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.

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.

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.

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.

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.

Caste-specific expression patterns of immune response and chemosensory related genes in the leaf-cutting ant, Atta vollenweideri.

Leaf-cutting ants are evolutionary derived social insects with elaborated division of labor and tremendous colony sizes with millions of workers. Their social organization is mainly based on olfactory communication using different pheromones and is promoted by a pronounced size-polymorphism of workers that perform different tasks within the colony. The size polymorphism and associated behaviors are correlated to distinct antennal lobe (AL) phenotypes. Two worker phenotypes differ in number of olfactory glomeruli in the AL and the presence or absence of an extremely large glomerulus (macroglomerulus), involved in trail-pheromone reception. The males' AL contains three macroglomeruli which are presumably involved in detection of sex-pheromone components. We investigated the antennal transcriptome data of all major castes (males, queens and workers) and two worker subcastes (large and tiny workers). In order to identify putative odorant receptor genes involved in pheromone detection, we identified differentially expressed odorant receptor genes (OR-genes) using custom microarrays. In total, we found 185 OR-gene fragments that are clearly related to ORs and we identified orthologs for 70 OR-genes. Among them one OR-gene differs in relative expression between the two worker subcastes by a factor of >3 and thus is a very promising candidate gene for the trail-pheromone receptor. Using the relative expression of OR-genes in males versus queens, we identified 2 candidates for sex-pheromone receptor genes in males. In addition, we identified genes from all other chemosensory related gene families (13 chemosensory protein genes, 8 odorant binding protein genes, 2 sensory-neuron membrane protein genes, 7 ionotropic receptor genes, 2 gustatory receptor genes), and we found ant-specific expansions in the chemosensory protein gene family. In addition, a large number of genes involved in immune defense exhibited differential expression across the three different castes, and some genes even between the two worker subcastes.

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.

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.

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.