Dumb and Lazy? A Comparison of Color Learning and Memory Retrieval in Drones and Workers of the Buff-Tailed Bumblebee, Bombus terrestris, by Means of PER Conditioning

More than 100 years ago, Karl von Frisch showed that honeybee workers learn and discriminate colors. Since then, many studies confirmed the color learning capabilities of females from various hymenopteran species. Yet, little is known about visual learning and memory in males despite the fact that in most bee species males must take care of their own needs and must find rewarding flowers to obtain food. Here we used the proboscis extension response (PER) paradigm to study the color learning capacities of workers and drones of the bumblebee, Bombus terrestris. Light stimuli were paired with sucrose reward delivered to the insects’ antennae and inducing a reflexive extension of the proboscis. We evaluated color learning (i.e. conditioned PER to color stimuli) in absolute and differential conditioning protocols and mid-term memory retention was measured two hours after conditioning. Different monochromatic light stimuli in combination with neutral density filters were used to ensure that the bumblebees could only use chromatic and not achromatic (e.g. brightness) information. Furthermore, we tested if bees were able to transfer the learned information from the PER conditioning to a novel discrimination task in a Y-maze. Both workers and drones were capable of learning and discriminating between monochromatic light stimuli and retrieved the learned stimulus after two hours. Drones performed as well as workers during conditioning and in the memory test, but failed in the transfer test in contrast to workers. Our data clearly show that bumblebees can learn to associate a color stimulus with a sugar reward in PER conditioning and that both workers and drones reach similar acquisition and mid-term retention performances. Additionally, we provide evidence that only workers transfer the learned information from a Pavlovian to an operant situation

Mechanismen der visuellen Gedächtnisbildung bei Bienen: Über unmittelbar früh exprimierte Gene und synaptische Plastizität

Animals form perceptual associations through processes of learning, and retain that information through mechanisms of memory. Honeybees and bumblebees are classic models for insect perception and learning, and despite their small brains with about one million neurons, they are organized in highly social colonies and possess an astonishing rich behavioral repertoire including navigation, communication and cognition. Honeybees are able to harvest hundreds of morphologically divergent flower types in a quick and efficient manner to gain nutrition and, back in the hive, communicate discovered food sources to nest mates. To accomplish such complex tasks, bees must be equipped with diverse sensory organs receptive to stimuli of different modalities and must be able to associatively learn and memorize the acquired information. Particularly color vision plays a prominent role, e.g. in navigation along landmarks and when bees identify inflorescences by their color signals. Once acquired, bees are known to retain visual information for days or even months. Numerous studies on visual perception and color vision have been conducted in the past decades and largely revealed the information processing pathways in the brain. In contrast, there are no data available on how the brain may change in the course of color learning experience and whether pathways differ for coarse and fine color learning. Although long-term memory (LTM) storage is assumed to generally include reorganization of the neuronal network, to date it is unclear where in the bee brain such changes occur in the course of color learning and whether visual memories are stored in one particular site or decentrally distributed over different brain domains. The present dissertation research aimed to dissect the visual memory trace in bees that is beyond mere stimulus processing and therefore two different approaches were elaborated: first, the application of immediate early genes (IEG) as genetic markers for neuronal activation to localize early processes underlying the formation of a stable LTM. Second, the analysis of late consequences of memory formation, including synaptic reorganization in central brain areas and dependencies of color discrimination complexity. Immediate early genes (IEG) are a group of rapidly and transiently expressed genes that are induced by various types of cellular stimulation. A great number of different IEGs are routinely used as markers for the localization of neuronal activation in vertebrate brains. The present dissertation research was dedicated to establish this approach for application in bees, with focus on the candidate genes Amjra and Amegr, which are orthologous to the two common vertebrate IEGs c-jun and egr-1. First the general requirement of gene transcription for visual LTM formation was proved. Bumblebees were trained in associative proboscis extension response (PER) conditioning to monochromatic light and subsequently injected with an inhibitor of gene transcription. Memory retention tests at different intervals revealed that gene transcription is not required for the formation of a mid-term memory, but for stable LTM. Next, the appliance of the candidate genes was validated. Honeybees were exposed to stimulation with either alarm pheromone or a light pulse, followed by qPCR analysis of gene expression. Both genes differed in their expression response to sensory exposure: Amjra was upregulated in all analyzed brain parts (antennal lobes, optic lobes and mushroom bodies, MB), independent from stimulus modality, suggesting the gene as a genetic marker for unspecific general arousal. In contrast, Amegr was not significantly affected by mere sensory exposure. Therefore, the relevance of associative learning on Amegr expression was assessed. Honeybees were trained in visual PER conditioning followed by a qPCR-based analysis of the expression of all three Amegr isoforms at different intervals after conditioning. No learning-dependent alteration of gene expression was observed. However, the presence of AmEgr protein in virtually all cerebral cell nuclei was validated by immunofluorescence staining. The most prominent immune-reactivity was detected in MB calyx neurons. Analysis of task-dependent neuronal correlates underlying visual long-term memory was conducted in free-flying honeybees confronted with either absolute conditioning to one of two perceptually similar colors or differential conditioning with both colors. Subsequent presentation of the two colors in non-rewarded discrimination tests revealed that only bees trained with differential conditioning preferred the previously learned color. In contrast, bees of the absolute conditioning group chose randomly among color stimuli. To investigate whether the observed difference in memory acquisition is also reflected at the level of synaptic microcircuits, so called microglomeruli (MG), within the visual domains of the MB calyces, MG distribution was quantified by whole-mount immunostaining three days following conditioning. Although learning-dependent differences in neuroarchitecture were absent, a significant correlation between learning performance and MG density was observed. Taken together, this dissertation research provides fundamental work on the potential use of IEGs as markers for neuronal activation and promotes future research approaches combining behaviorally relevant color learning tests in bees with examination of the neuroarchitecture to pave the way for unraveling the visual memory trace.Tiere erlangen Informationen über die Umwelt durch Lernprozesse und speichern diese Informationen durch Mechanismen der Gedächtnisbildung. Honigbienen und Hummeln stellen klassische Modellorganismen zur Untersuchung von sensorischer Perzeption und Lernvorgängen dar. Trotz ihres kleinen, lediglich etwa eine Millionen Nervenzellen umfassenden Gehirns sind diese hoch sozialen Bienen zu erstaunlichen Verhaltensleistungen fähig, welche komplexe Navigation, Kommunikation und Kognition einschließen. Auf der Suche nach Futterquellen navigieren Honigbienen über große Distanzen, ohne dabei die Lage ihres Nestes aus dem Gedächtnis zu verlieren. Außerdem sammeln sie hoch effizient Futter an zahlreichen morphologisch divergenten Blütentypen und kommunizieren neu erschlossene Futterstellen anderen Sammelbienen im Nest. Zur Bewältigung solch komplexer Aufgaben stehen Bienen diverse sensorische Organe zur Verfügung, womit sie Reize unterschiedlicher Modalitäten wahrnehmen und verarbeiten können. Außerdem sind sie zu assoziativem Lernen und dem Speichern und Abrufen von Informationen in der Lage. Insbesondere der Sehsinn spielt für Bienen eine große Rolle, wenn sie sich beispielsweise anhand von Landmarken orientieren oder farbige Blütensignale wahrnehmen. Einmal erlernte visuelle Informationen können mitunter über Tage und Monate hinweg gespeichert werden. Während die Aufnahme und Verarbeitung von Farbinformationen im Bienengehirn bereits gut untersucht wurde, ist über räumliche und zeitliche Abläufe der Speicherung solcher Informationen wenig bekannt. Mit der vorliegenden Arbeit wurde versucht, experimentellen Zugang zur visuellen Gedächtnisspur in Bienen zu bekommen. Die Bildung eines Langzeitgedächtnisses (LZG) geht im Allgemeinen mit Umstrukturierungsprozessen im neuronalen Netzwerk einher. Bislang ist es jedoch unklar, wo im Gehirn diese Veränderungen im Laufe des Farbenlernens stattfinden und ob Informationen in einem zentralen Bereich gespeichert oder dezentral über verschiedene Gehirndomänen verteilt werden. Unterschiedliche Verarbeitungsbahnen werden für das Erlernen grober und feiner Farbunterschiede vermutet. Mit der vorliegenden Arbeit wurden zwei Versuchsansätze gewählt, womit die Lage des visuellen Gedächtnisses untersucht werden sollte: Zum einen wurde die Eignung unmittelbar exprimierter Gene (immediate early genes, IEG) als genetische Marker für neuronale Aktivität untersucht, um damit frühe Prozesse der Bildung eines LZG lokalisieren zu können. Zum anderen wurden Spätfolgen der Bildung eines LZG auf die Organisation synaptischer Netzwerke im zentralen Gehirn untersucht und der Einfluss der Komplexität einer Aufgabenstellung auf diese Organisation betrachtet. IEGs sind eine Gruppe von Genen, die in Antwort auf zelluläre Stimulierung schnell und vorübergehend exprimiert werden. Zahlreiche IEGs werden bereits routinemäßig als Marker für neuronale Aktivierung im Gehirn von Vertebraten eingesetzt und mit der vorliegenden Arbeit sollten die Möglichkeiten evaluiert werden, diesen Ansatz auch in Bienen nutzbar zu machen. Hierzu wurde zunächst ermittelt, ob die Transkription von Genen überhaupt für die Ausbildung eines visuellen LZG von Nöten ist. Hummeln wurden mit Hilfe der Proboscis-Streckreaktion (PER) trainiert, monochromatisches Licht mit Zuckerbelohnung zu assoziieren. Nach erfolgtem Training wurde die Gentranskription pharmazeutisch gehemmt und die Gedächtnisleistung der Hummeln zu zwei Zeitpunkten ermittelt, die das Mittelzeitgedächtnis (MZG) bzw. LZG repräsentieren. Es zeigte sich, dass Gentranskription nicht für die Ausbildung des MZG, jedoch für die des LZG unabdingbar ist. Als nächstes wurden mögliche Kandidatengene validiert. Honigbienen wurden entweder mit Alarmpheromon oder einem Lichtimpuls stimuliert. Die Bienengehirne wurden anschließend seziert und mittels qPCR die Expression von Amjra und Amegr untersucht, zweier Gene, deren orthologe Vertreter c-jun bzw. egr-1 gebräuchliche IEGs in Vertebraten darstellen. Während durch beide Reize die Expression von Amjra in allen Gehirnbereichen (Antenalloben, optische Loben und Pilzkörper) induziert wurde, konnten keine Veränderungen in der Expression von Amegr festgestellt werden. Daraufhin wurde überprüft, ob die Induktion von Amegr möglicherweise abhängig von assoziativen Lernvorgängen ist. Honigbienen wurden mittels PER visuell konditioniert, bevor die Pilzkörper zu verschiedenen Zeiten nach dem Training isoliert und mittels qPCR auf die Expression von Amegr Isoformen untersucht wurden. Hierbei konnte kein Lerneffekt auf die Amegr-Expression nachgewiesen werden. Die Analyse Aufgaben-abhängiger neuronaler Korrelate, die der Bildung des visuellen LZG zugrunde liegen, wurde anhand frei-fliegender Honigbienen durchgeführt. Diese wurden entweder absolut konditioniert auf eine von zwei ähnlichen Farben, oder differentiell auf die Diskriminierung beider Farben. Bei der anschließenden unbelohnten Präsentation beider Farben bevorzugte nur die differentiell trainierte Gruppe die zuvor gelernte Farbe, während absolut konditionierte Bienen zufällig wählten. Um zu ermitteln, ob die beobachteten Unterschiede im Verhalten auch auf neuroanatomischer Ebene repräsentiert werden, wurden alle Bienen nach drei Tagen seziert und mittels Immunfärbung synaptische Komplexe, so genannte Microglomeruli, im visuelle Informationen verarbeitenden Bereich der Pilzkörper quantifiziert. Der Vergleich zwischen den Versuchsgruppen legte keine signifikanten Unterschiede in der neuronalen Architektur offen, jedoch wurden mögliche Zusammenhänge zwischen Lernleistung und Microglomeruli-Dichte gefunden. Die vorliegende Arbeit bietet grundlegende Ergebnisse zum Potential von IEGs als Marker neuronaler Aktivität und unterstreicht die Bedeutung integrativer Versuchsansätze, welche Verhaltensuntersuchungen mit der molekularen und histologischen Analyse des Nervensystems verbinden, um letztlich das visuelle Gedächtnis im Bienengehirn lokalisieren zu können

Impact of the conditioning protocol on performance in absolute color conditioning and memory retrieval.

<p>Learning curves of (A) workers and (B) drones during absolute conditioning. Bees were trained either with a paired (filled circles) or an unpaired (open circles) presentation of CS and US. Two groups of bees of each sex were trained with different conditioning protocols of the paired CS-US presentation: one group (<i>Paired 12</i> s) was presented in each trial with 12 s of CS, and 3 s of US 6 s after CS onset, which led to a 3s CS overhang after end of US; a second group (<i>Paired 9s</i>) received 9 s of CS and 3 s of US 6 s after CS onset. In the latter group, CS and US terminated simultaneously. Memory retrieval was tested by presenting the CS and a novel color stimulus (NCol) to the bees 2h after end of conditioning. <b>A</b>, <i>Paired 12 s/Unpaired</i>: MWU, p<0.001, Z = -3.587; <i>Paired 9 s/Unpaired</i>: MWU, p<0.001, Z = 3.587; <i>CS12 s/Ncol</i>: p = 0.003, chi² = 8.686; <i>CS9 s/Ncol</i>: p = 0.017, chi² = 5.729). <b>B</b>, <i>Paired 12 s/Unpaired</i>: MWU, p = 0.009, Z = -2.612; <i>Paired 9 s/Unpaired</i>: MWU, p = 0.009, Z = 2.612; <i>CS12 s/Ncol</i>: p = 0.017, chi² = 5.700; <i>CS9 s/Ncol</i>: p = 0.008, chi² = 7.125). *** P < 0.001; ** P < 0.01.</p

Differential color conditioning and memory retrieval in workers and drones.

<p>Three different monochromatic color stimuli combinations (435/528 nm; 435/488 nm and 435/455 nm) with different wavelength distances (93 nm; 53 nm and 20 nm) between stimuli were tested. Bumblebees were trained to discriminate the rewarded (CS+) and the unrewarded color stimulus (CS-). Each color stimulus combination was tested reciprocally. For the memory retrieval test the rewarded color stimulus (CS+: black bar) and the unrewarded color stimulus (CS-: gray bar) were presented to the bees 2 h after end of conditioning. Since no effects of asymmetrical discrimination between the two colors of each combination was found (except for 435/528 nm in workers, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134248#pone.0134248.g005" target="_blank">Fig 5</a>), data were pooled. <i>*** P</i> < 0.001; n.s.: not significant.</p

Does Fine Color Discrimination Learning in Free-Flying Honeybees Change Mushroom-Body Calyx Neuroarchitecture?

Honeybees learn color information of rewarding flowers and recall these memories in future decisions. For fine color discrimination, bees require differential conditioning with a concurrent presentation of target and distractor stimuli to form a long-term memory. Here we investigated whether the long-term storage of color information shapes the neural network of microglomeruli in the mushroom body calyces and if this depends on the type of conditioning. Free-flying honeybees were individually trained to a pair of perceptually similar colors in either absolute conditioning towards one of the colors or in differential conditioning with both colors. Subsequently, bees of either conditioning groups were tested in non-rewarded discrimination tests with the two colors. Only bees trained with differential conditioning preferred the previously learned color, whereas bees of the absolute conditioning group, and a stimuli-naïve group, chose randomly among color stimuli. All bees were then kept individually for three days in the dark to allow for complete long-term memory formation. Whole-mount immunostaining was subsequently used to quantify variation of microglomeruli number and density in the mushroom-body lip and collar. We found no significant differences among groups in neuropil volumes and total microglomeruli numbers, but learning performance was negatively correlated with microglomeruli density in the absolute conditioning group. Based on these findings we aim to promote future research approaches combining behaviorally relevant color learning tests in honeybees under free-flight conditions with neuroimaging analysis; we also discuss possible limitations of this approach.

Stimuli qualities and experimental setup.

<p>A: Spectral sensitivity of the three photoreceptor types in <i>Bombus terrestris</i> (data obtained from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134248#pone.0134248.ref041" target="_blank">41</a>]), overlaid by transmission of the four tested color filters (435 nm, 455 nm, 488 nm, and 528 nm). B: Intensities (photons per second and mm²) of the monochromatic light stimuli generated by means of different ND filters (13%, 51% and 100% transmission). C: Illustration of the set-up for visual PER conditioning. See text for description. fh, filter holder; s, movable sleigh. D: Y-maze set-up used for the transfer test after differential PER conditioning. The diffusor is omitted in the left arm to make the color filter (f) and the opening between the two chambers visible. See text for description. d, diffusor (parchment paper); fc, filter camber; f, color filter; tc, testing camber; ec, entrance chamber; dc, decision chamber.</p

Information transfer after PER conditioning to Y-maze.

<p>Workers (A; C) and drones (B; D) were tested 2 h after end of conditioning for transfer of the learned color information to a novel operant context. Proportion of first choice of the bumblebees towards the CS+ arm (A: workers; B: drones) for three different color combinations (435/528 nm; 435/488 nm and 435/455 nm) and time (C: workers; D: drones) spent in each arm (in seconds). Since there were no significant differences within the respective color combination regarding the rewarded stimulus during the first choice towards the CS+ arm and time spent in each arm, all data were pooled for each tested color combination. <i>*** P</i> < 0.001; <i>* P</i> < 0.05; n.s.: not significant.</p

Absolute color conditioning and memory retrieval in drones.

<p>Acquisition curves (in % PER) of drones during absolute conditioning of four different color stimuli (A: 435 nm, B: 455 nm, C: 488 nm and D: 528 nm). Drones were trained either to a paired (filled circles) or an unpaired (empty circles) presentation of CS and US. Memory retrieval was tested by presenting the CS (colored bar) and a novel color stimulus (NCol: gray bar) to the bees 2h after conditioning. <i>*** P</i> < 0.001; <i>** P</i> < 0.01.</p

Absolute color conditioning and memory retrieval in workers.

<p>Acquisition curves (proportion of bees that responded to the tested color stimulus by extending the proboscis [% PER]) of workers during absolute conditioning of four different color stimuli (A: 435 nm, B: 455 nm, C: 488 nm and D: 528 nm). Workers were trained either with a paired (filled circles) or an unpaired (empty circles) presentation of CS and US. Memory retrieval was tested by presenting the CS (colored bar) and a novel color stimulus (NCol: gray bar) to the bees 2h after conditioning. <i>*** P</i> < 0.001; <i>** P</i> < 0.01.</p