Infrastructure construction without information exchange: the trail clearing mechanism in Atta leafcutter ants.

A wide range of group-living animals construct tangible infrastructure networks, often of remarkable size and complexity. In ant colonies, infrastructure construction may require tens of thousands of work hours distributed among many thousand individuals. What are the individual behaviours involved in the construction and what level of complexity in inter-individual interaction is required to organize this effort? We investigate this question in one of the most sophisticated trail builders in the animal world: the leafcutter ants, which remove leaf litter, cut through overhangs and shift soil to level the path of trail networks that may cumulatively extend for kilometres. Based on obstruction experiments in the field and the laboratory, we identify and quantify different individual trail clearing behaviours. Via a computational model, we further investigate the presence of recruitment, which-through direct or indirect information transfer between individuals-is one of the main organizing mechanisms of many collective behaviours in ants. We show that large-scale transport networks can emerge purely from the stochastic process of workers encountering obstructions and subsequently engaging in removal behaviour with a fixed probability. In addition to such incidental removal, we describe a dedicated clearing behaviour in which workers remove additional obstructions independent of chance encounters. We show that to explain the dynamics observed in the experiments, no information exchange (e.g. via recruitment) is required, and propose that large-scale infrastructure construction of this type can be achieved without coordination between individuals.

Parallel processing via a dual olfactory pathway in the honeybee.

In their natural environment, animals face complex and highly dynamic olfactory input. Thus vertebrates as well as invertebrates require fast and reliable processing of olfactory information. Parallel processing has been shown to improve processing speed and power in other sensory systems and is characterized by extraction of different stimulus parameters along parallel sensory information streams. Honeybees possess an elaborate olfactory system with unique neuronal architecture: a dual olfactory pathway comprising a medial projection-neuron (PN) antennal lobe (AL) protocerebral output tract (m-APT) and a lateral PN AL output tract (l-APT) connecting the olfactory lobes with higher-order brain centers. We asked whether this neuronal architecture serves parallel processing and employed a novel technique for simultaneous multiunit recordings from both tracts. The results revealed response profiles from a high number of PNs of both tracts to floral, pheromonal, and biologically relevant odor mixtures tested over multiple trials. PNs from both tracts responded to all tested odors, but with different characteristics indicating parallel processing of similar odors. Both PN tracts were activated by widely overlapping response profiles, which is a requirement for parallel processing. The l-APT PNs had broad response profiles suggesting generalized coding properties, whereas the responses of m-APT PNs were comparatively weaker and less frequent, indicating higher odor specificity. Comparison of response latencies within and across tracts revealed odor-dependent latencies. We suggest that parallel processing via the honeybee dual olfactory pathway provides enhanced odor processing capabilities serving sophisticated odor perception and olfactory demands associated with a complex olfactory world of this social insect.

Glyphosate impairs collective thermoregulation in bumblebees.

Insects are facing a multitude of anthropogenic stressors, and the recent decline in their biodiversity is threatening ecosystems and economies across the globe. We investigated the impact of glyphosate, the most commonly used herbicide worldwide, on bumblebees. Bumblebee colonies maintain their brood at high temperatures via active thermogenesis, a prerequisite for colony growth and reproduction. Using a within-colony comparative approach to examine the effects of long-term glyphosate exposure on both individual and collective thermoregulation, we found that whereas effects are weak at the level of the individual, the collective ability to maintain the necessary high brood temperatures is decreased by more than 25% during periods of resource limitation. For pollinators in our heavily stressed ecosystems, glyphosate exposure carries hidden costs that have so far been largely overlooked.

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.

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.

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.

Moth pheromone-selective projection neurons with cell bodies in the antennal lobe lateral cluster exhibit diverse morphological and neurophysiological characteristics.

Olfactory projection neurons convey information from the insect antennal lobe (AL) to higher brain centers. Previous reports have demonstrated that pheromone-responsive projection neurons with cell bodies in the moth medial cell cluster (mcPNs) predominantly have dendritic arborizations in the sexually dimorphic macroglomerular complex (MGC) and send an axon from the AL to the calyces of the mushroom body (CA) as well as the lateral horn (LH) of the protocerebrum via the medial AL tract. These neurons typically exhibit a narrow odor tuning range related to the restriction of their dendritic arbors within a single glomerulus (uniglomerular). In this study, we report on the diverse physiological and morphological properties of a group of pheromone-responsive olfactory projection neurons with cell bodies in the AL lateral cell cluster (MGC lcPNs) of two closely related moth species. All pheromone-responsive lcPNs appeared to exhibit "basket-like" dendritic arborizations in two MGC compartments and made connections with various protocerebral targets including ventrolateral and superior neuropils via projections primarily through the lateral AL tract and to a lesser extent the mediolateral antennal lobe tract. Physiological characterization of MGC lcPNs also revealed a diversity of response profiles including those either enhanced by or reliant upon presentation of a pheromone blend. These responses manifested themselves as higher maximum firing rates and/or improved temporal resolution of pulsatile stimuli. MGC lcPNs therefore participate in conveying diverse olfactory information relating to qualitative and temporal facets of the pheromone stimulus to a more expansive number of protocerebral targets than their mcPN counterparts.

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.

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.

High Precision of Spike Timing across Olfactory Receptor Neurons Allows Rapid Odor Coding in Drosophila.

In recent years, it has become evident that olfaction is a fast sense, and millisecond short differences in stimulus onsets are used by animals to analyze their olfactory environment. In contrast, olfactory receptor neurons are thought to be relatively slow and temporally imprecise. These observations have led to a conundrum: how, then, can an animal resolve fast stimulus dynamics and smell with high temporal acuity? Using parallel recordings from olfactory receptor neurons in Drosophila, we found hitherto unknown fast and temporally precise odorant-evoked spike responses, with first spike latencies (relative to odorant arrival) down to 3 ms and with a SD below 1 ms. These data provide new upper bounds for the speed of olfactory processing and suggest that the insect olfactory system could use the precise spike timing for olfactory coding and computation, which can explain insects' rapid processing of temporal stimuli when encountering turbulent odor plumes.

Ultrastructure and electrophysiology of thermosensitive sensilla coeloconica in a tropical katydid of the genus Mecopoda (Orthoptera, Tettigoniidae).

In many acoustic insects, mate finding and mate choice are primarily based on acoustic signals. In several species with high-intensity calling songs, such as the studied katydid Mecopoda sp., males exhibit an increase in their thoracic temperature during singing, which is linearly correlated with the amount of energy invested in song production. If this increased body temperature is used by females as an additional cue to assess the male's quality during mate choice, as has been recently hypothesized ("hot-male" hypothesis), thermosensory structures would be required to evaluate this cue. In the present study, therefore, we investigated the ultrastructure and physiology of thermosensitive sensilla coeloconica on the antennal flagella of Mecopoda sp. using a combination of electron microscopy and electrophysiological recording techniques. We could identify three distinct types of sensilla coeloconica based on differences in the number and branching pattern of their dendrites. Physiological recordings revealed the innervation by antagonistically responding thermoreceptors (cold and warm) and bimodal hygro-/thermoreceptors (moist or dry) in various combinations. Our findings indicate that Mecopoda sp. females are capable of detecting a singing male from distances of at least several centimetres solely by assessing thermal cues.

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.

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.

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.

Two cold-sensitive neurons within one sensillum code for different parameters of the thermal environment in the ant Camponotus rufipes.

Ants show high sensitivity when responding to minute temperature changes and are able to track preferred temperatures with amazing precision. As social insects, they have to detect and cope with thermal fluctuations not only for their individual benefit but also for the developmental benefit of the colony and its brood. In this study we investigate the sensory basis for the fine-tuned, temperature guided behaviors found in ants, specifically what information about their thermal environment they can assess. We describe the dose-response curves of two cold-sensitive neurons, associated with the sensillum coelocapitulum on the antenna of the carpenter ant Camponotus rufipes.One cold-sensitive neuron codes for temperature changes, thus functioning as a thermal flux-detector. Neurons of such type continuously provide the ant with information about temperature transients (TT-neuron). The TT-neurons are able to resolve a relative change of 37% in stimulus intensity (ΔT) and antennal scanning of the thermal environment may aid the ant's ability to use temperature differences for orientation.The second cold-sensitive neuron in the S. coelocapitulum responds to temperature only within a narrow temperature range. A temperature difference of 1.6°C can be resolved by this neuron type. Since the working range matches the preferred temperature range for brood care of Camponotus rufipes, we hypothesize that this temperature sensor can function as a thermal switch to trigger brood care behavior, based on absolute (steady state) temperature.

Clearing pigmented insect cuticle to investigate small insects' organs in situ using confocal laser-scanning microscopy (CLSM).

Various microscopic techniques allow investigating structures from submicron to millimeter range, however, this is only possible if the structures of interest are not covered by pigmented cuticle. Here, we present a protocol that combines clearing of pigmented cuticle while preserving both, hard and soft tissues. The resulting transparent cuticle allows confocal laser-scanning microscopy (CLSM), which yields high-resolution images of e.g. the brain, glands, muscles and fine cuticular structures. Using a fluorescent dye, even single labeled neurons can be visualized and resolved up to an imaging depth of 150 µm through the cleared cuticle. Hydrogen-peroxide, which was used to clear the cuticle, does not preclude immunocytochemical techniques, shown by successful labeling of serotonin-immunoreactive neurons (5HT-ir) in the ants' brain. The 'transparent insect protocol' presented here is especially suited for small arthropods where dissection of organs is very demanding and difficult to achieve. Furthermore, the insect organs are preserved in situ thus allowing a more precise three-dimensional reconstruction of the structures of interest compared to, e.g., dissected or sectioned tissue.

Sexual dimorphism in the olfactory system of a solitary and a eusocial bee species.

Sexually dimorphic sensory systems are common in Hymenoptera and are considered to result from sex-specific selection pressures. An extreme example of sensory dimorphism is found in the solitary bee tribe Eucerini. Males of long-horned bees bear antennae that exceed body length. This study investigated the pronounced sexual dimorphism of the peripheral olfactory system and its representation in higher brain centers of the species Eucera berlandi. Eucera males have elongated antennae, with 10 times more pore plates and three times more olfactory receptor neurons than females. The male antennal lobe (AL) comprises fewer glomeruli than the female AL (∼100 vs. ∼130), of which four are male-specific macroglomeruli. No sex differences were found in the relative volume of the mushroom bodies, a higher order neuropil essential for learning and memory in Hymenoptera. Compared with the Western honeybee, the degree of sexual dimorphism in Eucera is more pronounced at the periphery. In contrast, sex differences in glomerular numbers are higher in the eusocial honeybee and a sexual dimorphism of the relative investment in mushroom body tissue is observed only in Apis. The observed differences between the eusocial and the solitary bee species may reflect differences in male-specific behavioral traits and associated selection pressures, which are discussed in brief.