Sparse and stereotyped encoding implicates a core glomerulus for ant alarm behavior.

Ants communicate via large arrays of pheromones and possess expanded, highly complex olfactory systems, with antennal lobes in the brain comprising up to ∼500 glomeruli. This expansion implies that odors could activate hundreds of glomeruli, which would pose challenges for higher-order processing. To study this problem, we generated transgenic ants expressing the genetically encoded calcium indicator GCaMP in olfactory sensory neurons. Using two-photon imaging, we mapped complete glomerular responses to four ant alarm pheromones. Alarm pheromones robustly activated ≤6 glomeruli, and activity maps for the three pheromones inducing panic alarm in our study species converged on a single glomerulus. These results demonstrate that, rather than using broadly tuned combinatorial encoding, ants employ precise, narrowly tuned, and stereotyped representations of alarm pheromones. The identification of a central sensory hub glomerulus for alarm behavior suggests that a simple neural architecture is sufficient to translate pheromone perception into behavioral outputs.

orco Mutagenesis Causes Loss of Antennal Lobe Glomeruli and Impaired Social Behavior in Ants.

Life inside ant colonies is orchestrated with diverse pheromones, but it is not clear how ants perceive these social signals. It has been proposed that pheromone perception in ants evolved via expansions in the numbers of odorant receptors (ORs) and antennal lobe glomeruli. Here, we generate the first mutant lines in the clonal raider ant, Ooceraea biroi, by disrupting orco, a gene required for the function of all ORs. We find that orco mutants exhibit severe deficiencies in social behavior and fitness, suggesting they are unable to perceive pheromones. Surprisingly, unlike in Drosophila melanogaster, orco mutant ants also lack most of the ∼500 antennal lobe glomeruli found in wild-type ants. These results illustrate that ORs are essential for ant social organization and raise the possibility that, similar to mammals, receptor function is required for the development and/or maintenance of the highly complex olfactory processing areas in the ant brain. VIDEO ABSTRACT.

The emergence of a collective sensory response threshold in ant colonies.

SignificanceIn this study, we ask how ant colonies integrate information about the external environment with internal state parameters to produce adaptive, system-level responses. First, we show that colonies collectively evacuate the nest when the ground temperature becomes too warm. The threshold temperature for this response is a function of colony size, with larger colonies evacuating the nest at higher temperatures. The underlying dynamics can thus be interpreted as a decision-making process that takes both temperature (external environment) and colony size (internal state) into account. Using mathematical modeling, we show that these dynamics can emerge from a balance between local excitatory and global inhibitory forces acting between the ants. Our findings in ants parallel other complex biological systems like neural circuits.

The Alarm Pheromone and Alarm Response of the Clonal Raider Ant.

Ants communicate via an arsenal of different pheromones produced in a variety of exocrine glands. For example, ants release alarm pheromones in response to danger to alert their nestmates and to trigger behavioral alarm responses. Here we characterize the alarm pheromone and the alarm response of the clonal raider ant Ooceraea biroi, a species that is amenable to laboratory studies but for which no pheromones have been identified. During an alarm response, ants quickly become unsettled, leave their nest pile, and are sometimes initially attracted to the source of alarm, but ultimately move away from it. We find that the alarm pheromone is released from the head of the ant and identify the putative alarm pheromone as a blend of two compounds found in the head, 4-methyl-3-heptanone and 4-methyl-3-heptanol. These compounds are sufficient to induce alarm behavior alone and in combination. They elicit similar, though slightly different behavioral features of the alarm response, with 4-methyl-3-heptanone being immediately repulsive and 4-methyl-3-heptanol being initially attractive before causing ants to move away. The behavioral response to these compounds in combination is dose-dependent, with ants becoming unsettled and attracted to the source of alarm pheromone at low concentrations and repulsed at high concentrations. While 4-methyl-3-heptanone and 4-methyl-3-heptanol are known alarm pheromones in other more distantly related ant species, this is the first report of the chemical identity of a pheromone in O. biroi, and the first alarm pheromone identified in the genus Ooceraea. Identification of a pheromone that triggers a robust, consistent, and conserved behavior, like the alarm pheromone, provides an avenue to dissect the behavioral and neuronal mechanisms underpinning chemical communication.

Genome-Wide Analysis of the Odorant Receptor Gene Family in Solenopsis invicta, Ooceraea biroi, and Monomorium pharaonis (Hymenoptera: Formicidae).

Olfactory systems in eusocial insects play a vital role in the discrimination of various chemical cues. Odorant receptors (ORs) are critical for odorant detection, and this family has undergone extensive expansion in ants. In this study, we re-annotated the OR genes from the most destructive invasive ant species Solenopsis invicta and 2 other Formicidae species, Ooceraea biroi and Monomorium pharaonis, with the aim of systematically comparing and analyzing the evolution and the functions of the ORs in ant species, identifying 356, 298, and 306 potential functional ORs, respectively. The evolutionary analysis of these ORs showed that ants had undergone chromosomal rearrangements and that tandem duplication may be the main contributor to the expansion of the OR gene family in S. invicta. Our further analysis revealed that 9-exon ORs had biased chromosome localization patterns in all three ant species and that a 9-exon OR cluster (SinvOR4-8) in S. invicta was under strong positive selection (Ka/Ks = 1.32). Moreover, we identified 5 S. invicta OR genes, namely SinvOR89, SinvOR102, SinvOR352, SinvOR327, and SinvOR135, with high sequence similarity (>70%) to the orthologs in O. biroi and M. pharaonis. An RT-PCR analysis was used to verify the antennal expression levels of these ORs, which showed caste-specific expression. The subsequent analysis of the antennal expression profiles of the ORs of the S. invicta workers from the polygyne and monogyne social forms indicated that SinvOR35 and SinvOR252 were expressed at much higher levels in the monogyne workers than in the polygyne workers and that SinvOR21 was expressed at higher levels in polygyne workers. Our study has contributed to the identification and analysis of the OR gene family in ants and expanded the understanding of the evolution and functions of the ORs in Formicidae species.

Globally invasive populations of the clonal raider ant are derived from Bangladesh.

Identifying the native range of invasive species is useful to understand their evolution and natural history, as well as to develop new methods to control potentially harmful introduced organisms. The clonal raider ant, Ooceraea biroi, is an introduced species and an increasingly important social insect model organism, but its native range remains unknown. Here, we report a new series of O. biroi collections from Bangladesh, Singapore, Vietnam and China. We use a molecular phylogeny constructed with five gene fragments from 27 samples to determine that invasive lineages of O. biroi originated in Bangladesh. These lineages may have spread from Bangladesh via the historically significant Bay of Bengal shipping ports. Ooceraea biroi shares multiple features of its biology with other introduced ants, including parthenogenesis, retention of heterozygosity and presence of multiple egg-layers in the colony. Using laboratory rearing and microsatellite markers, we show that colonies collected from disturbed habitat in Bangladesh have these traits in common with colonies from the invasive range. Ancestral populations with sexual reproduction in primary habitats either remain to be discovered or have gone extinct. Our findings advance our understanding of the global spread of the clonal raider ant and highlight a suite of general traits that make certain ants prone to becoming invasive.

Pheromone representation in the ant antennal lobe changes with age.

While the neural basis of age-related decline has been extensively studied (1-3), less is known about changes in neural function during the pre-senescent stages of adulthood. Adult neural plasticity is likely a key factor in social insect age polyethism, where individuals perform different tasks as they age and divide labor in an age-dependent manner (4-9). Primarily, workers transition from nursing to foraging tasks (5, 10), become more aggressive, and more readily display alarm behavior (11-16) as they get older. While it is unknown how these behavioral dynamics are neurally regulated, they could partially be generated by altered salience of behaviorally relevant stimuli (4, 6, 7). Here, we investigated how odor coding in the antennal lobe (AL) changes with age in the context of alarm pheromone communication in the clonal raider ant (Ooceraea biroi) (17). Similar to other social insects (11, 12, 16), older ants responded more rapidly to alarm pheromones, the chemical signals for danger. Using whole-AL calcium imaging (18), we then mapped odor representations for five general odorants and two alarm pheromones in young and old ants. Alarm pheromones were represented sparsely at all ages. However, alarm pheromone responses within individual glomeruli changed with age, either increasing or decreasing. Only two glomeruli became sensitized to alarm pheromones with age, while at the same time becoming desensitized to general odorants. Our results suggest that the heightened response to alarm pheromones in older ants occurs via increased sensitivity in these two core glomeruli, illustrating the importance of sensory modulation in social insect division of labor and age-associated behavioral plasticity.

Pheromone representation in the ant antennal lobe changes with age.

While the neural basis of age-related decline has been extensively studied,1,2,3 less is known about changes in neural function during the pre-senescent stages of adulthood. Adult neural plasticity is likely a key factor in social insect age polyethism, where individuals perform different tasks as they age and divide labor in an age-dependent manner.4,5,6,7,8,9 Primarily, workers transition from nursing to foraging tasks,5,10 become more aggressive, and more readily display alarm behavior11,12,13,14,15,16 as they get older. While it is unknown how these behavioral dynamics are neurally regulated, they could partially be generated by altered salience of behaviorally relevant stimuli.4,6,7 Here, we investigated how odor coding in the antennal lobe (AL) changes with age in the context of alarm pheromone communication in the clonal raider ant (Ooceraea biroi).17 Similar to other social insects,11,12,16 older ants responded more rapidly to alarm pheromones, the chemical signals for danger. Using whole-AL calcium imaging,18 we then mapped odor representations for five general odorants and two alarm pheromones in young and old ants. Alarm pheromones were represented sparsely at all ages. However, alarm pheromone responses within individual glomeruli changed with age, either increasing or decreasing. Only two glomeruli became sensitized to alarm pheromones with age, while at the same time becoming desensitized to general odorants. Our results suggest that the heightened response to alarm pheromones in older ants occurs via increased sensitivity in these two core glomeruli, illustrating the importance of sensory modulation in social insect division of labor and age-associated behavioral plasticity.

A caste differentiation mutant elucidates the evolution of socially parasitic ants.

Most ant species have two distinct female castes-queens and workers-yet the developmental and genetic mechanisms that produce these alternative phenotypes remain poorly understood. Working with a clonal ant, we discovered a variant strain that expresses queen-like traits in individuals that would normally become workers. The variants show changes in morphology, behavior, and fitness that cause them to rely on workers in wild-type (WT) colonies for survival. Overall, they resemble the queens of many obligately parasitic ants that have evolutionarily lost the worker caste and live inside colonies of closely related hosts. The prevailing theory for the evolution of these workerless social parasites is that they evolve from reproductively isolated populations of facultative intermediates that acquire parasitic phenotypes in a stepwise fashion. However, empirical evidence for such facultative ancestors remains weak, and it is unclear how reproductive isolation could gradually arise in sympatry. In contrast, we isolated these variants just a few generations after they arose within their WT parent colony, implying that the complex phenotype reported here was induced in a single genetic step. This suggests that a single genetic module can decouple the coordinated mechanisms of caste development, allowing an obligately parasitic variant to arise directly from a free-living ancestor. Consistent with this hypothesis, the variants have lost one of the two alleles of a putative supergene that is heterozygous in WTs. These findings provide a plausible explanation for the evolution of ant social parasites and implicate new candidate molecular mechanisms for ant caste differentiation.

Comparative Development of the Ant Chemosensory System.

The insect antennal lobe (AL) contains the first synapses of the olfactory system, where olfactory sensory neurons (OSNs) contact second-order projection neurons (PNs). In Drosophila melanogaster, OSNs expressing specific receptor genes send stereotyped projections to one or two of about 50 morphologically defined glomeruli [1-3]. The mechanisms for this precise matching between OSNs and PNs have been studied extensively in D. melanogaster, where development is deterministic and independent of neural activity [4-6]. However, a number of insect lineages, most notably the ants, have receptor gene repertoires many times larger than D. melanogaster and exhibit more structurally complex antennal lobes [7-12]. Moreover, perturbation of OSN function via knockout of the odorant receptor (OR) co-receptor, Orco, results in drastic AL reductions in ants [13, 14], but not in Drosophila [15]. Here, we characterize AL development in the clonal raider ant, Ooceraea biroi. We find that, unlike in Drosophila, ORs and Orco are expressed before the onset of glomerulus formation, and Orco protein is trafficked to developing axon terminals, raising the possibility that ORs play a role during ant AL development. Additionally, ablating ant antennae at the onset of pupation results in AL defects that recapitulate the Orco mutant phenotype. Thus, early loss of functional OSN innervation reveals latent structure in the AL that develops independently of peripheral input, suggesting that the AL is initially pre-patterned and later refined in an OSN-dependent manner. This two-step process might increase developmental flexibility and thereby facilitate the rapid evolution and expansion of the ant chemosensory system.