Worker-like behavioral and physiological phenotype in queens with removed wings in a ponerine ant.

Many highly eusocial insects are characterized by morphological differences between females, which are especially pronounced in ants. How these differences associate with particular behavioral and physiological phenotypes can illuminate early ant evolution. In ants, the morphological queen usually possesses a larger thorax with wings compared with a wingless worker. While queens specialize in reproduction, workers help with non-reproductive tasks and show various levels of reproductive degeneration. Here, we investigated the level of behavioral and physiological plasticity within queens in the ant species Harpegnathos saltator, which shows limited queen-worker dimorphism. We found that the experimental removal of wings led to the expression of worker behaviors and physiology, by examining young queens with wings, known as alate gynes, and those whose wings have been experimentally removed or naturally shed, known as dealate gynes. Compared with alate gynes, dealate gynes displayed higher frequencies of behaviors that are naturally shown by workers during reproductive competition. In addition, dealate gynes exhibited a worker-like range of ovarian activity. Like workers, they lacked the putative sex pheromones on their cuticle characteristic of dispersing gynes. Because gynes activate a worker-like phenotype after wing removal, the essential difference between the queen and worker in this species is a dispersal polyphenism. If the queen plasticity observed in H. saltator reflects the early stages of ant eusociality, a dispersal dimorphism rather than a distinct reproductive dimorphism might represent an early step in ant evolution.

Genetic and genomic architecture of species-specific cuticular hydrocarbon variation in parasitoid wasps.

Cuticular hydrocarbons (CHCs) serve two fundamental functions in insects: protection against desiccation and chemical signalling. How the interaction of genes shapes CHC profiles, which are essential for insect survival, adaptation and reproductive success, is still poorly understood. Here we investigate the genetic and genomic basis of CHC biosynthesis and variation in parasitoid wasps of the genus Nasonia. We mapped 91 quantitative trait loci (QTL) explaining the variation of a total of 43 CHCs in F2 hybrid males from interspecific crosses between three Nasonia species. To identify candidate genes, we localized orthologues of CHC biosynthesis-related genes in the Nasonia genomes. We discovered multiple genomic regions where the location of QTL coincides with the location of CHC biosynthesis-related candidate genes. Most conspicuously, on a region close to the centromere of chromosome 1, multiple CHC biosynthesis-related candidate genes co-localize with several QTL explaining variation in methyl-branched alkanes. The genetic underpinnings behind this compound class are not well understood so far, despite their high potential for encoding chemical information as well as their prevalence in hymenopteran CHC profiles. Our study considerably extends our knowledge on the genetic architecture governing this important compound class, establishing a model for methyl-branched alkane genetics in the Hymenoptera in general.

Genetic basis of chemical communication in eusocial insects.

Social behavior is one of the most fascinating and complex behaviors in humans and animals. A fundamental process of social behavior is communication among individuals. It relies on the capability of the nervous system to sense, process, and interpret various signals (e.g., pheromones) and respond with appropriate decisions and actions. Eusocial insects, including ants, some bees, some wasps, and termites, display intriguing cooperative social behavior. Recent advances in genetic and genomic studies have revealed key genes that are involved in pheromone synthesis, chemosensory perception, and physiological and behavioral responses to varied pheromones. In this review, we highlight the genes and pathways that regulate queen pheromone-mediated social communication, discuss the evolutionary changes in genetic systems, and outline prospects of functional studies in sociobiology.

Reversible plasticity in brain size, behaviour and physiology characterizes caste transitions in a socially flexible ant (Harpegnathos saltator).

Phenotypic plasticity allows organisms to respond to changing environments throughout their lifetime, but these changes are rarely reversible. Exceptions occur in relatively long-lived vertebrate species that exhibit seasonal plasticity in brain size, although similar changes have not been identified in short-lived species, such as insects. Here, we investigate brain plasticity in reproductive workers of the ant Harpegnathos saltator. Unlike most ant species, workers of H. saltator are capable of sexual reproduction, and they compete in a dominance tournament to establish a group of reproductive workers, termed 'gamergates'. We demonstrated that, compared to foragers, gamergates exhibited a 19% reduction in brain volume in addition to significant differences in behaviour, ovarian status, venom production, cuticular hydrocarbon profile, and expression profiles of related genes. In experimentally manipulated gamergates, 6-8 weeks after being reverted back to non-reproductive status their phenotypes shifted to the forager phenotype across all traits we measured, including brain volume, a trait in which changes were previously shown to be irreversible in honeybees and Drosophila. Brain plasticity in H. saltator is therefore more similar to that found in some long-lived vertebrates that display reversible changes in brain volume throughout their lifetimes.

Antennal Olfactory Physiology and Behavior of Males of the Ponerine Ant Harpegnathos saltator.

In comparison to the large amount of study on the communication abilities of females in ant societies and their associated chemical ecology and sensory physiology, such study of male ants has been largely ignored; accordingly, little is known about their olfactory sensory capabilities. To address this, we explored peripheral odor sensitivities in male Harpegnathos saltator by measuring the electrophysiological activity of olfactory sensory neurons within antennal trichoid and coeloconic sensilla using an extracellular recording technique. In an initial trial of 46 compounds, sensilla trichodea responded strongly to two alarm pheromone components, while a limited number of non-hydrocarbon odorants elicited strong responses in sensilla coeloconica. Both sensillar types responded indifferently to 31 cuticular hydrocarbons (CHCs) and synthetic long-chain hydrocarbons (HCs) typically found on insect cuticle. In a search for sensilla responding to CHCs and other compounds, we found some sensilla that responded to synthetic HCs and CHCs from virgin queen postpharyngeal glands that are potentially used in close range mate recognition. Olfactometer bioassays of male ants to 15 non-HCs correlated sensory responsiveness to the respective behavioral responses. Comparing olfactory responses between H. saltator males and females, we found that sensilla coeloconica and basiconica of workers showed greater responses and broader selectivity to all compounds. The rarity of CHC-responding trichoid sensilla in Harpegnathos males suggests a more specific role in sexual communication compared to that in females, which use CHCs in a broader communication context.

Incomplete Co-cladogenesis Between Zootermopsis Termites and Their Associated Protists.

Coevolution is a major driver of speciation in many host-associated symbionts. In the termite-protist digestive symbiosis, the protists are vertically inherited by anal feeding among nest mates. Lower termites (all termite families except Termitidae) and their symbionts have broadly co-diversified over ~170 million yr. However, this inference is based mainly on the restricted distribution of certain protist genera to certain termite families. With the exception of one study, which demonstrated congruent phylogenies for the protist Pseudotrichonympha and its Rhinotermitidae hosts, coevolution in this symbiosis has not been investigated with molecular methods. Here we have characterized the hindgut symbiotic protists (Phylum Parabasalia) across the genus Zootermopsis (Archotermopsidae) using single cell isolation, molecular phylogenetics, and high-throughput amplicon sequencing. We report that the deepest divergence in the Zootermopsis phylogeny (Zootermopsis laticeps [Banks; Isoptera: Termopsidae]) corresponds with a divergence in three of the hindgut protist species. However, the crown Zootermopsis taxa (Zootermopsis angusticollis [Hagen; Isoptera: Termopsidae], Z. nevadensis nevadensis [Hagen; Isoptera: Termopsidae], and Z. nevadensis nuttingi [Haverty & Thorne; Isoptera: Termopsidae]) share the same protist species, with no evidence of co-speciation under our methods. We interpret this pattern as incomplete co-cladogenesis, though the possibility of symbiont exchange cannot be entirely ruled out. This is the first molecular evidence that identical communities of termite-associated protist species can inhabit multiple distinct host species.

Eusociality outcompetes egalitarian and solitary strategies when resources are limited and reproduction is costly.

Explaining the evolution and maintenance of animal groups remains a challenge. Surprisingly, fundamental ecological factors, such as resource variance and competition for limited resources, tend to be ignored in models of cooperation. We use a mathematical model previously developed to quantify the influence of different group sizes on resource use efficiency in egalitarian groups and extend its scope to groups with severe reproductive skew (eusocial groups). Accounting for resource limitation, the model allows calculation of optimal group sizes (highest resource use efficiency) and equilibrium population sizes in egalitarian as well as eusocial groups for a broad spectrum of environmental conditions (variance of resource supply). We show that, in contrast to egalitarian groups, eusocial groups may not only reduce variance in resource supply for survival, thus reducing the risk of starvation, they may also increase variance in resource supply for reproduction. The latter effect allows reproduction even in situations when resources are scarce. These two facets of eusocial groups, resource sharing for survival and resource pooling for reproduction, constitute two beneficial mechanisms of group formation. In a majority of environmental situations, these two benefits of eusociality increase resource use efficiency and lead to supersaturation-a strong increase in carrying capacity. The increase in resource use efficiency provides indirect benefits to group members even for low intra-group relatedness and may represent one potential explanation for the evolution and especially the maintenance of eusociality and cooperative breeding.

An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants.

Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.

The Neuropeptide Corazonin Controls Social Behavior and Caste Identity in Ants.

Social insects are emerging models to study how gene regulation affects behavior because their colonies comprise individuals with the same genomes but greatly different behavioral repertoires. To investigate the molecular mechanisms that activate distinct behaviors in different castes, we exploit a natural behavioral plasticity in Harpegnathos saltator, where adult workers can transition to a reproductive, queen-like state called gamergate. Analysis of brain transcriptomes during the transition reveals that corazonin, a neuropeptide homologous to the vertebrate gonadotropin-releasing hormone, is downregulated as workers become gamergates. Corazonin is also preferentially expressed in workers and/or foragers from other social insect species. Injection of corazonin in transitioning Harpegnathos individuals suppresses expression of vitellogenin in the brain and stimulates worker-like hunting behaviors, while inhibiting gamergate behaviors, such as dueling and egg deposition. We propose that corazonin is a central regulator of caste identity and behavior in social insects.

Specialized odorant receptors in social insects that detect cuticular hydrocarbon cues and candidate pheromones.

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.Cuticular hydrocarbons (CHC) mediate the interactions between individuals in eusocial insects, but the sensory receptors for CHCs are unclear. Here the authors show that in ants such as H. saltator, the 9-exon subfamily of odorant receptors (HsOrs) responds to CHCs, and ectopic expression of HsOrs in Drosophila neurons imparts responsiveness to CHCs.

The evolution of cuticular fertility signals in eusocial insects.

A reproductive division of labor is a definitive characteristic of eusocial insect societies and it requires a means through which colony members can assess the presence and productivity of reproductive individuals. Cuticular hydrocarbons are the primary means of doing so across eusocial hymenopterans. However, recent experimental work presents conflicting views on how these chemical signals function, are interpreted by workers, and evolve. These recent advances include demonstrations of hydrocarbons as evolutionarily conserved 'queen pheromones' and as species-divergent 'fertility signals' used by both queens and workers. In this review, we synthesize conflicting studies into an evolutionary framework suggesting a transition of reproductive communication from cue-like signature mixtures, to learned fertility signals, to innate queen pheromones that evolved across eusocial insects.

Functional characterization of odorant receptors in the ponerine ant, Harpegnathos saltator.

Animals use a variety of sensory modalities-including visual, acoustic, and chemical-to sense their environment and interact with both conspecifics and other species. Such communication is especially critical in eusocial insects such as honey bees and ants, where cooperation is critical for survival and reproductive success. Various classes of chemoreceptors have been hypothesized to play essential roles in the origin and evolution of eusociality in ants, through their functional roles in pheromone detection that characterizes reproductive status and colony membership. To better understand the molecular mechanisms by which chemoreceptors regulate social behaviors, we investigated the roles of a critical class of chemoreceptors, the odorant receptors (ORs), from the ponerine ant Harpegnathos saltator in detecting cuticular hydrocarbon pheromones. In light of the massive OR expansion in ants (∼400 genes per species), a representative survey based on phylogenetic and transcriptomic criteria was carried out across discrete odorant receptor subfamilies. Responses to several classes of semiochemicals are described, including cuticular hydrocarbons and mandibular gland components that act as H. saltator pheromones, and a range of more traditional general odorants. When viewed through the prism of caste-specific OR enrichment and distinctive OR subfamily odorant response profiles, our findings suggest that whereas individual HsOrs appear to be narrowly tuned, there is no apparent segregation of tuning responses within any discrete HsOr subfamily. Instead, the HsOR gene family as a whole responds to a broad array of compounds, including both cuticular hydrocarbons and general odorants that are likely to mediate distinct behaviors.

Chemosensory sensitivity reflects reproductive status in the ant Harpegnathos saltator.

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.

The caste- and sex-specific DNA methylome of the termite Zootermopsis nevadensis.

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.

A Simple Behavioral Model Predicts the Emergence of Complex Animal Hierarchies.

Social dominance hierarchies are widespread, but little is known about the mechanisms that produce nonlinear structures. In addition to despotic hierarchies, where a single individual dominates, shared hierarchies exist, where multiple individuals occupy a single rank. In vertebrates, these complex dominance relationships are thought to develop from interactions that require higher cognition, but similar cases of shared dominance have been found in social insects. Combining empirical observations with a modeling approach, we show that all three hierarchy structures-linear, despotic, and shared-can emerge from different combinations of simple interactions present in social insects. Our model shows that a linear hierarchy emerges when a typical winner-loser interaction (dominance biting) is present. A despotic hierarchy emerges when a policing interaction is added that results in the complete loss of dominance status for an attacked individual (physical policing). Finally, a shared hierarchy emerges with the addition of a winner-winner interaction that results in a positive outcome for both interactors (antennal dueling). Antennal dueling is an enigmatic ant behavior that has previously lacked a functional explanation. These results show how complex social traits can emerge from simple behaviors without requiring advanced cognition.

Epigenetic (re)programming of caste-specific behavior in the ant Camponotus floridanus.

Eusocial insects organize themselves into behavioral castes whose regulation has been proposed to involve epigenetic processes, including histone modification. In the carpenter ant Camponotus floridanus, morphologically distinct worker castes called minors and majors exhibit pronounced differences in foraging and scouting behaviors. We found that these behaviors are regulated by histone acetylation likely catalyzed by the conserved acetyltransferase CBP. Transcriptome and chromatin analysis in brains of scouting minors fed pharmacological inhibitors of CBP and histone deacetylases (HDACs) revealed hundreds of genes linked to hyperacetylated regions targeted by CBP. Majors rarely forage, but injection of a HDAC inhibitor or small interfering RNAs against the HDAC Rpd3 into young major brains induced and sustained foraging in a CBP-dependent manner. Our results suggest that behavioral plasticity in animals may be regulated in an epigenetic manner via histone modification.

Larval regulation of worker reproduction in the polydomous ant Novomessor cockerelli.

Although workers in many ant species are capable of producing their own offspring, they generally rear the queen's offspring instead. There are various mechanisms that regulate worker reproduction including inhibitory effects of ant brood. Colonies of the ant Novomessor cockerelli are monogynous and polydomous resulting in a large portion of nest workers being physically isolated from the queen for extended periods of time. Some workers experimentally isolated from the queen in laboratory nests lay viable eggs, which develop into males. We investigate the mechanism that regulates worker fertility in subnests separated from the queen by giving queenless worker groups queen-produced larvae, queen-produced eggs, or no brood. Our findings show that larvae delay the time to worker egg-laying, but eggs have no effect. Larval inhibition is a likely mechanism that contributes to the regulation of worker reproduction in N. cockerellli because larvae are easily transported to subnests that do not contain a queen.

Chemoreceptor Evolution in Hymenoptera and Its Implications for the Evolution of Eusociality.

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.

Cuticular Hydrocarbon Pheromones for Social Behavior and Their Coding in the Ant Antenna.

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.

A genomic comparison of two termites with different social complexity.

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.