Behavioral flexibility promotes collective consistency in a social insect.

Deciphering the mechanisms that integrate individuals and their behavior into a functional unit is crucial for our understanding of collective behaviors. We here present empirical evidence for the impressive strength of social processes in this integration. We investigated collective temperature homeostasis in bumblebee (Bombus terrestris) colonies and found that bees are less likely to engage in thermoregulatory fanning and do so with less time investment when confronted with heat stress in a group setting than when facing the same challenge alone and that this down-regulation of individual stimulus-response behavior resulted in a consistent proportion of workers in a group engaged in the task of fanning. Furthermore, the bees that comprised the subset of fanning individuals changed from trial to trial and participation in the task was predominately unpredictable based on previous response behavior. Our results challenge basic assumptions in the most commonly used class of models for task allocation and contrast numerous collective behavior studies that emphasize the importance of fixed inter-individual variation for the functioning of animal groups. We demonstrate that bumblebee colonies maintain within-group behavioral heterogeneity and a consistent collective response pattern based on social responsiveness and behavioral flexibility at the individual level.

Learning Distinct Chemical Labels of Nestmates in Ants.

Colony coherence is essential for eusocial insects because it supports the inclusive fitness of colony members. Ants quickly and reliably recognize who belongs to the colony (nestmates) and who is an outsider (non-nestmates) based on chemical recognition cues (cuticular hydrocarbons: CHCs) which as a whole constitute a chemical label. The process of nestmate recognition often is described as matching a neural template with the label. In this study, we tested the prevailing view that ants use commonalities in the colony odor that are present in the CHC profile of all individuals of a colony or whether different CHC profiles are learned independently. We created and manipulated sub-colonies by adding one or two different hydrocarbons that were not present in the original colony odor of our Camponotus floridanus colony and later tested workers of the sub-colonies in one-on-one encounters for aggressive responses. We found that workers adjust their nestmate recognition by learning novel, manipulated CHC profiles, but still accept workers with the previous CHC profile. Workers from a sub-colony with two additional components showed aggression against workers with only one of the two components added to their CHC profile. Thus, additional components as well as the lack of a component can alter a label as "non-nestmate." Our results suggest that ants have multiple-templates to recognize nestmates carrying distinct labels. This finding is in contrast to what previously has been proposed, i.e., a widening of the acceptance range of one template. We conclude that nestmate recognition in ants is a partitioned (multiple-template) process of the olfactory system that allows discrimination and categorization of nestmates by differences in their CHC profiles. Our findings have strong implications for our understanding of the underlying mechanisms of colony coherence and task allocation because they illustrate the importance of individual experience and task associated differences in the CHC profiles that can be instructive for the organization of insect societies.

Social interactions promote adaptive resource defense in ants.

Social insects vigorously defend their nests against con- and heterospecific competitors. Collective defense is also seen at highly profitable food sources. Aggressive responses are elicited or promoted by several means of communication, e.g. alarm pheromones and other chemical markings. In this study, we demonstrate that the social environment and interactions among colony members (nestmates) modulates the propensity to engage in aggressive behavior and therefore plays an important role in allocating workers to a defense task. We kept Formica rufa workers in groups or isolated for different time spans and then tested their aggressiveness in one-on-one encounters with other ants. In groups of more than 20 workers that are freely interacting, individuals are aggressive in one-on-one encounters with non-nestmates, whereas aggressiveness of isolated workers decreases with increasing isolation time. We conclude that ants foraging collectively and interacting frequently, e.g. along foraging trails and at profitable food sources, remain in a social context and thereby maintain high aggressiveness against potential competitors. Our results suggest that the nestmate recognition system can be utilized at remote sites for an adaptive and flexible tuning of the response against competitors.

Distributed representation of social odors indicates parallel processing in the antennal lobe of ants.

In colonies of eusocial Hymenoptera cooperation is organized through social odors, and particularly ants rely on a sophisticated odor communication system. Neuronal information about odors is represented in spatial activity patterns in the primary olfactory neuropile of the insect brain, the antennal lobe (AL), which is analog to the vertebrate olfactory bulb. The olfactory system is characterized by neuroanatomical compartmentalization, yet the functional significance of this organization is unclear. Using two-photon calcium imaging, we investigated the neuronal representation of multicomponent colony odors, which the ants assess to discriminate friends (nestmates) from foes (nonnestmates). In the carpenter ant Camponotus floridanus, colony odors elicited spatial activity patterns distributed across different AL compartments. Activity patterns in response to nestmate and nonnestmate colony odors were overlapping. This was expected since both consist of the same components at differing ratios. Colony odors change over time and the nervous system has to constantly adjust for this (template reformation). Measured activity patterns were variable, and variability was higher in response to repeated nestmate than to repeated nonnestmate colony odor stimulation. Variable activity patterns may indicate neuronal plasticity within the olfactory system, which is necessary for template reformation. Our results indicate that information about colony odors is processed in parallel in different neuroanatomical compartments, using the computational power of the whole AL network. Parallel processing might be advantageous, allowing reliable discrimination of highly complex social odors.

Friends and foes from an ant brain's point of view--neuronal correlates of colony odors in a social insect.

BACKGROUND: Successful cooperation depends on reliable identification of friends and foes. Social insects discriminate colony members (nestmates/friends) from foreign workers (non-nestmates/foes) by colony-specific, multi-component colony odors. Traditionally, complex processing in the brain has been regarded as crucial for colony recognition. Odor information is represented as spatial patterns of activity and processed in the primary olfactory neuropile, the antennal lobe (AL) of insects, which is analogous to the vertebrate olfactory bulb. Correlative evidence indicates that the spatial activity patterns reflect odor-quality, i.e., how an odor is perceived. For colony odors, alternatively, a sensory filter in the peripheral nervous system was suggested, causing specific anosmia to nestmate colony odors. Here, we investigate neuronal correlates of colony odors in the brain of a social insect to directly test whether they are anosmic to nestmate colony odors and whether spatial activity patterns in the AL can predict how odor qualities like "friend" and "foe" are attributed to colony odors. METHODOLOGY/PRINCIPAL FINDINGS: Using ant dummies that mimic natural conditions, we presented colony odors and investigated their neuronal representation in the ant Camponotus floridanus. Nestmate and non-nestmate colony odors elicited neuronal activity: In the periphery, we recorded sensory responses of olfactory receptor neurons (electroantennography), and in the brain, we measured colony odor specific spatial activity patterns in the AL (calcium imaging). Surprisingly, upon repeated stimulation with the same colony odor, spatial activity patterns were variable, and as variable as activity patterns elicited by different colony odors. CONCLUSIONS: Ants are not anosmic to nestmate colony odors. However, spatial activity patterns in the AL alone do not provide sufficient information for colony odor discrimination and this finding challenges the current notion of how odor quality is coded. Our result illustrates the enormous challenge for the nervous system to classify multi-component odors and indicates that other neuronal parameters, e.g., precise timing of neuronal activity, are likely necessary for attribution of odor quality to multi-component odors.

Representation of thermal information in the antennal lobe of leaf-cutting ants.

Insects are equipped with various types of antennal sensilla, which house thermosensitive neurons adapted to receive different parameters of the thermal environment for a variety of temperature-guided behaviors. In the leaf-cutting ant Atta vollenweideri, the physiology and the morphology of the thermosensitive sensillum coeloconicum (Sc) has been thoroughly investigated. However, the central projections of its receptor neurons are unknown. Here we selectively stained the three neurons found in single Sc and tracked their axons into the brain of Atta vollenweideri workers. Each of the three axons terminates in a single glomerulus of the antennal lobe (Sc-glomeruli). Two of the innervated glomeruli are adjacent to each other and are located laterally, while the third one is clearly separated and located medially in the antennal lobe. Using two-photon Ca(2+) imaging of antennal lobe projection neurons, we studied where in the antennal lobe thermal information is represented. In the 11 investigated antennal lobes, we found up to 10 different glomeruli in a single specimen responding to temperature stimulation. Both, warm- and cold-sensitive glomeruli could be identified. The thermosensitive glomeruli were mainly located in the medial part of the antennal lobe. Based on the general representation of thermal information in the antennal lobe and functional data on the Sc-glomeruli we conclude that temperature stimuli received by Sc are processed in the medial of the three target glomeruli. The present study reveals an important role of the antennal lobe in temperature processing and links a specific thermosensitive neuron to its central target glomerulus.

Detection of minute temperature transients by thermosensitive neurons in ants.

The antennae of leaf-cutting ants are equipped with sensilla coeloconica that house three receptor neurons, one of which is thermosensitive. Using convective heat (air at different temperatures), we investigated the physiological characteristics of the thermosensitive neuron associated with the sensilla coeloconica in the leaf-cutting ant Atta vollenweideri. The thermosensitive neuron very quickly responds to a drop in temperature with a brief phasic increase (50 ms) in spike rate and thus classifies as cold receptor (ambient temperature = 24°C). The short latency and the brief phasic response enable the thermosensitive neuron to follow temperature transients up to an estimated frequency of around 5 Hz. Although the neuron responds as a cold receptor, it is extremely sensitive to warm stimuli. A temperature increase of only 0.005°C already leads to a pronounced decrease in the resting activity of the thermosensitive neuron. Through sensory adaptation, the sensitivity to temperature transients is maintained over a wide range of ambient temperatures (18-30°C). We conclude that the thermosensitive neuron of the sensilla coeloconica is adapted to detect minute temperature transients, providing the ants with thermal information of their microenvironment, which they may use for orientation.

Dummies versus air puffs: efficient stimulus delivery for low-volatile odors.

Aiming to unravel how animals perceive odors, a variety of neurophysiological techniques are used today. For olfactory stimulation, odors are commonly incorporated into a constant airstream that carries odor molecules to the receptor organ (air-delivered stimulation). Such odor delivery works well for odors of high volatility (naturally effective over long distances) but less or not at all for low-volatile odors (usually only received at short range). We developed a new odor stimulation technique especially suited for low-volatile odors and compared it with conventional air-delivered stimulation using 2 neurophysiological approaches. Odor-loaded dummies were moved into close vicinity of the receptor organs on the antenna of the Florida carpenter ant Camponotus floridanus (dummy-delivered stimulation). Neuronal activity was monitored either at receptor neuron level using electroantennography or in the first olfactory neuropile, the antennal lobes, using calcium imaging. We tested 3 odors of different volatility: C. floridanus' highly volatile alarm pheromone undecane, its low-volatile trail pheromone nerolic acid, and an even less volatile, behaviorally active C23 alkene, cis-9-tricosene. For low-volatile odors, dummy-delivered stimulation was particularly efficient. We conclude that dummy-delivered stimulation is advantageous compared to the commonly used air-delivered stimulation when studying an animal's detection and processing of low-volatile odors.

Phenotypic plasticity in number of glomeruli and sensory innervation of the antennal lobe in leaf-cutting ant workers (A. vollenweideri).

In the leaf-cutting ant Atta vollenweideri, the worker caste exhibits a pronounced size-polymorphism, and division of labor is dependent on worker size (alloethism). Behavior is largely guided by olfaction, and the olfactory system is highly developed. In a recent study, two different phenotypes of the antennal lobe of Atta vollenweideri workers were found: MG- and RG-phenotype (with/without a macroglomerulus). Here we ask whether the glomerular numbers are related to worker size. We found that the antennal lobes of small workers contain approximately 390 glomeruli (low-number; LN-phenotype), and in large workers we found a substantially higher number of approximately 440 glomeruli (high-number; HN-phenotype). All LN-phenotype workers and some small HN-phenotype workers do not possess an MG (LN-RG-phenotype and HN-RG-phenotype), and the remaining majority of HN-phenotype workers do possess an MG (HN-MG-phenotype). Using mass-staining of antennal olfactory receptor neurons we found that the sensory tracts divide the antennal lobe into six clusters of glomeruli (T1-T6). In LN-phenotype workers, approximately 50 glomeruli are missing in the T4-cluster. Selective staining of single sensilla and their associated receptor neurons revealed that T4-glomeruli are innervated by receptor neurons from the main type of olfactory sensilla, the Sensilla trichodea curvata. The other type of olfactory sensilla (Sensilla basiconica) exclusively innervates T6-glomeruli. Quantitative analyses of differently sized workers revealed that the volume of T6 glomeruli scales with the power of 2.54 to the number of Sensilla basiconica. The results suggest that developmental plasticity leading to antennal-lobe phenotypes promotes differences in olfactory-guided behavior and may underlie task specialization within ant colonies.

Preimaginal and adult experience modulates the thermal response behavior of ants.

Colonies of social insects display an amazing degree of flexibility in dealing with long-term and short-term perturbations in their environment. The key organizational element of insect societies is division of labor. Recent literature suggests that interindividual variability in response thresholds plays an important role in the emergence of division of labor among workers (reviewed in [1, 2]). Genetic variation can only partly explain the variability among workers. Here we document the effects of both preimaginal and adult thermal experience on the behavioral differentiation of Camponotus rufipes ant workers. We show that preimaginal temperature (22 degrees C or 32 degrees C during pupal stage) affects temperature-response thresholds and temperature preferences of adult brood-tending workers. We further show that brood-carrying experience gathered as adult during several repeated temperature increases modifies thermal behavior. Experienced workers showed a faster transition from first sensing the temperature stimulus to responding with brood translocation. Developmental plasticity of workers provides a colony with flexibility in dealing with thermal variations and constitutes an important mechanism underlying interindividual variability. Adult thermal experience further fine tunes the behavioral response thresholds and reinforces behavioral differentiation among workers.

The antennal lobes of fungus-growing ants (Attini): neuroanatomical traits and evolutionary trends.

Ants of the tribe Attini are characterized by their obligate cultivation of symbiotic fungi. In addition to the complex chemical communication system of ants in general, substrate selection and fungus cultivation pose high demands on the olfactory system of the Attini. Indeed, behavioral studies have shown a rich diversity of olfactory-guided behaviors and tremendous odor sensitivity has been demonstrated. To allow fine-tuned behavioral responses, adaptations within the olfactory system of the Attini are expected. We compared the number, volumes and position of the glomeruli (functional units) of the antennal lobe of 25 different species from all three major Attini groups (lower, higher and leaf-cutting Attini). The antennal lobes of all investigated Attini comprise a high number of glomeruli (>257). The highest number (630) was found in Apterostigma cf. mayri. This species is at a basal position within the Attini phylogeny, and we suggest that a high number of glomeruli might have been advantageous in the evolution of the advanced olfactory systems of the Attini. In the leaf-cutting Attini, an extremely large glomerulus (macroglomerulus) near the antennal nerve entrance was recently described in two species. Preliminary results show that this macroglomerulus is involved in processing of trail-pheromone information. In our comparative study, we find this macroglomerulus in all investigated leaf-cutting Attini, but in none of the lower and higher Attini species. It is found only in large workers, and for all investigated species it is located close to the entrance of the antennal nerve. Our results indicate that the presence of a macroglomerulus in large workers of leaf-cutting Attini is a derived over-expression of a trait in the polymorphic leaf-cutting species. It presumably represents an olfactory adaptation to elaborate foraging and mass recruitment systems, and adds to the complexity of division of labor and social organization known for this group.

Nestmate recognition in ants is possible without tactile interaction.

Ants of the genus Camponotus are able to discriminate recognition cues of colony members (nestmates) from recognition cues of workers of a different colony (non-nestmates) from a distance of 1 cm. Free moving, individual Camponotus floridanus workers encountered differently treated dummies on a T-bar and their behavior was recorded. Aggressive behavior was scored as mandibular threat towards dummies. Dummies were treated with hexane extracts of postpharyngeal glands (PPGs) from nestmates or non-nestmates which contain long-chain hydrocarbons in ratios comparable to what is found on the cuticle. The cuticular hydrocarbon profile bears cues which are essential for nestmate recognition. Although workers were prevented from antennating the dummies, they showed significantly less aggressive behavior towards dummies treated with nestmate PPG extracts than towards dummies treated with non-nestmate PPG extracts. In an additional experiment, we show that cis-9-tricosene, an alkene naturally not found in C. floridanus' cuticular profile, is behaviorally active and can interfere with nestmate recognition when presented together with a nestmate PPG extract. Our study demonstrates for the first time that the complex multi-component recognition cues can be perceived and discriminated by ants at close range. We conclude that contact chemosensilla are not crucial for nestmate recognition since tactile interaction is not necessary.

Organization of the olfactory pathway and odor processing in the antennal lobe of the ant Camponotus floridanus.

Ants rely heavily on olfaction for communication and orientation. Here we provide the first detailed structure-function analyses within an ant's central olfactory system asking whether in the carpenter ant, Camponotus floridanus, the olfactory pathway exhibits adaptations to processing many pheromonal and general odors. Using fluorescent tracing, confocal microscopy, and 3D-analyses we demonstrate that the antennal lobe (AL) contains up to approximately 460 olfactory glomeruli organized in seven distinct clusters innervated via seven antennal sensory tracts. The AL is divided into two hemispheres regarding innervation of glomeruli by either projection neurons (PNs) with axons leaving via the medial (m) or lateral (l) antennocerebral tract (ACT). M- and l-ACT PNs differ in their target areas in the mushroom-body calyx and lateral horn. Three additional ACTs project to the lateral protocerebrum only. We analyzed odor processing in AL glomeruli by retrograde loading of PNs with Fura-2 dextran and fluorimetric calcium imaging. Odor responses were reproducible and comparable across individuals. Calcium responses to pheromonal and nonpheromonal odors were very sensitive (10(-11) dilution) and patterns were partly overlapping, indicating that processing of both odor classes is not spatially segregated within the AL. Response patterns to the main trail-pheromone component nerolic acid remained stable over a wide range of intensities (7-8 log units), while response durations increased indicating that odor quality is maintained by a stable pattern and intensity is mainly encoded in response durations. The structure-function analyses contribute new insights into important aspects of odor processing in a highly advanced insect olfactory system.

Reformation process of the neuronal template for nestmate-recognition cues in the carpenter ant Camponotus floridanus.

Ants use cuticular hydrocarbons (CHC-profiles) as multicomponent recognition cues to identify colony members (nestmates). Recognition cues (label) are thought to be perceived during ant-ant encounters and compared to a neuronal template that represents the colony label. Over time, the CHC-profile may change, and the template is adjusted accordingly. A phenotype mismatch between label and template, as happens with CHC-profiles of foreign workers (non-nestmates), frequently leads to aggressive behavior. We investigated the template reformation in workers of the carpenter ant Camponotus floridanus by masking their antennae with postpharyngeal gland (PPG) extracts from nestmates or non-nestmates. The behavioral response of manipulated workers encountering unmanipulated workers was measured independently after 2 and after 15 h. After 2 h of incubation, workers treated with either of the two PPG-extracts showed low aggression towards nestmates and high aggression towards non-nestmates. In contrast, after 15 h of incubation, workers treated with non-nestmate PPG-extract showed low aggression towards both nestmates and non-nestmates. The slow (>2 h) adjustment of the template indicates a reformation localized in the central nervous system rather than in chemosensory neurons. In addition, our data show that template adjustment to a new CHC-profile does not impair the assessment of the old CHC-profile as nestmate label.

Dual olfactory pathway in the honeybee, Apis mellifera.

The antennal lobes (ALs) are the primary olfactory centers in the insect brain. In the AL of the honeybee, olfactory glomeruli receive input via four antennal sensory tracts (T1-4). Axons of projection neurons (PNs) leave the AL via several antenno-cerebral tracts (ACTs). To assign the input-output connectivity of all glomeruli, we investigated the spatial relationship of the antennal tracts and two prominent AL output tracts (medial and lateral ACT) mainly formed by uniglomerular (u) PNs using fluorescent tracing, confocal microscopy, and 3D analyses. Furthermore, we investigated the projections of all ACTs in higher olfactory centers, the mushroom-bodies (MB) and lateral horn (LH). The results revealed a clear segregation of glomeruli into two AL hemispheres specifically supplied by PNs of the medial and lateral ACT. PNs of the lateral ACT innervate glomeruli in the ventral-rostral AL and primarily receive input from T1 (plus a few glomeruli from T2 and T3). PNs of the medial ACT innervate glomeruli in the dorsal-caudal hemisphere, and mainly receive input from T3 (plus a few glomeruli from T2 and T4). The PNs of the m- and l-ACT terminate in different areas of the MB calyx and LH and remain largely segregated. Tracing of three mediolateral (ml) ACTs mainly formed by multiglomerular PNs revealed terminals in distinct compartments of the LH and in three olfactory foci within the lateral protocerebrum. The results indicate that olfactory input in the honeybee is processed via two separate, mainly uPN pathways to the MB calyx and LH and several pathways to the lateral protocerebrum.

Multiple olfactory receptor neurons and their axonal projections in the antennal lobe of the honeybee Apis mellifera.

The poreplate sensilla of honeybees are equipped with multiple olfactory receptor neurons (ORNs), which innervate glomeruli of the antennal lobe (AL). We investigated the axonal projection pattern in glomeruli of the AL (glomerular pattern), formed by the multiple ORNs of individual poreplate sensilla. We used the different glomerular patterns to draw conclusions about the equipment of poreplate sensilla with different ORN types. ORNs of single poreplate sensilla were stained and analyzed by laser-scanning confocal microscopy and 3D software (AMIRA). In 13 specimens we found between 7 and 23 ORNs. This is in accordance with data found in the literature (5-35 ORNs) suggesting that all ORNs of the single poreplate sensilla were stained. The ORNs innervate the AL via all four sensory tracts (T1-T4), and glomeruli of the anterior part of the AL are more often innervated. Each ORN innervates a single glomerulus (uniglomerular), and all ORNs of one poreplate sensillum project to different glomeruli. Visual inspection and individual identification of glomeruli, based on the honeybee digital AL atlas, were used to evaluate mapping of glomeruli by a rigid transformation of the experimental ALs onto a reference AL. ORNs belonging to individual poreplate sensilla form variable glomerular patterns, and we did not find a common organization of glomerular patterns. We conclude that poreplate sensilla are equipped with different ORN types but that the same ORN types can be found in different poreplate sensilla. The equipment of poreplate sensilla with ORNs is overlapping. The mapping of glomeruli by rigid transformation is revealed to be a powerful tool for comparative neuroanatomy.