Published: 4 January 2018

Genes and genomic processes underpinning the social lives of ants

EmelineFavreau, Carlos Martínez-Ruiz, Leandro Rodrigues Santiago, Robert L Hammond, YannickWurm

Current Opinion in Insect Science, 2018, Volume 25, Pages 83-90

Abstract

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The >15 000 ant species are all highly social and show great variation in colony organization, complexity and behavior. The mechanisms by which such sociality evolved, as well as those underpinning the elaboration of ant societies since their ∼140 million year old common ancestor, have long been pondered. Here, we review recent insights generated using various genomic approaches. This includes understanding the molecular mechanisms underlying caste differentiation and the diversity of social structures, studying the impact of eusociality on genomic evolutionary rates, and investigating gene expression changes associated with differences in lifespan between castes. Furthermore, functional studies involving RNAi and CRISPR have recently been successfully applied to ants, opening the door to exciting research that promises to revolutionize the understanding of the evolution and diversification of social living.

Introduction

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Ants show remarkable division of labor whereby males and queens reproduce while other females, the workers, build and defend the nest, rear brood and forage for food [1••]. Since their evolution from a solitary common ancestor ∼140 million years ago [2, 3••], the ants have radiated into >15 000 extant species. This ecologically dominant family exhibits an extraordinary diversity of social lifestyles, for example: obligatory fungus farming (leaf cutter ants), nomadic predatory lifestyles (army ants), slave-making and social parasitism [4]. Colonies vary greatly, with tens to several million individuals per colony, up to nine morphological worker castes within a species, as well as an array of mechanisms to resolve intra-colony conflicts [5].

While their behaviors, ecology, evolutionary contexts and morphologies have been extensively studied, it has only recently become possible to perform genome-scale analyses in ants [6], with the first seven ant genomes published in 2010 and 2011 [2]. Here, we review publications from the last few years on the genes and genomic processes underlying caste differentiation, the evolution of social organization, intraspecific variation in lifespan, symbiotic relationships, and chemical communication.

How the ants got their genomes

Publications of the first genome projects of humans and traditional laboratory organisms including Drosophila and Caenorhabditis alerted many biologists, including social insect researchers, to the power of genome knowledge. The development of post-Sanger sequencing technologies [7•] (see Glossary) made genome projects accessible to small teams interested in particular species. Within months of the 2009 ‘ant genomics’ meeting at Arizona State University [8], projects to sequence genomes of seven species were under way (Figure 1).

These first seven ant genomes [2] led to a broad range of studies on these species (some examples below), but also paved the way for the sequencing of genomes and ‘reference transcriptomes’ of additional species. As of writing, genomes of 23 ant species and transcriptomes of 26 additional species have been published (Table 1). Other studies have used reduced representation sequencing techniques such as restriction site associated DNA sequencing (RADseq, e.g. [6], see Glossary) or the sequencing of ultraconserved elements (UCEs, e.g. [9•], see Glossary) to identify genetic structures or to resolve phylogenies.

The coexistence of distinct female castes

Queen and worker ants are both female and do not differ genetically (with some exceptions [10]). Instead, caste (worker or queen) is irreversibly determined by environmental factors that trigger larvae to develop down either the worker or queen pathway [11]. Comparisons between queens and workers in multiple species demonstrate that many genes with strong caste-biased patterns of expression in one developmental stage show no such bias in other stages [12]. The differences in whole-body gene expression between castes are highest during the last larval stages [12, 13]. Interestingly, genes with high expression plasticity (i.e. with different expression levels in different castes) during larval development in Cardiocondyla obscurior also showed increased evolutionary rates [14•]. In adults, highly pleiotropic genes (i.e. under more phenotypic constraints, see Glossary) are consistently less likely to become caste-biased across a wide taxonomic range of ant species [15•]. Such findings support the idea that phenotypic plasticity along with genetic accommodation (see Glossary) are likely to play an essential role in caste differentiation [16, 17•].

Contrary to some expectations, genes expressed exclusively in a single caste are rare [18•, 19••]. Instead, transcriptome comparisons suggest that groups of genes have correlated expression profiles (i.e. ‘coexpression modules’) with consistent differential expression between adult queens and workers across 15 ant species [15•, 19••]. This suggests that caste-specific phenotypes can arise from modifications in gene regulatory networks (GRNs, see Glossary) (e.g. [18•]). However, other studies have found that taxonomically-restricted genes (see Glossary) are overrepresented among worker-biased genes [20]. These seemingly contradictory views are not mutually exclusive [21•] and the divergence between castes can involve differential usage of existing modules of genes as well as differential usage of fast-evolving genes with low connectivity [22, 23].

Because workers generally do not reproduce, selection acts only indirectly on worker phenotypes. All else being equal, selection is therefore expected to be less efficient on genes expressed in workers than on genes expressed in queens [24•, 25]. In agreement, expression profiling in Monomorium pharaonis reveals relaxed selection on worker-specific genes and faster evolution [26•]. In contrast, a study of 15 species [19••] shows no difference in evolutionary rates between castes, although this could be due to the use RNAseq from whole bodies. Indeed, allometric differences in tissues between castes are known to confound comparisons [27•]. However, although the M. pharaonis study used more specific samples (heads, gasters, larvae), these still include multiple tissues [26•]. Importantly, although the relaxed selection observed in M. pharaonis might be caused by indirect selection, an alternative is that relaxed selection facilitated the recruitment of genes to caste-biased roles [17•].

Many ant species have lost the queen caste but have both non-reproductive and reproductive workers (gamergates, see Glossary), comparisons between which can illuminate important molecular mechanisms. For example, this has allowed the identification of differentially expressed genes underpinning reproductive hierarchies [28•] and the role of specific proteins [29] in individual behavior.

Other ant species also display within-caste phenotypic plasticity not associated with reproduction, offering the chance to study several levels of variability within the same genome. Such studies confirm the important role of epigenetic cues in generating diversity [30]. For instance, experiments with juvenile hormone, known to regulate insect development, showed that the potential to produce super-soldiers is ancestral in many Pheidole species, and this facilitated the independent evolution of super-soldiers in two species [31]. Methylation patterns have been proposed as a mechanism to explain both between-caste and within-caste diversity [30], but the evidence so far is unclear [32], and suggests that methylation patterns may not be as important as previously thought in controlling ant polyphenism [33].

Genes and genetic architectures underlying social organization

Many species of ants include both single-queen and multiple-queen colonies. It was long thought that such variation is determined by environmental factors [34]. This idea was challenged in the late 1990s with the discovery that a genetic element, marked by alternate alleles of the odorant-binding protein (OBP) gene Gp-9, is responsible for different social organizations of the red fire ant Solenopsis invicta [35] (see Glossary for social polymorphism). Recent genome-wide analyses showed that the two Gp-9 alleles in fact mark alternate variants of a ‘supergene’ carried by a pair of ‘social chromosomes’. Recombination is suppressed between the two variants of the >19 Mb long supergene [36]. Intriguingly, the supergene variant responsible for multiple-queen colonies has much lower genetic diversity than the rest of the genome, consistent with the effect of a selective sweep or recent bottleneck [37•]. The two variants of the supergene carry alternate alleles for many of >400 protein-coding genes, including nine additional Gp-9-like OBPs. Six of these OBPs have differences in coding sequence between supergene variants [38•]. This work raised a question: is the situation in S. invicta an oddity? In short, no, because a pair of independently evolved social chromosomes, that bear almost no similarity to those in S. invicta, determine social form in the distantly related alpine silver ant, Formica selysi [39]. It is currently unknown whether such genetic architecture is required for the coexistence of social forms in other species, or for transitions between species from obligate single-queen colonies to obligate multiple-queen colonies.

Genetic changes underlying social organization can also be identified by comparing populations or species. For example, young Pogonomyrmex californicus queens predisposed to found nests either alone or in groups show fundamental differences in gene expression profiles of heads [40]. Similarly, in socially parasitic species, one can predict that the loss of the worker caste leads to a loss in the genome of potential ‘worker genes’. Instead, comparisons between the genomes of three social parasite species and genomes of non-parasitic host species found almost no differences in gene presence/absence [18•]. This is consistent with the idea that genes used in workers also have roles in other castes.

Investigating intraspecific variation in lifespan

The intraspecific lifespan of ants is extremely variable and in the same colony queens can live for years while workers typically live a few months and males a few weeks, despite sharing the same genome [41, 42]. Such disparity is fascinating yet challenges the application of classic theories of aging [43] and we have limited understanding of the mechanisms involved. A transcriptomic analysis of Lasius ants at three time points found that DNA repair genes are more highly expressed in both brain and leg tissues of queens than age-matched workers [44•], pointing at an association between greater lifespan and somatic repair. Because queens are long lived but mate shortly after emergence from the pupae, the long-term storage of viable sperm in the queen’s specialized organ (spermatheca) is another conundrum that is only now being investigated. Recent comparative transcriptomic analysis of spermatheca in virgin and mated Crematogaster queens identified a number of differentially expressed genes including some associated with antioxidizing function [45•].

Genes and mechanisms underlying the evolution of symbiotic relationships

The intricate symbiotic relationships (e.g. mutualism, parasitism) that many ants have with other taxa can now be scrutinized using genomics. For example, analysis of UCEs in 18 Pseudomyrmex species determined that after the ant-acacia plant mutualism evolved in one lineage, a second independent Pseudomyrmex lineage colonized plants that were involved in the first mutualism [9•]. A different study identified protein-coding genes with signatures of adaptive evolution in three ant-plant mutualistic Pseudomyrmex in comparison with four generalist species [46•]. In the attine ants, mutualistic fungus farming is associated with clear large-scale genomic changes including genomic rearrangements, loss of the arginine biosynthesis and positive selection on chitinase pathways, with complementary changes in the fungus [47].

Transcriptomics analyses are also beginning to illuminate the mechanisms by which parasites manipulate ant host behavior. For instance, hundreds of genes are differentially expressed in brains of Temnothorax workers infected with the cestode Anomotaenia compared to controls [48•]. Similarly, large-scale changes in gene expression occur in heads of Camponotus carpenter ants when their behavior is manipulated by the Ophiocordyceps fungus. Interestingly, many of the fungal genes up-regulated during manipulated biting behavior are absent from non-parasitic relatives [49].

Whole genome sequencing has also helped reveal new mutualisms. Sequencing of C. obscurior queens [50•] revealed a previously unknown intracellular bacterium symbiont, Westeberhardia. Analysis of its genome and titers in the host suggest this bacterium may assist cuticle synthesis. Finally, there is also an interest to identify ant microbiomes (e.g. in Formica [51]) and, motivated by pest control efforts, transcriptomic analyses have identified viruses of invasive ants (e.g. S. invicta [52], Linepithema humile [53], Nylanderia spp. [54, 55], Anoplolepis gracilipes [56]).

Roles of chemical communication genes in colony life

Ants have a fascinating communication system based on the production and perception of pheromones. In line with this, the first ant genome projects discovered particularly high number of genes involved in chemical communication, with for example more than 400 putative olfactory receptors in the fire ant [57]. Some chemical communication genes likely have highly conserved functions as indicated by conserved sequences and antennal expression of core sets of single-ortholog odorant binding proteins (OBPs) and chemosensory proteins (CSPs) [58, 59]. However, among the 330 ± 26 (mean ± standard deviation) odorant and the 80 ± 67 gustatory receptors identified in each of eight ant species, >30% were taxon-specific [60]. Frequent gene duplications and losses suggest that many chemical communication genes are involved in adaptive processes in response to changing environments or arms races. This is in line with analyses of a clade of CSPs with high turnover and extensive evidence of adaptive evolution [59]. There is even variation in olfactory genes within species: the multiple-queen form of S. invicta includes an OBP absent from single-queen colonies [38•].

CRISPR transgenics recently confirmed that the highly conserved orco gene, required for proper functioning of other olfactory receptors in Drosophila, plays a similar role in ants: ants with broken orco lacked antennal glomeruli and their colonies were dysfunctional, highlighting the importance of such chemical communication genes for social organization [61••, 62••].

Recent analyses have shown that non-olfactory communication is also important: trophallaxis fluid (see Glossary) is used to transfer compounds including proteins, microRNAs and juvenile hormone between colony members [63].

Concluding remarks

The continued improvements in low-cost sequencing technologies are making it possible to pursue analyses that would have been difficult to imagine only few years ago. Focusing on individual species, this includes in-depth tissue-specific profiling of RNAs or epigenetic marks [64•], as well as the sequencing of whole genomes of many individuals to enable population genomics approaches [65•]. Large whole genome sequencing initiatives including large number of related species, or one species for many genera — such as in the Global Ant Genomics Alliance [3••] — are already under way. Such datasets will deepen our understanding of the specific mechanisms and evolutionary forces involved in the evolution of complex phenotypes, symbiotic interactions and social behavior. Furthermore, analyses of such data will make it possible to pinpoint key candidate genes. Detailed analyses which involve artificially modifying the activity or sequence of genes have already begun, with several recent studies demonstrating the effectiveness of temporarily switching genes off using RNA interference [66, 67] — which can propagate by trophallaxis through the colony — of changing the reproductive status of an adult worker injecting specific neuropeptides [29] or of permanently modifying gene sequences using CRISPR transgenics [68]. Such approaches pave the way for many new breakthroughs bridging our ultimate and proximate understanding of social evolution.

Conflict of interest statement

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None declared.

Acknowledgements

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The authors thank Rodrigo Pracana, Karina Zile and Gino Brignoli for comments on earlier drafts of this manuscript. This work was supported by the Biotechnology and Biological Sciences Research Council [grant BB/K004204/1] and the Natural Environment Research Council [grant NE/L00626X/1 and NE/L002485/1] and Conselho Nacional de Desenvolvimento Científico e Tecnológico [grant 248391/2013-5].

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