Fast learning but coarse discrimination of colours in restrained honeybees.

addresses: Department of Neurobiology, Institute of Biology, Free University of Berlin, 14195 Berlin, Germany.types: Journal Article; Research Support, Non-U.S. Gov't© 2009 Company of Biologists. Post print version deposited in accordance with SHERPA RoMEO guidelines. The definitive version is available at: http://jeb.biologists.org/content/212/9/1344Colours are quickly learnt by free-moving bees in operant conditioning settings. In the present study, we report a method using the classical conditioning of the proboscis extension response (PER) in restrained honeybees (Apis mellifera), which allows bees to learn colours after just a few training trials. We further analysed how visual learning and discrimination is influenced by the quality of a stimulus by systematically varying the chromatic and achromatic properties of the stimuli. Using differential conditioning, we found that faster colour discrimination learning was correlated with reduced colour similarity between stimuli. In experiments with both absolute and differential conditioning, restrained bees showed poor colour discrimination and broad generalisation. This result is in strong contrast to the well-demonstrated ability of bees to finely discriminate colours under free-flight conditions and raises further questions about the temporal and perceptual processes underlying the ability of bees to discriminate and learn colours in different behavioural contexts

Visual associative learning in wood ants

Wood ants are a model system for studying visual learning and navigation. They can forage for food and navigate to their nests effectively by forming memories of visual features in their surrounding environment. Previous studies of freely behaving ants have revealed many of the behavioural strategies and environmental features necessary for successful navigation. However, little is known about the exact visual properties of the environment that animals learn or the neural mechanisms that allow them to achieve this. As a first step towards addressing this, we developed a classical conditioning paradigm for visual learning in harnessed wood ants that allows us to control precisely the learned visual cues. In this paradigm, ants are fixed and presented with a visual cue paired with an appetitive sugar reward. Using this paradigm, we found that visual cues learnt by wood ants through Pavlovian conditioning are retained for at least one hour. Furthermore, we found that memory retention is dependent upon the ants’ performance during training. Our study provides the first evidence that wood ants can form visual associative memories when restrained. This classical conditioning paradigm has the potential to permit detailed analysis of the dynamics of memory formation and retention, and the neural basis of learning in wood ants

Visual Associative Learning in Restrained Honey Bees with Intact Antennae

A restrained honey bee can be trained to extend its proboscis in response to the pairing of an odor with a sucrose reward, a form of olfactory associative learning referred to as the proboscis extension response (PER). Although the ability of flying honey bees to respond to visual cues is well-established, associative visual learning in restrained honey bees has been challenging to demonstrate. Those few groups that have documented vision-based PER have reported that removing the antennae prior to training is a prerequisite for learning. Here we report, for a simple visual learning task, the first successful performance by restrained honey bees with intact antennae. Honey bee foragers were trained on a differential visual association task by pairing the presentation of a blue light with a sucrose reward and leaving the presentation of a green light unrewarded. A negative correlation was found between age of foragers and their performance in the visual PER task. Using the adaptations to the traditional PER task outlined here, future studies can exploit pharmacological and physiological techniques to explore the neural circuit basis of visual learning in the honey bee

Mechanisms, functions and ecology of colour vision in the honeybee.

notes: PMCID: PMC4035557types: Journal Article© The Author(s) 2014.This is an open access article that is freely available in ORE or from Springerlink.com. Please cite the published version available at: http://link.springer.com/article/10.1007%2Fs00359-014-0915-1Research in the honeybee has laid the foundations for our understanding of insect colour vision. The trichromatic colour vision of honeybees shares fundamental properties with primate and human colour perception, such as colour constancy, colour opponency, segregation of colour and brightness coding. Laborious efforts to reconstruct the colour vision pathway in the honeybee have provided detailed descriptions of neural connectivity and the properties of photoreceptors and interneurons in the optic lobes of the bee brain. The modelling of colour perception advanced with the establishment of colour discrimination models that were based on experimental data, the Colour-Opponent Coding and Receptor Noise-Limited models, which are important tools for the quantitative assessment of bee colour vision and colour-guided behaviours. Major insights into the visual ecology of bees have been gained combining behavioural experiments and quantitative modelling, and asking how bee vision has influenced the evolution of flower colours and patterns. Recently research has focussed on the discrimination and categorisation of coloured patterns, colourful scenes and various other groupings of coloured stimuli, highlighting the bees' behavioural flexibility. The identification of perceptual mechanisms remains of fundamental importance for the interpretation of their learning strategies and performance in diverse experimental tasks.Biotechnology and Biological Sciences Research Council (BBSRC

Toxic but Drank: Gustatory Aversive Compounds Induce Post-ingestional Malaise in Harnessed Honeybees

BACKGROUND: Deterrent substances produced by plants are relevant due to their potential toxicity. The fact that most of these substances have an unpalatable taste for humans and other mammals contrasts with the fact that honeybees do not reject them in the range of concentrations in which these compounds are present in flower nectars. Here we asked whether honeybees detect and ingest deterrent substances and whether these substances are really toxic to them. RESULTS: We show that pairing aversive substances with an odor retards learning of this odor when it is subsequently paired with sucrose. Harnessed honeybees in the laboratory ingest without reluctance a considerable volume (20 µl) of various aversive substances, even if some of them induce significant post-ingestional mortality. These substances do not seem, therefore, to be unpalatable to harnessed bees but induce a malaise-like state that in some cases results in death. Consistently with this finding, bees learning that one odor is associated with sugar, and experiencing in a subsequent phase that the sugar was paired with 20 µl of an aversive substance (devaluation phase), respond less than control bees to the odor and the sugar. Such stimulus devaluation can be accounted for by the malaise-like state induced by the aversive substances. CONCLUSION: Our results indicate that substances that taste bitter to humans as well as concentrated saline solutions base their aversive effect on the physiological consequences that their ingestion generates in harnessed bees rather than on an unpalatable taste. This conclusion is only valid for harnessed bees in the laboratory as freely-moving bees might react differently to aversive compounds could actively reject aversive substances. Our results open a new possibility to study conditioned taste aversion based on post-ingestional malaise and thus broaden the spectrum of aversive learning protocols available in honeybees

Insect Bio-inspired Neural Network Provides New Evidence on How Simple Feature Detectors Can Enable Complex Visual Generalization and Stimulus Location Invariance in the Miniature Brain of Honeybees

This work was supported by a Human Frontier Science Program Grant RGP0022/2014 to LC and Queen Mary University of London Scholarship to MR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

from photoreceptor to behaviour

Ziel der vorliegenden Dissertation war es, zum Verständnis des visuellen Systems der Honigbiene Apis mellifera beizutragen. Dazu wurde die Expression eines Opsins mit Hilfe der Methode RNA Interferenz (RNAi) spezifisch herunterreguliert. Opsine stellen den Proteinanteil des photorezeptiven visuellen Pigments dar und determinieren die spektrale Empfindlichkeit der Photorezeptoren. Von den drei Rezeptortypen der Retina wurde der für langwelliges Licht empfindliche Photorezeptortyp (L-Rezeptor) als Untersuchungsobjekt gewählt, da er in der aufgabenspezifischen Kodierung von chromatischer und achromatischer Information eine wichtige Rolle spielt. Der Erfolg der Inhibition wurde mit molekularbiologischen und elektrophysiologischen Methoden untersucht. Zusätzlich wurde ein verhaltensbiologischer Test entwickelt, um das Farbensehen und –lernen in fixierten Tieren untersuchen zu können. Die Evaluation der Wirkung von RNAi auf die mRNA-Menge des für langwelliges Licht empfindlichen Opsins (LW-Opsin) mit Real Time PCR ergab, dass eine Hemmung um bis zu 60% erreicht werden konnte. Die Wirkung war zeitlich auf einige Stunden und lokal auf die injizierte Retina begrenzt. Überdies wurde eine Mindestmenge an doppelsträngiger RNA benötigt, damit eine Hemmung detektiert werden konnte. Der Gehalt an LW-Opsin Protein wurde mit Hilfe eines für das LW-Opsin der Hummel entwickelten Antiköpers durch SDS-PAGE und Western Blot quantifiziert. Die Untersuchung von unbehandelten Tieren offenbarte eine natürliche Schwankung der LW-Opsin Proteinmenge im Tagesverlauf, mit einem Maximum acht Stunden nach Beginn der Photoperiode und einen folgenden Abfall um fast 50%. Zwölf Stunden nach der Injektion von spezifischer doppelsträngiger RNA war die Proteinmenge relativ zu Kontrolltieren um ca. 25% reduziert wenn die Injektion am Morgen stattfand. Eine Injektion am Abend rief dagegen keine statistisch signifikante Wirkung hervor, ebenso war in späteren Messungen keine Reduktion des Proteins messbar. In der Messung retinaler Summenpotentiale (Elektroretinogramme, ERGs) wirkte sich die RNAi 12 und 24 Stunden nach Injektion nicht nachweisbar auf die Amplitude der Reizantwort auf langwelliges (grünes) Licht in Relation zur Antwort auf mittel- und kurzwellige Stimulation (blau und UV) aus. Der Vergleich der Form der Reizantworten zwischen der abendlichen und der morgendlichen Messung offenbarte jedoch eine Veränderung in der Relation der ERG-Komponenten zueinander. Diese Veränderung fiel in Experimental- und Kontrolltieren unterschiedlich aus. Die Ergebnisse erweitern und bestätigen die bisher bekannten Hinweise, dass das visuelle System der Honigbiene einem zirkadianen Rhythmus unterliegt, charakterisiert durch ein tageszeitabhängiges Volumen der Proteinmenge des LW-Opsins sowie die Ausprägung verschiedener Komponenten des ERGs. Auch die unterschiedliche Wirksamkeit der RNAi zu verschiedenen Zeitpunkten kann möglicherweise auf den zirkadianen Rhythmus zurückgeführt werden. Ferner wurde im Rahmen dieser Arbeit das Farbenlernen der Honigbiene durch die Weiterentwicklung einer Dressurmethode für fixierte Tiere untersucht. Die Fixierung der Tiere ist von Vorteil für kombinierte Untersuchungen auf physiologischer und Verhaltensebene. Bei der klassischen Konditionierung auf visuelle Stimuli lernten die Tiere die Farbe des belohnten Stimulus, nicht jedoch dessen Intensität. Es zeigte sich, dass die Diskriminationsleistung in fixierten Tieren schwächer war als in frei fliegenden Tieren. Die Art der Konditionierung (absolut oder differentiell) beeinflusste die Stärke der Generalisierung. Für die gelernte Farbe bildeten die Tiere ein stabiles Gedächtnis, das mindestens eine Stunde anhielt.The present thesis aims to widen our understanding of the visual system of the honeybee Apis mellifera by new molecular biological and behavioral methods. RNA Interference (RNAi) was used to specifically down-regulate the expression of an opsin being the protein component of the photosensitive visual pigment in the photoreceptor membrane and determining the receptor’s spectral sensitivity. There are three different types of photoreceptors in the bee retina. The L-receptor, sensitive to long wavelength, was chosen as target because of its main role in the coding of chromatic and achromatic visual information. The inhibition was assessed by molecular biological and electrophysiological methods. In addition a behavioral test was developed to investigate color perception and learning in restrained animals. The effect of RNAi on the expression of the long-wavelength sensitive opsin (LW opsin) mRNA was evaluated by Real Time PCR revealing a down-regulation of up to 60%. The effect was transient and restricted to the injected retina. A minimum amount of 5µg of double-stranded RNA was needed to detect a significant inhibition. The amount of LW opsin protein was quantified by means of an antibody developed for the LW opsin of a bumblebee using SDS PAGE and Western Blots. Repetitive sampling of non-injected animals revealed a natural oscillation of the protein within 24 hours, with a peak eight hours after beginning of the light period and a subsequent decrease of almost 50%. Twelve hours after the injection of double-stranded RNA a reduction effect of about 25% was measured when the injection occurred at the beginning of the light phase. If the treatment was administered at the end of the light phase, no down-regulation was detected after the same period of time of 12 hours, nor after longer time periods. The effect of RNAi on the signal responses in the bee retina was assessed through recording electroretinograms (ERG) 12 and 24 hours after a morning injection. No reduction was detectable when the amplitude of responses to long wavelength (green light) were normalized against the responses to short and medium wavelength (UV and blue light). However, the shape of the ERG response to the light stimuli changed between the two measurements through the differential expression of the transient components of the ERG. These changes were different in experimental and control groups. These results supplement and confirm earlier established findings that indicate influences of circadian rhythm on visual functions in the visual system of the honeybee. These effects have been characterized here by a change in the amount of LW-opsin protein and the shape of the ERG over the day. The difference in the efficiency of the RNAi is most likely also related to the circadian rhythm. In addition, I further developed a behavioral test for the investigation of color learning in restrained honeybees. Restraining the animals bears the possibility of simultaneous investigation of physiological processes and behavior. The classical conditioning to colored light stimuli revealed that the animals learned the colour of a light stimulus but not its intensity. Comparing the results obtained to the performance of free-flying bees, colour discrimination was poor. In addition, a strong tendency for colour generalization was revealed. This generalization depended partly on the conditioning procedure, e.g. whether bees were conditioned absolutely or differentially. Bees formed a stable memory for the learned colour that lasted at least for one hour

Colour-dependent target detection by bees

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