The effects of genotype, foraging role, and sucrose responsiveness on the tactile learning performance of honey bees (Apis mellifera L.)

We analyzed sucrose responsiveness and associative tactile learning in two genetic strains of honey bees under laboratory conditions. These strains differ in their foraging behavior. Bees of the "high" strain preferentially collect pollen. "Low"-strain bees mainly forage for nectar. Responsiveness to different sucrose concentrations and tactile learning were examined using the proboscis extension reflex. Acquisition, extinction of conditioned responses, and responses to an alternative tactile stimulus were tested. High-strain bees are more responsive to sucrose than low-strain bees. Regardless of genotype, pollen foragers are more responsive to sucrose than nectar foragers. In bees of both strains we find the same relationship between responsiveness to sucrose and acquisition. Bees responding to low sucrose concentrations show more often the conditioned response during acquisition than those responding only to higher sucrose concentrations. Extinction of conditioned responses depends on the response probability during acquisition. Discrimination between the two tactile stimuli is affected by genotype but not by responsiveness to sucrose. High-strain bees discriminate better than low-strain bees. Our experiments thus establish links between division of labor, responsiveness to sucrose, and associative learning in honey bees.     

Learning in honey bees with brain lesions: how partial mushroom-body ablations affect sucrose responsiveness and tactile antennal learning

Honey bees are ideal organisms for studying associative learning, because they can rapidly learn different sensory cues, even under laboratory conditions. Classical olfactory learning experiments have shown that the mushroom bodies (MBs), a prominent neuropil of the central nervous system of the bee, are involved in olfactory learning and memory formation. We tested whether the MBs are also involved in tactile antennal learning. As in olfactory learning, bees use the antennae during tactile learning to sense tactile cues. We produced specific MB ablations by applying the mitotic blocker hydroxyurea (HU). In Drosophila, HU-induced brain lesions of the MBs strongly impaired olfactory memory formation. As treatment with HU might also interfere with the processing of the reward stimulus, sucrose, we measured the responsiveness to sucrose stimuli in these bees. Treatment with HU led to partial ablations of the median MB sub-units on one or both sides of the brain. We analysed side-specific effects in double-blind tests, testing sucrose responsiveness separately for each antenna, and conditioning first one antenna and then the other in a reversal learning assay. HU-treated bees without detectable ablations were less responsive to sucrose and had a poorer learning performance than untreated controls. Partial MB ablation did not additionally affect responsiveness to sucrose or tactile antennal learning. Interestingly, bees with bilateral MB ablations did not differ from untreated controls in their learning performance during the first learning phase. During reversal learning, acquisition in these bees was significantly lower than that in untreated controls. It is concluded that HU treatment affects sucrose responsiveness and tactile learning even without detectable ablation of neuropils. The effects of MB ablations on tactile learning are not side-specific and not correlated with the volume of the ablated neuropil. Accepted after revision: 15 January 2001 ❚ Electronic Publication     

Peak shift in honey bee olfactory learning

If animals are trained with two similar stimuli such that one is rewarding (S+) and one punishing (S?), then following training animals show a greatest preference not for the S+, but for a novel stimulus that is slightly more different from the S? than the S+ is. This peak shift phenomenon has been widely reported for vertebrates and has recently been demonstrated for bumblebees and honey bees. To explore the nature of peak shift in invertebrates further, here we examined the properties of peak shift in honey bees trained in a free-flight olfactory learning assay. Hexanal and heptanol were mixed in different ratios to create a continuum of odour stimuli. Bees were trained to artificial flowers such that one odour mixture was rewarded with 2 molar sucrose (S+), and one punished with distasteful quinine (S?). After training, bees were given a non-rewarded preference test with five different mixtures of hexanal and heptanol. Following training bees’ maximal preference was for an odour mixture slightly more distinct from the S? than the trained S+. This effect was not seen if bees were initially trained with two distinct odours, replicating the classic features of peak shift reported for vertebrates. We propose a conceptual model of how peak shift might occur in honey bees. We argue that peak shift does not require any higher level of processing than the known olfactory learning circuitry of the bee brain and suggest that peak shift is a very general feature of discrimination learning.     

Color modulates olfactory learning in honeybees by an occasion-setting mechanism

A sophisticated form of nonelemental learning is provided by occasion setting. In this paradigm, animals learn to disambiguate an uncertain conditioned stimulus using alternative stimuli that do not enter into direct association with the unconditioned stimulus. For instance, animals may learn to discriminate odor rewarded from odor nonrewarded trials if these two situations are indicated by different colors that do not themselves become associated with the reward. Despite a growing interest in nonelemental learning in insects, no study has so far attempted to study occasion setting in restrained honeybees, although this would allow direct access to the neural basis of nonelemental learning. Here we asked whether colors can modulate olfactory conditioning of the proboscis extension reflex (PER) via an occasion-setting mechanism. We show that intact, harnessed bees are not capable of learning a direct association between color and sucrose. Despite this incapacity, bees solved an occasion-setting discrimination in which colors set the occasion for appropriate responding to an odor that was rewarded or nonrewarded depending on the color. We therefore provide the first controlled demonstration of bimodal (color-odor) occasion setting in harnessed honeybees, which opens the door for studying the neural basis of such bimodal, nonelemental discriminations in insects.     

Associative mechanosensory conditioning of the proboscis extension reflex in honeybees

The present work introduces a form of associative mechanosensory conditioning of the proboscis extension reflex (PER) in honeybees. In our paradigm, harnessed honeybees learn the elemental association between mechanosensory, antennal stimulation and a reward of sucrose solution delivered to the proboscis. Thereafter, bees extend their proboscis to the antennal mechanosensory stimulation alone. We show that bees can learn such an association in a side-specific manner, that is, they learn the association on the antennal side that was rewarded and not on the side that was not rewarded. Responding produced by the paired training does likely contain a substantial Pavlovian component. Responding is only elicited by mechanosensory stimulation and not by spurious cues such as olfactory, visual, and contextual ones. The interstimulus interval (ISI) affects one-trial mechanosensory learning: a bell-shaped curve with a maximum of responding approximately 4 sec ISI was obtained. Mechanosensory memory is still operative 24 h after conditioning. Apart from absolute conditioning in which mechanosensory stimulation of one antenna is paired with sucrose, differential, side-specific, mechanosensory conditioning using two mechanosensory stimulations, one rewarded and the other not, is also possible. This paradigm constitutes, therefore, a new standard procedure for further learning studies in honeybees.     

Differences in performance on a reversal learning test and division of labor in honey bee colonies

We studied the association between honey bee (Apis mellifera) division of labor and performance on an olfactory reversal-learning test. Manipulations of colony age structure and flight experience were used to test whether differences in performance are associated with age, current behavioral state, or flight experience. Nurse bees showed significantly faster rates of extinction to a learned odor than did foragers. This difference was associated primarily with differences in behavioral state, rather than age; it was seen when comparing nurses and foragers from typical colonies and normal-age nurses and precocious foragers from single-cohort colonies. Differences in extinction rate were not related to differences in flight experience; there was no difference between foragers and foraging-age bees denied flight experience. These results suggest that changes in learning and memory occur in association with division of labor. We speculate on the possible functional significance of the difference in extinction rate between nurses and foragers. Received: 15 January 2000 / Accepted: 22 August 2000     

Development of an ethanol model using social insects: III. Preferences for ethanol solutions

Experiments are designed to assess whether free-flying honey bees have an aversion to an ethanol solution when given a choice between targets containing an ethanol solution in sucrose or sucrose only. Animals given a choice between a 1% ethanol solution and sucrose only show no aversion to the ethanol solution either in acquisition or extinction. Honey bees given a choice between a 5% ethanol solution and sucrose only show no differences in the initial choice of targets but some ees do switch over to the sucrose-only target. Performance during extinction indicates that bees landed on the previously reinforced sucrose-only target more than the target previously containing the 5% ethanol solution. An experiment in which bees were given a single 5%, ethanol target showed that of 20 bees, 11 returned for the entire 12 trials of the experiment. All bees returned at least 6 times to the 5% ethanol target. Additional experiments were run on harnessed foragers in a palatability study of alcoholic beverages consumed by humans. The results of the palatability experiment indicate that in general, bees prefer more sweet drinks with less alcohol.     

The glass is not yet half empty: agitation but not <Emphasis Type="Italic">Varroa</Emphasis> treatment causes cognitive bias in honey bees

Honey bees (Apis mellifera) are prone to judge an ambiguous stimulus negatively if they had been agitated through shaking which simulates a predator attack. Such a cognitive bias has been suggested to reflect an internal emotional state analogous to humans who judge more pessimistically when they do not feel well. In order to test cognitive bias experimentally, an animal is conditioned to respond to two different stimuli, where one is punished while the other is rewarded. Subsequently a third, ambiguous stimulus is presented and it is measured whether the subject responds as if it expects a reward or a punishment. Generally, it is assumed that negative experiences lower future expectations, rendering the animals more pessimistic. Here we tested whether a most likely negatively experienced formic acid treatment against the parasitic mite Varroa destructor also affects future expectations of honey bees. We applied an olfactory learning paradigm (i.e., conditioned proboscis extension response) using two odorants and blends of these odorants as the ambiguous stimuli. Unlike agitating honey bees, exposure to formic acid did not significantly change the response to the ambiguous stimuli in comparison with untreated bees. Overall evidence suggests that the commonest treatment against one of the most harmful bee pests has no detrimental effects on cognitive bias in honey bees.     

Acute ethanol ingestion impairs appetitive olfactory learning and odor discrimination in the honey bee

Invertebrates are valuable models for increasing our understanding of the effects of ethanol on the nervous system, but most studies on invertebrates and ethanol have focused on the effects of ethanol on locomotor behavior. In this work we investigate the influence of an acute dose of ethanol on appetitive olfactory learning in the honey bee (Apis mellifera), a model system for learning and memory. Adult worker honey bees were fed a range of doses (2.5%, 5%, 10%, or 25%) of ethanol and then conditioned to associate an odor with a sucrose reward using either a simple or differential conditioning paradigm. Consumption of ethanol before conditioning significantly reduced both the rate of acquisition and the asymptotic strength of the association. Honey bees also exhibited a dose dependent reduction in arousal/attention during conditioning. Consumption of ethanol after conditioning did not affect recall 24h later. The observed deficits in acquisition were not due to the affect of ethanol on gustatory sensitivity or motor function. However, honey bees given higher doses of ethanol had difficulty discriminating amongst different odors suggesting that ethanol consumption influences olfactory processing. Taken together, these results demonstrate that an acute dose of ethanol affects appetitive learning and olfactory perception in the honey bee.     

Side-specificity of olfactory learning in the honeybee: generalization between odors and sides

Honeybees (Apis mellifera) can be trained to associate an odor stimulus with a sucrose reward. The neural structures involved in the detection and integration of olfactory stimuli are represented bilaterally in the brain. Little is known about the respective roles of the two sides of the brain in olfactory learning. Does each side learn independently of the other, or do they communicate, and if so, to what extent and at what level of neural integration? We addressed these questions using the proboscis extension response (PER) conditioning paradigm applied in a preparation that allows the separation of the two input sides during olfactory stimulations. Bees conditioned to two odorants A and B, one being learned on each side (A+/B+ training), showed in extinction tests rather unspecific responses: They responded to both odorants on both sides. This could be attributable to either a transfer of the learned information between sides, or to a generalization between odorants on each side. By subjecting bees to conditioning on one side only (A+/0 training), we found that the learned information is indeed transferred between sides. However, when bees were trained explicitly to give opposite values to the two odorants on the two sides (A+B-/B+A- training), they showed clear side-specific response patterns to these odorants. These results are used in the elaboration of a functional model of laterality of olfactory learning and memory processing in the honeybee brain.     

Decision-making and associative color learning in harnessed bumblebees (Bombus impatiens)

In honeybees, the conditioning of the proboscis extension response (PER) has provided a powerful tool to explore the mechanisms underlying olfactory learning and memory. Unfortunately, PER conditioning does not work well for visual stimuli in intact honeybees, and performance is improved only after antennal amputation, thus limiting the analysis of visual learning and multimodal integration. Here, we study visual learning using the PER protocol in harnessed bumblebees, which exhibit high levels of odor learning under restrained conditions. We trained bumblebees in a differential task in which two colors differed in their rewarding values. We recorded learning performance as well as response latency and accuracy. Bumblebees rapidly learned the task and discriminated the colors within the first two trials. However, performance varied between combinations of colors and was higher when blue or violet was associated with a high reward. Overall, accuracy and speed were negatively associated, but both components increased during acquisition. We conclude that PER conditioning is a good tool to study visual learning, using Bombus impatiens as a model, opening new possibilities to analyze the proximate mechanisms of visual learning and memory, as well as the process of multimodal integration and decision-making.     

Configural olfactory learning in honeybees: negative and positive patterning discrimination

In an appetitive context, honeybees (Apis mellifera) learn to associate odors with a reward of sucrose solution. If an odor is presented immediately before the sucrose, an elemental association is formed that enables the odor to release the proboscis extension response (PER). Olfactory conditioning of PER was used to study whether, beyond elemental associations, honeybees are able to process configural associations. Bees were trained in a positive and anegative patterning discrimination problem. In the first problem, single odorants were nonreinforced whereas the compound was reinforced. In the second problem, single odorants were reinforced whereas the compound was nonreinforced. We studied whether bees can solve these problems and whether the ratio between the number of presentations of the reinforced stimuli and the number of presentations of the nonreinforced stimuli affects discrimination. Honeybees differentiated reinforced and nonreinforced stimuli in positive and negative patterning discriminations. They thus can process configural associations. The variation of the ratio of reinforced to nonreinforced stimuli modulated the amount of differentiation. The assignment of singular codes to complex odor blends could be implemented at the neural level: When bees are stimulated with odor mixtures, the activation patterns evoked at the primary olfactory neuropile, the antennal lobe, may be combinations of the single odorant responses that are not necessarily fully additive.     

Sampling and decision rules used by honey bees in a foraging arena

Animals must continuously choose among various available options to exploit the most profitable resource. They also need to keep themselves updated about the values of all available options, since their relative values can change quickly due to depletion or exploitation by competitors. While the sampling and decision rules by which foragers profitably exploit a flower patch have attracted a great deal of attention in theory and experiments with bumble bees, similar rules for honey bee foragers, which face similar foraging challenges, are not as well studied. By presenting foragers of the honey bee Apis cerana with choice tests in a foraging arena and recording their behavior, we investigate possible sampling and decision rules that the foragers use to choose one option over another and to track other options. We show that a large part of the sampling and decision-making process of a foraging honey bee can be explained by decomposing the choice behavior into dichotomous decision points and incorporating the cost of sampling. The results suggest that a honey bee forager, by using a few simple rules as part of a Bayesian inference process, is able to effectively deal with the complex task of successfully exploiting foraging patches that consist of dynamic and multiple options.     

Drosophila Mushroom Bodies Are Dispensable for Visual, Tactile, and Motor Learning

A total of 18 associative learning/memory tests have been applied to Drosophila melanogaster flies lacking mushroom bodies. Only in paradigms involving chemosensory cues as conditioned stimuli have flies been found to be compromised by a block in the mushroom body pathway. Among the learning tasks not requiring these structures are a case of motor learning (yaw torque/heat), a test of the fly’s spatial orientation in total darkness, conditioned courtship suppression by mated females, and nine different examples of visual learning. The latter used the reinforcers of heat, visual oscillations, mechanical shaking, or sucrose, and as conditioned stimuli, color, intensity contrast, as well as stationary and moving visual patterns. No forms of consolidated memory have been tested in mushroom body-less flies. With respect to short-term memory the mushroom bodies of Drosophila are specially required for chemosensory learning tasks, but not for associative learning and memory in general.     

Brain composition and olfactory learning in honey bees

Correlations between brain or brain component size and behavioral measures are frequently studied by comparing different animal species, which sometimes introduces variables that complicate interpretation in terms of brain function. Here, we have analyzed the brain composition of honey bees (Apis mellifera) that have been individually tested in an olfactory learning paradigm. We found that the total brain size correlated with the bees’ learning performance. Among different brain components, only the mushroom body, a structure known to be involved in learning and memory, showed a positive correlation with learning performance. In contrast, visual neuropils were relatively smaller in bees that performed better in the olfactory learning task, suggesting modality-specific behavioral specialization of individual bees. This idea is also supported by inter-individual differences in brain composition. Some slight yet statistically significant differences in the brain composition of European and Africanized honey bees are reported. Larger bees had larger brains, and by comparing brains of different sizes, we report isometric correlations for all brain components except for a small structure, the central body.     

Comparative evaluations of reward dimensions in honey bees: evidence from two-alternative forced choice proboscis-extension conditioning

A major challenge in understanding choice behaviour is determining how subjects evaluate alternatives that differ along multiple dimensions. Of particular interest is whether similar dimensions are compared to each other or whether each alternative is assigned an absolute value (utility). We assumed that choice proportions would follow Weber’s effect, according to which discrimination is proportional to relative difference (difference/mean). We tested honey bees in a two-alternative forced choice (2AFC) paradigm of proboscis-extension response (PER) conditioning. Subjects were conditioned over six trials to associate each of two odours with sucrose solution rewards and then tested in a choice trial between the two alternatives. Each group of subjects was tested in one treatment, and there were four treatments in each of six experiments. Rewards differed in delay, duration of feeding, and sucrose concentration. In each treatment, the high-profitability alternative was better than the low-profitability alternative along a single dimension, but between treatments of each experiment values in another dimension monotonically increased. Proboscis-response proportions during the conditioning phase tended to be greater for the high-profitability alternative, and choice proportions for it in the choice phase ranged between 0.72 and 0.89 in the 24 treatments. We show for the first time that harnessed bees are sensitive to reward delay. Preferences did not differ statistically between the different treatments of any of the experiments. The results support comparative evaluation of alternatives and are pertinent to multi-attribute choice, with implications for context-dependent preferences. We also discuss the potential advantages of the 2AFC PER simultaneous choice assay.     

A test of transitive inferences in free-flying honeybees: unsuccessful performance due to memory constraints

We asked whether honeybees, Apis mellifera, could solve a transitive inference problem. Individual free-flying bees were conditioned with four overlapping premise pairs of five visual patterns in a multiple discrimination task (A+ vs. B-, B+ vs. C-, C+ vs. D-, D+ vs. E-, where + and - indicate sucrose reward or absence of it, respectively). They were then tested with the nonadjacent pairs A vs. E and B vs. D. Preference of B to D is consistent with the use of the implicit hierarchy A > B > C > D > E. Equal choice of B and D supports choice based on the associative strength of the stimuli. The bees' choice was determined by their memory constraints: experience with the last premise pair (D+ vs. E-) predominated. In the tests, bees preferred A to E and chose equally B and D. An analysis of the performance in terms of a reward/penalty ratio showed that B had a higher associative strength than D. Thus, bees do not establish transitive inferences but, rather, guide their choices by the joint action of a recency effect and the associative strength of the stimuli. The former supports choice of D, whereas the latter supports choice of B, thus determining equal choice of B and D in the tests.     

Interaction of the Dopaminergic System With Mechanisms of Associative Learning and Cognition:Implications for Drug Abuse

The possible role of mechanisms of associative learning in drug abuse and addiction is considered with respect to psychomotor stimulant drugs such as cocaine and amphetamine. Although the initial sites of the reinforcing effects of these drugs are associated with the mesolimbic dopamine projection to the nucleus accumbens, other important neuromodulatory influences are identified. These include the amygdala, which appears to mediate the associative processes by which environmental stimuli come to control drug-seeking behavior. Possible parallel mechanisms by which stimulant drugs enhance effects of aversive as well as rewarding stimuli are discussed. The importance of attribution and context in determining relative rewarding and aversive effects is pointed out. These attributional and contextual factors may be a function of cortical regions that may themselves be impaired by chronic drug abuse, leading to further dysexecutive control over cognition and behavior.     

Associative recognition processes are modulated by the semantic unitizability of memoranda

Although memory of episodic associations is generally considered to be recollective in nature, it has been suggested that when stimuli are experienced as a unit, familiarity processes might contribute to their subsequent associative recognition. To investigate the effect of semantic relatedness during episodic encoding on the processes of retrieval of associative information, we had participants interactively encode pairs of object pictures, vertically arranged so as to suggest a functional or configural relationship between them. Half the pairs were independently judged to be of related objects (e.g., a lamp over a table) and half of unrelated objects (e.g., a key-ring over an apple). At test, participants discriminated between intact, recombined, and new pairs while event related potentials (ERPs) were recorded. In an early ERP marker of retrieval success generally associated with familiarity processes, differences related to associative memory only emerged for related pairs, while differences associated with item memory emerged for both related and unrelated pairs. In contrast, in a later ERP effect associated with recollection, differences related to associative memory emerged for both related and unrelated pairs. These findings may indicate that retrieval of episodic associations formed between two semantically related visual stimuli can be supported by familiarity-related processes.     

Do inexperienced bumblebee foragers use scent marks as social information?

Bumblebees (Bombus spp.) foraging in the field typically reject flowers where they detect the olfactory footprints of previous visitors and hence avoid recently emptied inflorescences. A growing number of studies have begun to illustrate that associative learning shapes the development of this process, in both bumblebees and other bee species. This raises the question of what the default response to such marks is, but little is known about how inexperienced foragers use social information. Here, we offered flower-naive bees a choice between scent-marked flowers and unmarked alternatives and found that individuals neither avoided nor preferred marked flowers. Our findings provide no support for ‘hard-wired’ responses to scent marks in bumblebees and highlight the importance of associative learning in shaping social information use to match local circumstances.