can be detected by many insects, context-dependent increased costs of high sensitivity might lead to plasticity in sensillum responsiveness. We have functionally characterized basiconic sensilla of the ant Harpegnathos saltator for responses to general odors in comparison to cuticular hydrocarbons which can act as fertility signals emitted by the principal reproductive(s) of a colony to inhibit reproduction by worker colony members. When released from inhibition workers may become reproductive gamergates. We observed plasticity in olfactory sensitivity after transition to reproductive status with significant reductions in electrophysiological responses to several long-chained cuticular hydrocarbons. Although gamergates lived on average five times longer than non-reproductive workers, the shift to reproductive status rather than age differences matched the pattern of changes in olfactory sensitivity. Decreasing sensillum responsiveness to cuticular hydrocarbons could potentially reduce mutually inhibitory or self-inhibitory effects on gamergate reproduction.

Eusocial insects, mostly Hymenoptera, have evolved unique colonial lifestyles that rely on the perception of social context mainly through pheromones, and chemoreceptors are hypothesized to have played important adaptive roles in the evolution of sociality. However, because chemoreceptor repertoires have been characterized in few social insects and their solitary relatives, a comprehensive examination of this hypothesis has not been possible. Here, we annotate ∼3,000 odorant and gustatory receptors in recently sequenced Hymenoptera genomes and systematically compare >4,000 chemoreceptors from 13 hymenopterans, representing one solitary lineage (wasps) and three independently evolved eusocial lineages (ants and two bees). We observe a strong general tendency for chemoreceptors to expand in Hymenoptera, whereas the specifics of gene gains/losses are highly diverse between lineages. We also find more frequent positive selection on chemoreceptors in a facultative eusocial bee and in the common ancestor of ants compared with solitary wasps. Our results suggest that the frequent expansions of chemoreceptors have facilitated the transition to eusociality. Divergent expression patterns of odorant receptors between honeybee and ants further indicate differential roles of chemoreceptors in parallel trajectories of social evolution.

Gut-associated microbiota of ants include Rhizobiales bacteria with affiliation to the genus Bartonella. These bacteria may enable the ants to fix atmospheric nitrogen, but no genomes have been sequenced yet to test the hypothesis. Sequence reads from a member of the Rhizobiales were identified in the data collected in a genome project of the ant Harpegnathos saltator. We present an analysis of the closed 1.86 Mb genome of the ant-associated bacterium, for which we suggest the species name Candidatus Tokpelaia hoelldoblerii. A phylogenetic analysis reveals a relationship to Bartonella and Brucella, which infect mammals. Novel gene acquisitions include a gene for a putative extracellular protein of more than 6,000 amino acids secreted by the type I secretion system, which may be involved in attachment to the gut epithelium. No genes for nitrogen fixation could be identified, but genes for a multi-subunit urease protein complex are present in the genome. The urease genes are also present in Brucella, which has a fecal-oral transmission pathway, but not in Bartonella, which use blood-borne transmission pathways. We hypothesize that the gain and loss of the urease function is related to transmission strategies and lifestyle changes in the host-associated members of the Rhizobiales.

Epigenetic inheritance plays an important role in mediating alternative phenotype in highly social species. In order to gain a greater understanding of epigenetic effects in societies, we investigated DNA methylation in the termite Zootermopsis nevadensis. Termites are the most ancient social insects, and developmentally distinct from highly-studied, hymenopteran social insects. We used replicated bisulfite-sequencing to investigate patterns of DNA methylation in both sexes and among castes of Z. nevadensis. We discovered that Z. nevadensis displayed some of the highest levels of DNA methylation found in insects. We also found strong differences in methylation between castes. Methylated genes tended to be uniformly and highly expressed demonstrating the antiquity of associations between intragenic methylation and gene expression. Differentially methylated genes were more likely to be alternatively spliced than not differentially methylated genes, and possessed considerable enrichment for development-associated functions. We further observed strong overrepresentation of multiple transcription factor binding sites and miRNA profiles associated with differential methylation, providing new insights into the possible function of DNA methylation. Overall, our results show that DNA methylation is widespread and associated with caste differences in termites. More generally, this study provides insights into the function of DNA methylation and the success of insect societies.