Delayed symbolic matching in honeybees (Apis mellifera)

In a recent experiment on short-term memory (P. A. Couvillon, T. P. Ferreira, & M. E. Bitterman, 2003), honeybees (Apis mellifera) learned to choose between 2 colors on the basis of immediately preceding experience with 1 of them. Some learned to choose the same color as the sample (perseveration or matching), others to choose the alternative color (alternation or nonmatching). Performance in the 2 problems was very much the same. In the present experiment, honeybees learned no less readily to choose between the 2 colors on the basis of sample stimuli that were different from the colors (symbolic matching). A simple associative interpretation of the results is proposed.     

Simultaneous conditioning in honeybees (Apis mellifera).

Honeybees (Apis mellifera) were classically conditioned with odor as conditioned stimulus (CS), sucrose as unconditioned stimulus (US), and proboscis extension as response. The purpose of Experiment 1 (Ns = 26 and 27) was to look for facilitation of forward conditioning by CS-US overlap, but rapid conditioning without overlap left little room for improvement. In 2 further experiments, CS and US were simultaneous, and response to odor alone was measured in subsequent tests. In Experiment 2, a Simultaneous group (N = 25) responded more to the training odor than did an Unpaired control group (N = 25). In Experiment 3, a differentially conditioned Simultaneous group (N = 29) responded more to an odor paired with sucrose in training (S+) than to an odor presented alone (S-). The implications of the results for the problem of the role of amount of reward in honeybee learning are considered.     

Learning in honeybees (Apis mellifera) as a function of sucrose concentration.

Foraging honeybees (Apis mellifera) were trained with 2 successively presented targets differing in color or odor, one of which always contained a 5-microliters drop of 50% sucrose solution and the other, a 5-microliters drop of 20% sucrose solution. Latency of response to each target was measured during the training, and at the conclusion, preference was measured in an unrewarded choice test. Analysis of the latencies showed both a prospective effect (faster response to the 50% target than to the 20% target) and a nonassociative retrospective effect (faster response after leaving the 20% target than after leaving the 50% target) reminiscent of the frustration effect in rats. The results both for prospective latency and for choice can be understood on the simple theory that the attractiveness of a target depends on the strength of its association with sucrose and that the effect of concentration is on asymptotic strength.     

Conditional withholding of proboscis extension in honeybees (Apis mellifera) during discriminative punishment.

Proboscis extension conditioning of honeybee workers was used to test the ability of bees to respond to appetitive and aversive stimuli while restrained in a harness that allows subjects to move their antennae and mouthparts (Kuwabara, 1957; Menzel, Erber, & Masuhr, 1974). Subjects were conditioned to discriminate between two odors, one associated with sucrose feeding and the other associated with a 10 V AC shock if they responded to the sucrose unconditioned stimulus (US) in the context of that odor. Most Ss readily learned to respond to the odor followed by sucrose feeding and not to the odor associated with sucrose stimulation plus shock. Furthermore, in the context of the odor associated with shock, significantly more subjects withheld or delayed proboscis extension on stimulation with the sucrose US than they did in the context of the odor associated with feeding. Thus, restrained honeybees can readily learn to avoid shock according to an odor context by withholding proboscis extension to a normally powerful releaser. Analysis of individual learning curves revealed that subjects differed markedly in performance on this task. Some learn the discrimination quickly, whereas others show different kinds of response patterns.     

One retrieval trial induces reconsolidation in an appetitive learning paradigm in honeybees (Apis mellifera)

Combining memory retrieval with the application of a protein synthesis-inhibitor leads to an amnestic effect that is referred to as the reconsolidation phenomenon. Several behavioural studies demonstrate that only a few or weak retrieval trials (that do not result in significant extinction) lead to this phenomenon. In contrast, many trials (that result in significant extinction) combined with a protein synthesis inhibitor result in an inhibition of the extinction memory. Based on these findings it was suggested that extinction is the boundary condition for reconsolidation: when extinction is induced the consolidation of the extinction memory is the dominant process. Recently we were not able to confirm this hypothesis in the honeybee (Apis mellifera): We did not find the reconsolidation phenomenon after one retrieval trial, but demonstrated reconsolidation after five retrieval trials that led to extinction. To exclude that this observation resembles a special case in insects we here wanted to know if one retrieval trial induces reconsolidation as it has been demonstrated before in many other species. To do so we used experimental parameters that had been used before to demonstrate consolidation in the honeybee with the exception that this time the protein synthesis-inhibitor was applied 1 h after one memory retrieval instead after acquisition. We thereby demonstrate the reconsolidation phenomenon after one retrieval trial but only when using the doubled dose of protein synthesis-inhibitor that has been used to inhibit consolidation.     

Contrasting effects of imidacloprid on habituation in 7- and 8-day-old honeybees (Apis mellifera)

We examined the effects of sublethal doses (0.1, 1, and 10 ng per animal) of a new neonicotinoid insecticide, Imidacloprid, on habituation of the proboscis extension reflex (PER) in honeybees (Apis mellifera) reared under laboratory conditions. In untreated honeybees, the habituation of the proboscis extension reflex is age-dependent and there is a significant increase in the number of trials required for habituation in older bees (8-10 days old) as compared to very young bees (4-7 days old). Imidacloprid alters the number of trials needed to habituate the honeybee response to multiple sucrose stimulation. In 7-day-old bees, treatment with Imidacloprid leads to an increase in the number of trials necessary to abolish the response, whereas in 8-day-old bees, it leads to a reduction in the number of trials for habituation (15 min and 1 h after treatment), and to an increase 4 h after treatment. The temporal effects of Imidacloprid in both 7- and 8-day-old bees suggest that 4h after treatment the observed effects are due to a metabolite of Imidacloprid, rather than to Imidacloprid itself. Our results suggest the existence of two distinct subtypes of nicotinic receptors in the honeybee that have different affinities to Imidacloprid and are differentially expressed in 7- and 8-day-old individuals.     

Learning by honeybees (Apis mellifera) on arrival at and departure from a feeding place

The question of when in the course of a visit to a feeding place foraging honeybees (Apis mellifera) learn about its location was studied in Experiment 1 by moving the animals a short distance after they arrived and began to feed. A preference for the arrival place developed, although less rapidly than in control animals for which the arrival and departure places were the same. In Experiments 2-5, a distinctive object was used to define the location of the feeding place. When the object was removed after arrival or introduced only after arrival, the animals learned less about its color and shape than did control animals for which it was present throughout each visit. The results contradict the claim that honeybees learn about certain characteristics of a feeding place only on arrival and about others only on departure.     

Heritable variation for latent inhibition and its correlation with reversal learning in honeybees (Apis mellifera)

Latent inhibition (LI) in honeybees (Apis mellifera) was studied by using a proboscis extension response conditioning procedure. Individual queens, drones, and workers differed in the degree to which they revealed LI. The authors hypothesized that individual differences would have a substantial genetic basis. Two sets of progeny were established by crossing virgin queens and individual drones, both of which had been selected for differential expression of inhibition. LI was stronger in the progeny from the queens and drones that had shown greater inhibition. The expression of LI was also dependent on environmental factors that are most likely associated with age, foraging experience outside of the colony, or both. Furthermore, there was a correlated response in the speed at which progeny reversed a learned discrimination of 2 odors. These genetic analyses may reveal underlying mechanisms that these 2 learning paradigms have in common.     

Quantitative analysis of risk sensitivity in honeybees (Apis mellifera) with variability in concentration and amount of reward

Free-flying honeybees (Apis mellifera) were trained in a series of experiments designed to look for evidence of risk sensitivity in foraging for sucrose solution. The suitability of the choice method used was established in 3 preliminary experiments with differences in concentration, amount, and probability of reward. Of 5 subsequent experiments in which 2 alternatives provided the same mean concentration of sucrose solution with different variance, 3 showed risk indifference, and 2 showed risk aversion (preference for consistent reward). Of 2 final experiments in which the alternatives provided the same mean amount of sucrose solution with different variance, both showed risk aversion. Performance could be simulated quantitatively with a simple choice model developed by P. A. Couvillon and M. E. Bitterman (1991) to account for the results of a wide range of previous experiments on discriminative learning in honeybees.     

Delayed alternation in the pigeon

Pigeons were studied in a delayed-response task requiring alternation of key pecks on two response keys. Blackouts of from 1 to 10 sec intervened between successive choices on the two keys.

The following results were obtained: (1) Birds performed at well above chance accuracy on all the delays tested. Accuracy was generally lowest at 1- and 10-sec delays. (2) Overt postural orientations during the delay interval appeared to mediate accurate key-pecking behavior. (3) The shape of the delay vs. accuracy function was discussed in terms of the possibly confounding influences of (a) stimulus “trace” variables, and (b) aversive effects of the time outs produced by incorrect responding.

     

Evidence for associative learning in newly emerged honey bees (Apis mellifera)

The honey bee is a model organism for studies on the neural substrates of learning and memory. Associative olfactory learning using sucrose rewards is fast and reliable in foragers and older hive bees. However, researchers have so far failed to show any significant learning in newly emerged bees. It is generally argued that in these bees only part of the brain structures important for learning are fully developed. Here we show for the first time that newly emerged honey bees are capable of associative learning, if they are sufficiently responsive to sucrose. Responsiveness to sucrose, which can be measured using the proboscis extension response (PER), increases with age. Newly emerged bees are on average very unresponsive to sucrose. We show that if newly emerged bees displaying a PER to 10% sucrose or lower sucrose concentrations are conditioned to an odour, they show significant associative learning and early long-term memory. Nevertheless, the level of acquisition is still lower than in foragers. The general assumption that newly emerged honey bees are incapable of associative learning must therefore be reconsidered. Further, our study suggests that an age-dependent increase in responsiveness to rewarding stimuli is directly related to the development of early learning abilities. The decisive influence of responsiveness to rewarding stimuli in associative learning of newly emerged bees has far reaching consequences for studies on the development of associative learning capabilities in insects and vertebrates.     

Involvement of alpha-bungarotoxin-sensitive nicotinic receptors in long-term memory formation in the honeybee (Apis mellifera)

In the honeybee Apis mellifera, multiple-trial olfactory conditioning of the proboscis extension response specifically leads to long-term memory (LTM) which can be retrieved more than 24 h after learning. We studied the involvement of nicotinic acetylcholine receptors in the establishment of LTM by injecting the nicotinic antagonists mecamylamine (1 mM), alpha-bungarotoxin (alpha-BGT, 0.1 mM) or methyllycaconitine (MLA, 0.1 mM) into the brain through the median ocellus 20 min before or 20 min after multiple-trial learning. The retention tests were performed 1, 3, and 24 h after learning. Pre-training injections of mecamylamine induced a lower performance during conditioning but had no effect on LTM formation. Post-training injections of mecamylamine did not affect honeybees' performances. Pre-training injections of MLA or post-training injection of alpha-BGT specifically induced LTM impairment whereas acquisition as well as memory retrieval tested 1 or 3 h after learning was normal. This indicates that brain injections of alpha-BGT and MLA did not interfere with learning or medium-term memory. Rather, these blockers affect the LTM. To explain these results, we advance the hypothesis that honeybee alpha-BGT-sensitive acetylcholine receptors are also sensitive to MLA. These receptors could be essential for triggering intracellular mechanisms involved in LTM. By contrast, medium-term memory is not dependent upon these receptors but is affected by mecamylamine.     

Categorization of visual stimuli in the honeybee Apis mellifera

Categorization refers to the classification of perceptual input into defined functional groups. We present and discuss evidence suggesting that stimulus categorization can also be found in an invertebrate, the honeybee Apis mellifera, thus underlining the generality across species of this cognitive process. Honeybees show positive transfer of appropriate responding from a trained to a novel set of visual stimuli. Such a transfer was demonstrated for specific isolated features such as symmetry or orientation, but also for assemblies (layouts) of features. Although transfer from training to novel stimuli can be achieved by stimulus generalization of the training stimuli, most of these transfer tests involved clearly distinguishable stimuli for which generalization would be reduced. Though in most cases specific experimental controls such as stimulus balance and discriminability are still required, it seems appropriate to characterize the performance of honeybees as reflecting categorization. Further experiments should address the issue of which categorization theory accounts better for the visual performances of honeybees.This contribution is part of the special issue “Animal Logics” (Watanabe and Huber 2006).     

Honeybees (Apis mellifera) holding on to memories: response competition causes retroactive interference effects

Five experiments on honeybees examined how the learning of a second task interferes with what was previously learned. Free flying bees were tested for landmark-based memory in variations on a paradigm of retroactive interference. Bees first learned Task 1, were tested on Task 1 (Test 1), then learned Task 2, and were tested again on Task 1 (Test 2). A 60-min delay (waiting in a box) before Test 2 caused no performance decrements. If the two tasks had conflicting response requirements, (e.g., target right of a green landmark in Task 1 and left of a blue landmark in Task 2), then a strong decrement on Test 2 was found (retroactive interference effect). When response competition was minimised during training or testing, however, the decrement on Test 2 was small or nonexistent. The results implicate response competition as a major contributor to the retroactive interference effect. The honeybee seems to hold on to memories; new memories do not wipe out old ones.     

Pattern vision of the honeybee (Apis mellifera): blue and green receptors in the discrimination of translocation

The visual discrimination of horizontal gratings by the honeybee (Apis mellifera) was studied in a Y-choice apparatus with fixed patterns presented vertically at a set range. Translocation in this context is the exchange of the positions of two different colored or black areas. This paper investigates what cues the bees have learned in this task. The patterns, made from combinations of calibrated colored papers, are designed to explore the parts played by the blue and green receptors when the boundary between the two colors provides contrast to only one receptor type. Horizontal translocation is not discriminated without contrast to the green receptors, but up/down translocation can be discriminated whatever the contrast at the boundary. The trained bees were tested on the same patterns made with different papers that included extreme changes in contrast. The results show that discrimination of up/down translocation involves green receptors and also blue receptors. When bees discriminate a translocation that shows contrast to only one type of receptor, they do not use the apparent brightness or the direction of the contrast to that receptor type acting alone. Instead, they discriminate the locations of colored areas irrespective of intensity differences or directions of contrasts. They use some measure of the photon flux at both receptor types and remember the difference between the colors and their locations.     

The Attraction of Africanized Honey Bees (Apis mellifera L.) to Soft Drinks and Perfumes

The attraction of Africanized bees to soft drinks and perfumes was investigated. In the first experiment, the ability of bees to discriminate between two perfumes (Realm for Men and Realm for Women) was assessed. The results indicated that the scents could be used as conditioned stimuli. In the second experiment, bees were divided into individual groups and confined to small metal tubes. Groups were fed either Diet Pepsi, spring water (control), Diet Guaraná, Diet Coca-Cola, Coca-Cola, Sprite, Pepsi, Guaraná, Sukita Orange, Fanta Orange, Fanta Grape, or sucrose (control). The dependent variable was the amount of time the bee's proboscis was in contact with a strip of filter paper saturated with the substance. The results indicated little or no contact with the diet soft drinks and spring water, 45–65 s of contact with Coca-Cola, Sprite, Pepsi, Guaraná, and Sukita Orange, and 75–140 s of contact with Fanta Orange, Fanta Grape, and sucrose (sucrose elicited the most contact of the substances tested). In a third experiment, the ability of bees to associate an odor with a soft drink was assessed in a discrimination task. With Citral and Geraniol as conditioned stimuli, conditioning was best to sucrose, Guaraná, and Fanta Orange. Little conditioning was observed with Pepsi, and no conditioning was observed with Diet Pepsi.     

Blocking and pseudoblocking: the reply of Rattus norvegicus to Apis mellifera

Blaser, Couvillon, and Bitterman (2006) presented data obtained with honeybees that in principle challenged all traditional interpretations of blocking. They administered A + followed by either A + or + alone (where + indicates an unconditioned stimulus) and then tested on X. They observed less responding to X when they administered A + than when + alone was administered, a phenomenon they called "pseudoblocking". Here we examined pseudoblocking in a rat fear-conditioning preparation. In Experiment 1, using a control procedure that was similar to our usual blocking control, we obtained conventional blocking but failed to observe pseudoblocking in our analogue to Blaser et al.'s procedure. In Experiment 2, we used Blaser et al.'s control procedure and again failed to observe the pseudoblocking effect with rats when we used the experimental context as an analogue to the honeybee feeder used by Blaser et al. After reviewing their protocol and previously published studies from their laboratory, we hypothesized that the feeder that they treated as a training context probably served as a punctate cue. We also tested this possibility in Experiment 2, using a punctate cue as a surrogate feeder, and were now able to reproduce their pseudoblocking phenomena. Our results are consistent with a simple overshadowing account of pseudoblocking, within the framework of existing theories of associative learning, which is not applicable to the conventional blocking paradigm. Thus, blocking remains a real phenomenon that must be addressed by models of associative learning.     

Blocking and pseudoblocking: The reply of Rattus norvegicus to Apis mellifera

Blaser, Couvillon, and Bitterman (2006) presented data obtained with honeybees that in principle challenged all traditional interpretations of blocking. They administered A + followed by either A + or + alone (where + indicates an unconditioned stimulus) and then tested on X. They observed less responding to X when they administered A + than when + alone was administered, a phenomenon they called “pseudoblocking”. Here we examined pseudoblocking in a rat fear-conditioning preparation. In Experiment 1, using a control procedure that was similar to our usual blocking control, we obtained conventional blocking but failed to observe pseudoblocking in our analogue to Blaser et al.'s procedure. In Experiment 2, we used Blaser et al.'s control procedure and again failed to observe the pseudoblocking effect with rats when we used the experimental context as an analogue to the honeybee feeder used by Blaser et al. After reviewing their protocol and previously published studies from their laboratory, we hypothesized that the feeder that they treated as a training context probably served as a punctate cue. We also tested this possibility in Experiment 2, using a punctate cue as a surrogate feeder, and were now able to reproduce their pseudoblocking phenomena. Our results are consistent with a simple overshadowing account of pseudoblocking, within the framework of existing theories of associative learning, which is not applicable to the conventional blocking paradigm. Thus, blocking remains a real phenomenon that must be addressed by models of associative learning.     

Delayed alternation in the rat: Effect of contextual stimuli on proactive interference

The effect of contextual stimuli on proactive interference in the rat was investigated in three experiments. The task was a modified delayed alternation procedure in which the rat was initially forced to one side of a T maze (the interfering forcing) followed by a forcing to the opposite side (the target forcing). A free choice run followed the target forcing in which a turn in either direction could be made. In order to obtain reinforcement on the free choice run, the rat was required to turn in the direction opposite that of the target forcing (i.e., in the same direction as the interfering forcing). Manipulation of the context prevailing during the interfering forcing, target forcing, and free choice runs revealed that free choice accuracy was (a) greater when the interfering and target forcings were conducted in different contexts than when they were conducted in the same context and (b) influenced only slightly, if at all, by whether the free choice context coincided with that prevailing during the interfering forcing or with that prevailing during the target forcing.