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

Neuronale Korrelate der Nestgenossen-Erkennung bei der Rossameise, Camponotus floridanus

Cooperation is beneficial for social groups and is exemplified in its most sophisticated form in social insects. In particular, eusocial Hymenoptera, like ants and honey bees, exhibit a level of cooperation only rarely matched by other animals. To assure effective defense of group members, foes need to be recognized reliably. Ants use low-volatile, colony-specific profiles of cuticular hydrocarbons (colony odor) to discriminate colony members (nestmates) from foreign workers (non-nestmates). For colony recognition, it is assumed that multi-component colony odors are compared to a neuronal template, located in a so far unidentified part of the nervous system, where a mismatch results in aggression. Alternatively, a sensory filter in the periphery of the nervous system has been suggested to act as a template, causing specific anosmia to nestmate colony odor due to sensory adaptation and effectively blocking perception of nestmates. Colony odors are not stable, but change over time due to environmental influences. To adjust for this, the recognition system has to be constantly updated (template reformation). In this thesis, I provide evidence that template reformation can be induced artificially, by modifying the sensory experience of carpenter ants (Camponotus floridanus; Chapter 1). The results of the experiments showed that template reformation is a relatively slow process taking several hours and this contradicts the adaptation-based sensory filter hypothesis. This finding is supported by first in-vivo measurements describing the neuronal processes underlying template reformation (Chapter 5). Neurophysiological measurements were impeded at the beginning of this study by the lack of adequate technical means to present colony odors. In a behavioral assay, I showed that tactile interaction is not necessary for colony recognition, although colony odors are of very low volatility (Chapter 2). I developed a novel stimulation technique (dummy-delivered stimulation) and tested its suitability for neurophysiological experiments (Chapter 3). My experiments showed that dummy-delivered stimulation is especially advantageous for presentation of low-volatile odors. Colony odor concentration in headspace was further increased by moderately heating the dummies, and this allowed me to measure neuronal correlates of colony odors in the peripheral and the central nervous system using electroantennography and calcium imaging, respectively (Chapter 4). Nestmate and non-nestmate colony odor elicited strong neuronal responses in olfactory receptor neurons of the antenna and in the functional units of the first olfactory neuropile of the ant brain, the glomeruli of the antennal lobe (AL). My results show that ants are not anosmic to nestmate colony odor and this clearly invalidates the previously suggested sensory filter hypothesis. Advanced two-photon microscopy allowed me to investigate the neuronal representation of colony odors in different neuroanatomical compartments of the AL (Chapter 5). Although neuronal activity was distributed inhomogeneously, I did not find exclusive representation restricted to a single AL compartment. This result indicates that information about colony odors is processed in parallel, using the computational power of the whole AL network. In the AL, the patterns of glomerular activity (spatial activity patterns) were variable, even in response to repeated stimulation with the same colony odor (Chapter 4&5). This finding is surprising, as earlier studies indicated that spatial activity patterns in the AL reflect how an odor is perceived by an animal (odor quality). Under natural conditions, multi-component odors constitute varying and fluctuating stimuli, and most probably animals are generally faced with the problem that these elicit variable neuronal responses. Two-photon microscopy revealed that variability was higher in response to nestmate than to non-nestmate colony odor (Chapter 5), possibly reflecting plasticity of the AL network, which allows template reformation. Due to their high variability, spatial activity patterns in response to different colony odors were not sufficiently distinct to allow attribution of odor qualities like ‘friend’ or ‘foe’. This finding challenges our current notion of how odor quality of complex, multi-component odors is coded. Additional neuronal parameters, e.g. precise timing of neuronal activity, are most likely necessary to allow discrimination. The lower variability of activity patterns elicited by non-nestmate compared to nestmate colony odor might facilitate recognition of non-nestmates at the next level of the olfactory pathway. My research efforts made the colony recognition system accessible for direct neurophysiological investigations. My results show that ants can perceive their own nestmates. The neuronal representation of colony odors is distributed across AL compartments, indicating parallel processing. Surprisingly, the spatial activity patterns in response to colony are highly variable, raising the question how odor quality is coded in this system. The experimental advance presented in this thesis will be useful to gain further insights into how social insects discriminate friends and foes. Furthermore, my work will be beneficial for the research field of insect olfaction as colony recognition in social insects is an excellent model system to study the coding of odor quality and long-term memory mechanisms underlying recognition of complex, multi-component odors.Kooperation innerhalb sozialer Gruppen ist vorteilhaft und zeigt sich bei sozialen Insekten in seiner am höchsten entwickelten Form. Besonders eusoziale Hymenopteren, wie Ameisen und Honigbienen, zeigen ein Maß an Kooperation, das nur selten von anderen Tierarten erreicht wird. Um eine effektive Verteidigung der Gruppenmitglieder sicher zu stellen, ist die zuverlässige Erkennung von Feinden unerlässlich. Ameisen verwenden schwerflüchtige, koloniespezifische Profile kutikulärer Kohlenwasserstoffe (Kolonieduft) zur Unterscheidung zwischen Gruppenmitgliedern (Nestgenossen) und fremden Arbeiterinnen (Nestfremdlinge). Man geht davon aus, dass die aus einer Vielzahl von Komponenten bestehenden Koloniedüfte zum Zweck der Kolonieerkennung mit einer neuronalen Schablone, welche sich an bisher unbestimmter Stelle im Nerven-system befindet, abgeglichen werden. Dabei führt eine Diskrepanz zwischen Schablone und Kolonieduft zu Aggression. Eine alternative Hypothese besagt, dass ein sensorischer Filter in der Peripherie des Nervensystems die Aufgabe einer neuronalen Schablone übernimmt. Dies würde mittels sensorischer Adaptation zu spezifischer Anosmie gegenüber Nestgenossen-Kolonieduft führen, so dass die Wahrnehmung von Nestgenossen effektiv verhindert wäre. Allerdings sind Koloniedüfte nicht stabil, sondern verändern sich im Lauf der Zeit aufgrund von Umwelteinflüssen. Um dies zu kompensieren, muss das Erkennungssystem fortwährend aktualisiert werden (Schablonenerneuerung). In dieser Arbeit erbringe ich den Nachweis, dass bei Rossameisen (Camponotus floridanus) die Schablonenerneuerung artifiziell durch Modifizierung der sensorischen Erfahrung induziert werden kann (Kapitel 1). Die Ergebnisse der in Kapitel 1 beschriebenen Experimente zeigen, dass die Schablonenerneuerung ein relativ langsamer Prozess ist, der mehrere Stunden in Anspruch nimmt. Dies widerspricht der Hypothese eines sensorischen Filters, welcher auf sensorischer Adaptation beruht. Dieser Befund konnte mittels erster in-vivo Messungen bestätigt werden, mit Hilfe derer die der Schablonenerneuerung zugrunde liegenden neuronalen Prozesse beschrieben wurden (Kapitel 5). Die neurophysiologischen Messungen wurden zu Beginn dieser Studie durch das Fehlen eines adäquaten Mittels zur Präsentation von Koloniedüften erschwert. In einem Verhaltensversuch konnte ich zeigen, dass taktile Interaktionen für die Kolonieerkennung nicht notwendig sind (Kapitel 2). Ich entwickelte eine neuartige Stimulierungsmethode (Dummy-vermittelte Stimulierung) und testete deren Eignung für neurophysiologische Experimente (Kapitel 3). Meine Experimente zeigten, dass die Dummy-vermittelte Stimulierung besonders für die Präsentation von schwerflüchtigen Düften geeignet ist. Die Konzentration von Koloniedüften im Gasraum konnte durch moderates Aufheizen der Dummys weiter gesteigert werden. Dies erlaubte mir, die neuronalen Korrelate von Koloniedüften im peripheren und im zentralen Nervensystem mittels Elektroantennographie bzw. funktionaler Bildgebung (Calcium Imaging) zu messen (Kapitel 4). Nestgenossen- und Nestfremdlings-Koloniedüfte riefen starke neuronale Antworten in den olfaktorischen Rezeptorneuronen der Antenne und in den funktionalen Einheiten des ersten olfaktorischen Neuropils des Ameisengehirns, den Glomeruli des Antennallobus (AL), hervor. Meine Ergebnisse zeigen, dass Ameisen nicht anosmisch gegenüber Nestgenossen-Koloniedüften sind, womit die vorgeschlagene Hypothese eines sensorischen Filters eindeutig für ungültig erklärt werden kann. Mittels fortschrittlicher Zwei-Photonen-Mikroskopie konnte ich die neuronale Repräsentation von Koloniedüften in verschiedenen neuroanatomischen Kompartimenten des AL messen (Kapitel 5). Obgleich die neuronale Aktivität inhomogen verteilt war, konnte ich keine exklusive Repräsentation finden, die auf ein einzelnes AL-Kompartiment beschränkt gewesen wäre. Dieses Ergebnis weist darauf hin, dass Informationen über Koloniedüfte parallel verarbeitet werden und dies erlaubt die Nutzung der Rechenleistung des kompletten AL-Netzwerkes. Im AL waren die Muster glomerulärer Aktivität (räumliche Aktivitätsmuster) variabel, selbst wenn sie durch wiederholte Stimulierung mit dem gleichen Kolonieduft hervorgerufen wurden (Kapitel 4&5). Dieser Befund ist insofern überraschend, als frühere Studien darauf hinwiesen, dass die räumlichen Aktivitätsmuster im AL widerspiegeln, wie ein Duft von einem Tier wahrge¬nommen wird (Duftqualität). Unter natürlichen Bedingungen stellen Düfte, die aus einer Vielzahl von Komponenten bestehen, variable und fluktuierende Stimuli dar. Höchstwahrscheinlich sind Tiere generell mit dem Problem konfrontiert, dass solche Düfte variable neuronale Antworten hervorrufen. Mittels Zwei-Photonen-Mikroskopie konnte ich zeigen, dass die Variabilität in Antwort auf Nestgenossen-Kolonieduft höher war als in Antwort auf Nestfremdlings-Kolonieduft (Kapitel 5). Möglicherweise spiegelt dies jene Plastizität im AL-Netzwerk wider, welche die Schablonenerneuerung ermöglicht. Aufgrund ihrer hohen Variabilität waren die von verschiedenen Koloniedüften hervorgerufenen räumlichen Aktivierungsmuster nicht hinreichend unterschiedlich, um eine Zuordnung von Duft-qualitäten wie ‚Freund‘ oder ‚Feind‘ zu erlauben. Dieser Befund stellt unsere momentane Auffassung in Frage, wie die Duftqualität komplexer, aus vielen Komponenten bestehender Düfte kodiert wird. Höchstwahrscheinlich sind zusätzliche neuronale Parameter, wie z.B. die präzise, zeitliche Koordinierung neuronaler Aktivität, zur Diskriminierung notwendig. Die geringere Variabilität der von Nestfremdlings-Kolonieduft hervorgerufenen Aktivitätsmuster könnte die Erkennung von Nestfremdlingen auf der nächsten Ebene der olfaktorischen Bahn begünstigen. Meine Forschungsarbeit hat das Kolonieerkennungssystem für direkte neurophysiologische Untersuchungen zugänglich gemacht. Meine Ergebnisse zeigen, dass Ameisen ihre eigenen Nest-genossen wahrnehmen können. Die neuronale Repräsentation von Koloniedüften ist über die AL-Kompartimente verteilt, was auf eine parallele Verarbeitung hinweist. Desweiteren könnte die geringere Variabilität der von Nestfremdlings-Kolonieduft hervorgerufenen Aktivitätsmuster die Erkennung von Nestfremdlingen auf der nächsten Ebene der olfaktorischen Bahn begünstigen. Erstaunlicherweise sind die räumlichen Aktivitätsmuster in Antwort auf Koloniedüfte hochvariabel. Die wirft die Frage auf, wie in diesem System die Duftqualität kodiert wird. Der experimentelle Fortschritt, den ich in dieser Doktorarbeit vorstelle, wird nützlich sein, um weitere Erkenntnisse zu gewinnen, wie soziale Insekten Freunde von Feinden unterscheiden. Desweiteren wird meine Arbeit dem Forschungsbereich Insektenolfaktion zuträglich sein, da die Kolonieerkennung bei sozialen Insekten ein hervorragendes Modelsystem darstellt, um die Kodierung von Duftqualität zu erforschen, sowie Langzeitmechanismen, die der Erkennung komplexer, aus vielen Komponenten bestehender Düfte zugrunde liegen

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

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 multicomponent 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

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

Ants use cuticular hydrocarbons (CHC-proWles) 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-proWle may change, and the template is adjusted accordingly. A phenotype mismatch between label and template, as happens with CHC-proWles of foreign workers (nonnestmates), frequently leads to aggressive behavior. We investigated the template reformation in workers of the carpenter ant Camponotus Xoridanus 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-proWle does not impair the assessment of the old CHC-proWle as nestmate label