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The superiority of spaced over massed learning is an established fact in the formation of long-term memories (LTM). Here we addressed the cellular processes and the temporal demands of this phenomenon using a weak spatial object recognition (wSOR) training, which induces short-term memories (STM) but not LTM. We observed SOR-LTM promotion when two identical wSOR training sessions were spaced by an inter-trial interval (ITI) ranging from 15 min to 7 h, consistently with spaced training. The promoting effect was dependent on neural activity, protein synthesis and ERKs1/2 activity in the hippocampus. Based on the "behavioral tagging" hypothesis, which postulates that learning induces a neural tag that requires proteins to induce LTM formation, we propose that retraining will mainly retag the sites initially labeled by the prior training. Thus, when weak, consecutive training sessions are experienced within an appropriate spacing, the intracellular mechanisms triggered by each session would add, thereby reaching the threshold for protein synthesis required for memory consolidation. Our results suggest in addition that ERKs1/2 kinases play a dual role in SOR-LTM formation after spaced learning, both inducing protein synthesis and setting the SOR learning-tag. Overall, our findings bring new light to the mechanisms underlying the promoting effect of spaced trials on LTM formation.

Honeybees are a standard model for the study of appetitive learning and memory. Yet, fewer attempts have been performed to characterize aversive learning and memory in this insect and uncover its molecular underpinnings. Here, we took advantage of the positive phototactic behavior of bees kept away from the hive in a dark environment and established a passive-avoidance task in which they had to suppress positive phototaxis. Bees placed in a two-compartment box learned to inhibit spontaneous attraction to a compartment illuminated with blue light by associating and entering into that chamber with shock delivery. Inhibitory learning resulted in an avoidance memory that could be retrieved 24 h after training and that was specific to the punished blue light. The memory was mainly operant but involved a Pavlovian component linking the blue light and the shock. Coupling conditioning with transcriptional analyses in key areas of the brain showed that inhibitory learning of phototaxis leads to an up-regulation of the dopaminergic receptor gene Amdop1 in the calyces of the mushroom bodies, consistently with the role of dopamine signaling in different forms of aversive learning in insects. Our results thus introduce new perspectives for uncovering further cellular and molecular underpinnings of aversive learning and memory in bees. Overall, they represent an important step toward comparative learning studies between the appetitive and the aversive frameworks., (© 2019 Marchal et al.; Published by Cold Spring Harbor Laboratory Press.)

Ants have recently emerged as useful models for the study of olfactory learning. In this framework, the development of a protocol for the appetitive conditioning of the maxilla-labium extension response (MaLER) provided the possibility of studying Pavlovian odor-food learning in a controlled environment. Here we extend these studies by introducing the first Pavlovian aversive learning protocol for harnessed ants in the laboratory. We worked with carpenter ants Camponotus aethiops and first determined the capacity of different temperatures applied to the body surface to elicit the typical aversive mandible opening response (MOR). We determined that 75°C is the optimal temperature to induce MOR and chose the hind legs as the stimulated body region because of their high sensitivity. We then studied the ability of ants to learn and remember odor-heat associations using 75°C as the unconditioned stimulus. We studied learning and short-term retention after absolute (one odor paired with heat) and differential conditioning (a punished odor versus an unpunished odor). Our results show that ants successfully learn the odor-heat association under a differential-conditioning regime and thus exhibit a conditioned MOR to the punished odor. Yet, their performance under an absolute-conditioning regime is poor. These results demonstrate that ants are capable of aversive learning and confirm previous findings about the different attentional resources solicited by differential and absolute conditioning in general., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Honeybees defend their colonies aggressively against intruders and release a potent alarm pheromone to recruit nestmates into defensive tasks. The effect of floral odours on this behaviour has never been studied, despite the relevance of these olfactory cues for the biology of bees. Here we use a novel assay to investigate social and olfactory cues that drive defensive behaviour in bees. We show that social interactions are necessary to reveal the recruiting function of the alarm pheromone and that specific floral odours-linalool and 2-phenylethanol-have the surprising capacity to block recruitment by the alarm pheromone. This effect is not due to an olfactory masking of the pheromone by the floral odours, but correlates with their appetitive value. In addition to their potential applications, these findings provide new insights about how honeybees make the decision to engage into defence and how conflicting information affects this process.

The ability of animals to adjust their behaviour according to seasonal changes in their ecology is crucial for their fitness. Eusocial insects display strong collective behavioural seasonality, yet the mechanisms underlying such changes are poorly understood. We show that nest preference by emigrating Temnothorax albipennis ant colonies is influenced by a season-specific modulatory pheromone that may help tune decision-making according to seasonal constraints. The modulatory pheromone triggers aversion towards low-quality nests and enhances colony cohesion in summer and autumn, but not after overwintering-in agreement with reports that field colonies split in spring and reunite in summer. Interestingly, we show that the pheromone acts by downgrading the perceived value of marked nests by informed and naive individuals. This contrasts with theories of collective intelligence, stating that accurate collective decision-making requires independent evaluation of options by individuals. The violation of independence highlighted here was accordingly shown to increase error rate during emigrations. However, this is counterbalanced by enhanced cohesion and the transmission of valuable information through the colony. Our results support recent claims that optimal decisions are not necessarily those that maximize accuracy. Other criteria-such as cohesion or reward rate-may be more relevant in animal decision-making.

Social insects possess remarkable learning capabilities, which are crucial for their ecological success. They also exhibit interindividual differences in responsiveness to environmental stimuli, which underlie task specialization and division of labor. Here we investigated for the first time the relationships between sucrose responsiveness, behavioral specialization, and appetitive olfactory learning in ants, including reproductive castes. We show that castes of the ant Camponotus aethiops differ in their responsiveness to sucrose and in their learning success in olfactory conditioning experiments in which sucrose is used as reward. Olfactory learning was better in foragers than in nurses, in agreement with their higher sucrose responsiveness. Interindividual variation in stimulus responsiveness and in learning may be, therefore, a crucial factor for division of labor in social insects.

Insects possess miniature brains but exhibit a sophisticated behavioral repertoire. Recent studies have reported the existence of unsuspected cognitive capabilities in various insect species that go beyond the traditionally studied framework of simple associative learning. Here, I focus on capabilities such as attentional modulation and concept learning and discuss their mechanistic bases. I analyze whether these behaviors, which appear particularly complex, can be explained on the basis of elemental associative learning and specific neural circuitries or, by contrast, require an explanatory level that goes beyond simple associative links. In doing this, I highlight experimental challenges and suggest future directions for investigating the neurobiology of higher-order learning in insects, with the goal of uncovering the basic neural architectures underlying cognitive processing., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The honey bee Apis mellifera has emerged as a robust and influential model for the study of classical conditioning thanks to the existence of a powerful Pavlovian conditioning protocol, the olfactory conditioning of the proboscis extension response (PER). In 2011, the olfactory PER conditioning protocol celebrated its 50 years since it was first introduced by Kimihisa Takeda in 1961. In this protocol, individually harnessed honey bees are trained to associate an odor with sucrose solution. The resulting olfactory learning is fast and induces robust olfactory memories that have been characterized at the behavioral, neuronal and molecular levels. Despite the success of this protocol for studying the bases of learning and memory at these different levels, innumerable procedural variants have arisen throughout the years, which render comparative analyses of behavioral performances difficult. Moreover, because even slight variations in conditioning procedures may introduce significant differences in acquisition and retention performances, we revisit olfactory PER conditioning and define here a standardized framework for experiments using this behavioral protocol. To this end, we present and discuss all the methodological steps and details necessary for successful implementation of olfactory PER conditioning., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The honeybee Apis mellifera has emerged as a robust and influential model for the study of classical conditioning, thanks to the existence of a powerful Pavlovian conditioning protocol, the olfactory conditioning of the proboscis extension response (PER). In 2011, the olfactory PER conditioning protocol celebrates 50 years since it was first introduced by Kimihisa Takeda in 1961. Here, we review its origins, developments, and perspectives in order to define future research avenues and necessary methodological and conceptual evolutions. We show that olfactory PER conditioning has become a versatile tool for the study of questions in extremely diverse fields in addition to the study of learning and memory and that it has allowed behavioral characterizations, not only of honeybees, but also of other insect species, for which the protocol was adapted. We celebrate, therefore, Takeda's original work and prompt colleagues to conceive and establish further robust behavioral tools for an accurate characterization of insect learning and memory at multiple levels of analysis.

In Pavlovian conditioning, an originally neutral stimulus (conditioned stimulus or CS) gains control over an animal's reflex after its association with a biologically relevant stimulus (unconditioned stimulus or US). As a consequence, a conditioned response is emitted by the animal upon further CS presentations. In such a situation, the subject exhibits a reflex response, so that whether the CS thereby acquires a positive or a negative value for the animal is difficult to assess. In honeybees, Apis mellifera, an odour (CS) can be associated either with sucrose solution (US) in the appetitive conditioning of the proboscis extension reflex (PER), or with an electric shock (US) in the aversive conditioning of the sting extension reflex (SER). The term ;aversive' may not apply to the latter as bees do not suppress SER as a consequence of learning but, on the contrary, start emitting SER to the CS. To determine whether the CS acquires a positive or a negative value in these conditioning forms, we compared the orientation behaviour of freely walking honeybees in an olfactory-cued Y-maze after training them with an odour-sucrose association (PER conditioning) or an odour-shock association (SER conditioning). We show that the same odours can acquire either a positive value when associated to sucrose, or a negative value when associated to an electric shock, as bees respectively approach or avoid the CS in the Y-maze. Importantly, these results clearly establish the aversive nature of SER conditioning in honeybees.

Equipped with a mini brain smaller than one cubic millimeter and containing only 950,000 neurons, honeybees could be indeed considered as having rather limited cognitive abilities. However, bees display a rich and interesting behavioral repertoire, in which learning and memory play a fundamental role in the framework of foraging activities. We focus on the question of whether adaptive behavior in honeybees exceeds simple forms of learning and whether the neural mechanisms of complex learning can be unraveled by studying the honeybee brain. Besides elemental forms of learning, in which bees learn specific and univocal links between events in their environment, bees also master different forms of non-elemental learning, including categorization, contextual learning and rule abstraction, both in the visual and in the olfactory domain. Different protocols allow accessing the neural substrates of some of these learning forms and understanding how complex problem solving can be achieved by a relatively simple neural architecture. These results underline the enormous richness of experience-dependent behavior in honeybees, its high flexibility, and the fact that it is possible to formalize and characterize in controlled laboratory protocols basic and higher-order cognitive processing using an insect as a model.

Biogenic amines like dopamine or octopamine modify neural function at multiple levels, sensitizing or depressing behaviour. Recent studies in insects have now shown that, besides a role in motivational modulation, biogenic amines substitute the reinforcer function in associative learning, thus instructing the nervous system about the relevance of external events.

This review provides evidence for the enormous richness of insect behavior, its high flexibility, and the cross-talk between different behavioral routines. The memory structure established by multiple forms of learning represents sensory inputs and relates behaviors in such a way that representations of complex environmental conditions are formed. Navigation and communication in social hymenoptera are particularly telling examples in this respect, but it is fair to conclude that similar integrated forms of dealing with the environment will be found in other insects when they are studied more closely. In this sense, research addressing behavioral complexity and its underlying neural substrates is necessary to characterize the real potential of insect learning and memory. Usually, such an approach has been used to characterize behavioral simplicity rather than complexity. It seems therefore timely to focus on the latter by studying problem solving alongside and in addition to elemental forms of learning.

We combined behavioural and electrophysiological experiments to study whether bitter taste is perceived at the antennal level in honeybees, Apis mellifera. Our behavioural studies showed that neither quinine nor salicin delivered at one antenna at different concentrations induced a retraction of the proboscis once it was extended in response to 1 M sucrose solution delivered to the opposite antenna. Bees that extended massively their proboscis to 1 M sucrose responded only partially when stimulated with a mixture of 1 M sucrose and 100 mM quinine. The mixture of 1 m sucrose and 100 mM salicin had no such suppressive effect. No behavioural suppression was found for mixtures of salt solution and either bitter substance. Electrophysiological recordings of taste sensillae at the antennal tip revealed sensillae that responded specifically either to sucrose or salt solutions, but none responded to the bitter substances quinine and salicin at the different concentrations tested. The electrophysiological responses of sensillae to 15 mM sucrose solution were inhibited by a mixture of 15 mM sucrose and 0.1 mM quinine, but not by a mixture of 15 mM sucrose and 0.1 mM salicin. The responses of sensillae to 50 mM NaCl were reduced by a mixture of 50 mm NaCl and 1 mM quinine but not by a mixture of 50 mM NaCl and 1 mM salicin. We concluded that no receptor cells for the bitter substances tested, exist at the level of the antennal tip of the honeybee and that antennal bitter taste is not represented as a separate perceptual quality.

The brain of a honeybee contains only 960,000 neurons and its volume represents only 1 mm3. However, it supports impressive behavioral capabilities. Honeybees are equipped with sophisticated sensory systems and have well developed learning and memory capacities, whose essential mechanisms do not differ drastically from those of vertebrates. Here, I focus on non-elemental forms of learning by honeybees. I show that bees exhibit learning abilities that have been traditionally ascribed to a restricted portion of vertebrates, as they go beyond simple stimulus-stimulus or response-stimulus associations. To relate these abilities to neural structures and functioning in the bee brain we focus on the antennal lobes and the mushroom bodies. We conclude that there is a fair chance to understand complex behavior in bees, and to identify the potential neural substrates underlying such behavior by adopting a cognitive neuroethological approach. In such an approach, behavioral and neurobiological studies are combined to understand the rules and mechanisms of plastic behavior in a natural context.

We addressed the question of whether the amount of individual experience determines the use of elemental or configural visual discrimination strategies in free-flying honeybees Apis mellifera. We trained bees to fly into a Y-maze to collect sucrose solution on a rewarded stimulus presented in one of the arms of the maze. Stimuli were colour disks, violet (V), green (G) or yellow (Y), which were of equal psychophysical salience for honeybees. Training followed an A+, BC+ design, followed by an AC versus BC test. Training consisted of 6 (3 A+ and 3 BC+), 20 (10 A+ and 10 BC+) or 40 (20 A+ and 20 BC+) acquisition trials. Elemental models of compound processing predict a preference for the non-trained stimulus AC while configural models predict a preference for the trained stimulus BC. Our results show that increasing the number of acquisition trials results in a change of the internal representation of stimuli. After six training trials, bees favoured an elemental strategy and preferred AC to BC during the tests. Generally, increasing the number of training trials resulted in an increase of the choice of BC. Thus, short training favoured processing of the compound as the sum of its elements (elemental account) while long training favoured its processing as being different from the sum of its elements (configural account). Additionally, we observed that the change in stimulus processing was also influenced by stimulus similarity. Colour perceptual similarity favoured configural processing with increasing experience.

We investigated the capability of honeybees to discriminate between single odorants, binary olfactory mixtures, and ternary olfactory mixtures in olfactory conditioning of the proboscis extension reflex. In Experiment 1, three single odorants (A+, B+, and C+) and three binary mixtures of these odors (AB+, AC+, and BC+) were reinforced while the ternary compound, consisting of all three odors (ABC-), was nonreinforced. In Experiment 2, only one single odorant (A+) and one binary olfactory compound (BC+) were reinforced while the ternary compound (ABC-) consisting of the single odor and the binary compound was nonreinforced. We studied whether bees can solve these problems and whether the course of differentiation can be predicted by the unique cue theory, a modified unique cue theory, or Pearce's configural theory. Honeybees were not able to differentiate reinforced from nonreinforced stimuli in Experiment 1. However, summation to ABC observed at the beginning of training contradicts the predictions of Pearce's configural theory. In Experiment 2, differentiation between the single odorant A and the ternary compound developed more easily than between the binary compound BC and ABC. This pattern of differentiation is in line with a modified unique cue theory and Pearce's configural theory. Summation to ABC at the beginning of training, however, again was at odds with Pearce's configural theory. Thus, olfactory compound processing in honeybees can best be explained by a modified unique cue theory.