Appetitive Olfactory Learning Research Articles

Microplastics reach the brain and interfere with honey bee cognition

Scientific research on the impact of microplastics (MPs) in terrestrial systems is still emerging, but it has confirmed adverse health effects in organisms exposed to plastics. Although recent studies have shown the toxicological effects of individual MPs polymers on honey bees, the effects of different polymer combinations on cognitive and behavioural performance remain unknown. To fill this knowledge gap, we investigated the effects of oral exposure to spherical MPs on cognitive performance and brain accumulation in the honey bee Apis mellifera. We evaluated the acute toxicity, after a two-day exposure, of polystyrene (PS - 4.8-5.8 μm) and plexiglass (Poly(methyl methacrylate), or PMMA - 1-40 μm) MPs, and a combination of the two (MIX), at two environmentally relevant and one higher concentration (0.5, 5 and 50 mg L−1) and analysed their effects on sucrose responsiveness and appetitive olfactory learning and memory. We also used fluorescent thermoset amino formaldehyde MPs (1–5 μm) to explore whether microspheres of this diameter could penetrate the insect blood-brain barrier (BBB), using Two-Photon Fluorescence Microscopy (TPFM) in combination with an optimized version of the DISCO clearing technique. The results showed that PS reduced sucrose responsiveness, while PMMA had no significant effect; however, the combination had a marked negative effect on sucrose responsiveness. PMMA, PS, and MIX impaired bee learning and memory in bees, with PS showing the most severe effects. 3D brain imaging analysis using TFPM showed that 1–5 μm MPs penetrated and accumulated in the brain after only three days of oral exposure. These results raise concerns about the potential mechanical, cellular, and biochemical damage that MPs may cause to the central nervous system.

Behavioral Tests for Associative Learning in Caenorhabditis elegans.

Learning is critical for survival as it provides the capacity to adapt to a changing environment. At the molecular and cellular level, learning leads to alterations within neural circuits that include synaptic rewiring, synaptic plasticity, and protein level/gene expression changes. There has been substantial progress in recent years on dissecting how learning and memory is regulated at the molecular and cellular level, including the use of compact invertebrate nervous systems as experimental models. This progress has been facilitated by the establishment of robust behavioral assays that generate a quantifiable readout of the extent to which animals learn and remember. This chapter will focus on protocols of behavioral tests for associative learning using the nematode Caenorhabditis elegans, with its unparalleled genetic tractability, compact nervous system of ~300 neurons, high level of conservation with mammalian systems, and amenability to a suite of behavioral tools and analyses. Specifically, we will provide a detailed description of the methods for two behavioral assays that model associative learning, one measuring appetitive olfactory learning and the other assaying aversive gustatory learning.

Visual learning in tethered bees modifies flight orientation and is impaired by epinastine

Visual-orientation learning of a tethered flying bee was investigated using a flight simulator and a novel protocol in which orientation preference toward trained visual targets was assessed in tests performed before and after appetitive conditioning. Either a blue or a green rectangle (conditioned stimulus, CS) was associated with 30% sucrose solution (unconditioned stimulus, US), whereas the other rectangle was not paired with US. Bees were tested in a closed-looped flight simulator 5 min after ten pairings of the US and CS. Conditioned bees were preferentially oriented to the CS after such training. This increase in preference for CS was maintained for 24 h, indicating the presence of long-term memory. Because the total orienting time was not altered by conditioning, conditioning did not enhance orientation activity itself but increased the relative time for orientation to CS. When 0.4 or 4 mM epinastine (an antagonist of octopamine receptors) was injected into the bee’s head 30 min prior to the experiment, both short- and long-term memory formation were significantly impaired, suggesting that octopamine, which is crucial for appetitive olfactory learning in insects, is also involved in visual orientation learning.

Acute and chronic ingestion of polyethylene (PE) microplastics has mild effects on honey bee health and cognition

The massive use of plastic has contributed to huge quantities of hazardous refuse at a global scale and represents one of the most prominent issues of the Anthropocene. Microplastics (MPs) have been detected in almost all environments and pose a potential threat to a variety of plant and animal species. Many studies have reported a variety of effects, from negligible to detrimental, of MPs to aquatic organisms. Conversely, much less is known about their effect on terrestrial biota, and particularly on animal behavior and cognition. We assessed the oral toxicity of polyethylene (PE) MPs at three different concentrations (0.5, 5, and 50 mg L−1), and at different timescales (1 day and 7 days of exposure) and tested for their effects on survival, food intake, sucrose responsiveness, habituation to sucrose and appetitive olfactory learning and memory in the honey bee Apis mellifera. We found that workers were not completely unaffected by acute and prolonged ingestion of this polymer. A significant effect of PE on bee mortality was found for the highest concentration but not for lower ones. PE affected feeding behavior in a concentration-dependent manner, with bees consuming more food than controls when exposed to low concentration PE. Regarding our behavioral and cognitive experiments, the high concentration PE was found to affect only bees’ ability to respond consistently to sucrose but not sucrose sensitivity, habituation to sucrose or learning and memory abilities, even for prolonged exposure to PE. While these last results may look somewhat encouraging, we discussed why caution is warranted before ruling out the possibility that PE particles at environmental concentrations are harmful to honey bees.

Differential gene expression analysis following olfactory learning in honeybee (Apis mellifera L.).

Insects change their stimulus-response through the perception of associating these stimuli with important survival events such as rewards, threats, and mates. Insects develop strong associations and relate them to their experiences through several behavioral procedures. Among the insects, Apis species, Apis mellifera ligustica are known for their outstanding ability to learn with tremendous economic importance. Apis mellifera ligustica has a strong cognitive ability and promising model species for investigating the neurobiological basis of remarkable olfactory learning abilities. Here we evaluated the olfactory learning ability of A. mellifera by using the proboscis extension reflex (PER) protocol. The brains of the learner and failed-learner bees were examined for comparative transcriptome analysis by RNA-Seq to explain the difference in the learning capacity. In this study, we used an appetitive olfactory learning paradigm in the same age of A. mellifera bees to examine the differential gene expression in the brain of the learner and failed-learner. Bees that respond in 2nd and 3rd trials or only responded to 3rd trials were defined as learned bees, failed-learner individuals were those bees that did not respond in all learning trials The results indicate that the learning ability of learner bees was significantly higher than failed-learner bees for 12 days. We obtained approximately 46.7 and 46.4 million clean reads from the learner bees failed-learner bees, respectively. Gene expression profile between learners’ bees and failed-learners bees identified 74 differentially expressed genes, 57 genes up-regulated in the brains of learners and 17 genes were down-regulated in the brains of the bees that fail to learn. The qRT-PCR validated the differently expressed genes. Transcriptome analyses revealed that specific genes in learner and failed-learner bees either down-regulated or up-regulated play a crucial role in brain development and learning behavior. Our finding suggests that down-regulated genes of the brain involved in the integumentary system, storage proteins, brain development, sensory processing, and neurodegenerative disorder may result in reduced olfactory discrimination and olfactory sensitivity in failed-learner bees. This study aims to contribute to a better understanding of the olfactory learning behavior and gene expression information, which opens the door for understanding of the molecular mechanism of olfactory learning behavior in honeybees.

Appetitive olfactory learning suffers in ants when octopamine or dopamine receptors are blocked.

Associative learning relies on the detection of coincidence between a stimulus and a reward or punishment. In the insect brain, this process is carried out in the mushroom bodies under the control of octopaminergic and dopaminergic neurons. It was assumed that appetitive learning is governed by octopaminergic neurons, while dopamine is required for aversive learning. This view has recently been challenged: both neurotransmitters are involved in both types of learning in bees and flies. Here, we tested which neurotransmitters are required for appetitive learning in ants. We trained Lasius niger workers to discriminate two mixtures of linear hydrocarbons and to associate one of them with a sucrose reward. We analysed the walking paths of the ants using machine learning and found that the ants spent more time near the rewarded odour than near the other, a preference that was stable for at least 24 h. We then treated the ants before learning with either epinastine, an octopamine receptor blocker, or flupentixol, a dopamine receptor blocker. Ants with blocked octopamine receptors did not prefer the rewarded odour. Octopamine signalling is thus necessary for appetitive learning of olfactory cues, probably because it signals information about odours or reward to the mushroom body. In contrast, ants with blocked dopamine receptors initially learned the rewarded odour but failed to retrieve this memory 24 h later. Dopamine is thus probably required for long-term memory consolidation, independent of short-term memory formation. Our results show that appetitive olfactory learning depends on both octopamine and dopamine signalling in ants.

Effects of sequential exposures of sub-lethal doses of amitraz and thiacloprid on learning and memory of honey bee foragers, Apis mellifera

Pollinators, honey bees in particular, are continuously exposed to various mixtures of pesticides, which contribute to their population decline. Both amitraz and thiacloprid have been proven less toxic to honey bees and are frequently applied in- and out-hive, respectively. We examined the sub-lethal effects of amitraz, thiacloprid and their sequential exposure on learning, memory and sugar responsiveness in Apis mellifera using the Proboscis extension response (PER). Sub-lethal doses of amitraz (0.1, 0.2 and 0.4 µg/bee) and thiacloprid (0.05, 0.1 and 0.2 µg/bee) were tested. Sub-lethal effects were observed only at the highest doses of each pesticide treatment; amitraz (0.4 µg/bee) and thiacloprid (0.2 µg/bee) but not in lower doses. In sequential treatment of amitraz and thiacloprid, reduced acquisition and memory retention were significant across all tested doses. The same profile was also obtained on sugar responsiveness of foragers. Our results suggest that the sequential exposure would pose higher risk to honey bee compared to single pesticide exposure by reducing the bees’ appetitive olfactory learning, memory and sugar acuity more than individual pesticide exposures.

Serotonin receptor 5-HT7 in Drosophila mushroom body neurons mediates larval appetitive olfactory learning

Serotonin (5-HT) and dopamine are critical neuromodulators known to regulate a range of behaviors in invertebrates and mammals, such as learning and memory. Effects of both serotonin and dopamine are mediated largely through their downstream G-protein coupled receptors through cAMP-PKA signaling. While the role of dopamine in olfactory learning in Drosophila is well described, the function of serotonin and its downstream receptors on Drosophila olfactory learning remain largely unexplored. In this study we show that the output of serotonergic neurons, possibly through points of synaptic contacts on the mushroom body (MB), is essential for training during olfactory associative learning in Drosophila larvae. Additionally, we demonstrate that the regulation of olfactory associative learning by serotonin is mediated by its downstream receptor (d5-HT7) in a cAMP-dependent manner. We show that d5-HT7 expression specifically in the MB, an anatomical structure essential for olfactory learning in Drosophila, is critical for olfactory associative learning. Importantly our work shows that spatio-temporal restriction of d5-HT7 expression to the MB is sufficient to rescue olfactory learning deficits in a d5-HT7 null larvae. In summary, our results establish a critical, and previously unknown, role of d5-HT7 in olfactory learning.

NPY/NPF-Related Neuropeptide FLP-34 Signals from Serotonergic Neurons to Modulate Aversive Olfactory Learning in Caenorhabditis elegans.

Aversive learning is fundamental for animals to increase chances of survival. In addition to classical neurotransmitters, neuropeptides have emerged to modulate such complex behaviors. Among them, neuropeptide Y (NPY) is well known to promote aversive memory acquisition in mammals. Here we identify an NPY/neuropeptide F (NPF)-related neuropeptide system in Caenorhabditis elegans and show that this FLP-34/NPR-11 system is required for learning negative associations, a process that is reminiscent of NPY signaling in mammals. The Caenorhabditis elegans NPY/NPF ortholog FLP-34 displays conserved structural hallmarks of bilaterian-wide NPY/NPF neuropeptides. We show that it is required for aversive olfactory learning after pairing diacetyl with the absence of food, but not for appetitive olfactory learning in response to butanone. To mediate diacetyl learning and thus integrate the aversive food context with the diacetyl odor, FLP-34 is released from serotonergic neurons and signals through its evolutionarily conserved NPY/NPF GPCR, NPR-11, in downstream AIA interneurons. NPR-11 activation in the AIA integration center results in avoidance of a previously attractive stimulus. This study opens perspectives for a deeper understanding of stress conditions in which aversive learning results in excessive avoidance.SIGNIFICANCE STATEMENT Aversive learning evolved early in evolution to promote avoidance of dangerous and stressful situations. In addition to classical neurotransmitters, neuropeptides are emerging as modulators of complex behaviors, including learning and memory. Here, we identified the evolutionary ortholog of neuropeptide Y/neuropeptide F in the nematode Caenorhabditis elegans, and we discovered that it is required for olfactory aversive learning. In addition, we elucidated the neural circuit underlying this avoidance behavior, and we discovered a novel coordinated action of Caenorhabditis elegans neuropeptide Y/neuropeptide F and serotonin that could aid in our understanding of the molecular mechanisms underlying stress disorders in which excessive avoidance results in maladaptive behaviors.

Reward signaling in a recurrent circuit of dopaminergic neurons and peptidergic Kenyon cells

Dopaminergic neurons in the brain of the Drosophila larva play a key role in mediating reward information to the mushroom bodies during appetitive olfactory learning and memory. Using optogenetic activation of Kenyon cells we provide evidence that recurrent signaling exists between Kenyon cells and dopaminergic neurons of the primary protocerebral anterior (pPAM) cluster. Optogenetic activation of Kenyon cells paired with odor stimulation is sufficient to induce appetitive memory. Simultaneous impairment of the dopaminergic pPAM neurons abolishes appetitive memory expression. Thus, we argue that dopaminergic pPAM neurons mediate reward information to the Kenyon cells, and in turn receive feedback from Kenyon cells. We further show that this feedback signaling is dependent on short neuropeptide F, but not on acetylcholine known to be important for odor-shock memories in adult flies. Our data suggest that recurrent signaling routes within the larval mushroom body circuitry may represent a mechanism subserving memory stabilization.

Appetitive olfactory learning and memory in the honeybee depend on sugar reward identity

One of the most important tasks of the brain is to learn and remember information associated with food. Studies in mice and Drosophila have shown that sugar rewards must be metabolisable to form lasting memories, but few other animals have been studied. Here, we trained adult, worker honeybees (Apis mellifera) in two olfactory tasks (massed and spaced conditioning) known to affect memory formation to test how the schedule of reinforcement and the nature of a sugar reward affected learning and memory. The antennae and mouthparts of honeybees were most sensitive to sucrose but glucose and fructose were equally phagostimulatory. Whether or not bees could learn the tasks depended on sugar identity and concentration. However, only bees rewarded with glucose or sucrose formed robust long-term memory. This was true for bees trained in both the massed and spaced conditioning tasks. Honeybees fed with glucose or fructose exhibited a surge in haemolymph sugar of greater than 120mM within 30s that remained elevated for as long as 20min after a single feeding event. For bees fed with sucrose, this change in haemolymph glucose and fructose occurred with a 30s delay. Our data showed that olfactory learning in honeybees was affected by sugar identity and concentration, but that olfactory memory was most strongly affected by sugar identity. Taken together, these data suggest that the neural mechanisms involved in memory formation sense rapid changes in haemolymph glucose that occur during and after conditioning.

Appetitive Olfactory Learning and Long-Term Associative Memory in Caenorhabditis elegans.

Because of the relative simplicity of its nervous system, Caenorhabditis elegans is a useful model organism to study learning and memory at cellular and molecular levels. For appetitive conditioning in C. elegans, food has exclusively been used as an unconditioned stimulus (US). It may be difficult to analyze neuronal circuits for associative memory since food is a multimodal combination of olfactory, gustatory, and mechanical stimuli. Here, we report classical appetitive conditioning and associative memory in C. elegans, using 1-nonanol as a conditioned stimulus (CS), and potassium chloride (KCl) as a US. Before conditioning, C. elegans innately avoided 1-nonanol, an aversive olfactory stimulus, and was attracted by KCl, an appetitive gustatory stimulus, on assay agar plates. Both massed training without an intertrial interval (ITI) and spaced training with a 10-min ITI induced significant levels of memory of association regarding the two chemicals. Memory induced by massed training decayed within 6 h, while that induced by spaced training was retained for more than 6 h. Animals treated with inhibitors of transcription or translation formed the memory induced by spaced training less efficiently than untreated animals, whereas the memory induced by massed training was not significantly affected by such treatments. By definition, therefore, memories induced by massed training and spaced training are classified as short-term memory (STM) and long-term memory (LTM), respectively. When animals conditioned by spaced training were exposed to 1-nonanol alone, their learning index was lower than that of untreated animals, suggesting that extinction learning occurs in C. elegans. In support of these results, C. elegans mutants defective in nmr-1, encoding an NMDA receptor subunit, formed both STM and LTM less efficiently than wild-type animals, while mutations in crh-1, encoding a ubiquitous transcription factor CREB required for memory consolidation, affected LTM, but not STM. The paradigm established in the present study should allow us to elucidate neuronal circuit plasticity for appetitive learning and memory in C. elegans.

Suppression of Dopamine Neurons Mediates Reward.

Massive activation of dopamine neurons is critical for natural reward and drug abuse. In contrast, the significance of their spontaneous activity remains elusive. In Drosophila melanogaster, depolarization of the protocerebral anterior medial (PAM) cluster dopamine neurons en masse signals reward to the mushroom body (MB) and drives appetitive memory. Focusing on the functional heterogeneity of PAM cluster neurons, we identified that a single class of PAM neurons, PAM-γ3, mediates sugar reward by suppressing their own activity. PAM-γ3 is selectively required for appetitive olfactory learning, while activation of these neurons in turn induces aversive memory. Ongoing activity of PAM-γ3 gets suppressed upon sugar ingestion. Strikingly, transient inactivation of basal PAM-γ3 activity can substitute for reward and induces appetitive memory. Furthermore, we identified the satiety-signaling neuropeptide Allatostatin A (AstA) as a key mediator that conveys inhibitory input onto PAM-γ3. Our results suggest the significance of basal dopamine release in reward signaling and reveal a circuit mechanism for negative regulation.

Effects of Caffeine on Olfactory Learning in Crickets.

Caffeine is a plant-derived alkaloid that is generally known as a central nervous system (CNS) stimulant. In order to examine the effects of caffeine on higher CNS functions in insects, we used an appetitive olfactory learning paradigm for the cricket Gryllus bimaculatus. Crickets can form significant long-term memories (LTMs) after repetitive training sessions, during which they associate a conditioned stimulus (CS: odor) with an unconditioned stimulus (US: reward). Administration of hemolymphal injections of caffeine established LTM after only single-trial conditioning over a wide range of caffeine dosages (1.6 µµg/kg to 39 mg/kg). We investigated the physiological mechanisms underlying this enhancement of olfactory learning performance pharmacologically, focusing on three major physiological roles of caffeine: 1) inhibition of phosphodiesterase (PDE), 2) agonism of ryanodine receptors, and 3) antagonism of adenosine receptors. Application of drugs relevant to these actions resulted in significant effects on LTM formation. These results suggest that externally applied caffeine enhances LTM formation in insect olfactory learning via multiple cellular mechanisms.

Honey Bee Allatostatins Target Galanin/Somatostatin-Like Receptors and Modulate Learning: A Conserved Function?

Sequencing of the honeybee genome revealed many neuropeptides and putative neuropeptide receptors, yet functional characterization of these peptidic systems is scarce. In this study, we focus on allatostatins, which were first identified as inhibitors of juvenile hormone synthesis, but whose role in the adult honey bee (Apis mellifera) brain remains to be determined. We characterize the bee allatostatin system, represented by two families: allatostatin A (Apime-ASTA) and its receptor (Apime-ASTA-R); and C-type allatostatins (Apime-ASTC and Apime-ASTCC) and their common receptor (Apime-ASTC-R). Apime-ASTA-R and Apime-ASTC-R are the receptors in bees most closely related to vertebrate galanin and somatostatin receptors, respectively. We examine the functional properties of the two honeybee receptors and show that they are transcriptionally expressed in the adult brain, including in brain centers known to be important for learning and memory processes. Thus we investigated the effects of exogenously applied allatostatins on appetitive olfactory learning in the bee. Our results show that allatostatins modulate learning in this insect, and provide important insights into the evolution of somatostatin/allatostatin signaling.

Neuropeptide F neurons modulate sugar reward during associative olfactory learning of Drosophila larvae

AbstractAll organisms continuously have to adapt their behavior according to changes in the environment in order to survive. Experience‐driven changes in behavior are usually mediated and maintained by modifications in signaling within defined brain circuits. Given the simplicity of the larval brain of Drosophila and its experimental accessibility on the genetic and behavioral level, we analyzed if Drosophila neuropeptide F (dNPF) neurons are involved in classical olfactory conditioning. dNPF is an ortholog of the mammalian neuropeptide Y, a highly conserved neuromodulator that stimulates food‐seeking behavior. We provide a comprehensive anatomical analysis of the dNPF neurons on the single‐cell level. We demonstrate that artificial activation of dNPF neurons inhibits appetitive olfactory learning by modulating the sugar reward signal during acquisition. No effect is detectable for the retrieval of an established appetitive olfactory memory. The modulatory effect is based on the joint action of three distinct cell types that, if tested on the single‐cell level, inhibit and invert the conditioned behavior. Taken together, our work describes anatomically and functionally a new part of the sugar reinforcement signaling pathway for classical olfactory conditioning in Drosophila larvae. J. Comp. Neurol. 523:2637–2664, 2015. © 2015 Wiley Periodicals, Inc.

Neuropeptide F neurons modulate sugar reward during associative olfactory learning of Drosophila larvae.

All organisms continuously have to adapt their behavior according to changes in the environment in order to survive. Experience-driven changes in behavior are usually mediated and maintained by modifications in signaling within defined brain circuits. Given the simplicity of the larval brain of Drosophila and its experimental accessibility on the genetic and behavioral level, we analyzed if Drosophila neuropeptide F (dNPF) neurons are involved in classical olfactory conditioning. dNPF is an ortholog of the mammalian neuropeptide Y, a highly conserved neuromodulator that stimulates food-seeking behavior. We provide a comprehensive anatomical analysis of the dNPF neurons on the single-cell level. We demonstrate that artificial activation of dNPF neurons inhibits appetitive olfactory learning by modulating the sugar reward signal during acquisition. No effect is detectable for the retrieval of an established appetitive olfactory memory. The modulatory effect is based on the joint action of three distinct cell types that, if tested on the single-cell level, inhibit and invert the conditioned behavior. Taken together, our work describes anatomically and functionally a new part of the sugar reinforcement signaling pathway for classical olfactory conditioning in Drosophila larvae.

Characterization of the octopaminergic and tyraminergic neurons in the central brain of Drosophila larvae.

Drosophila larvae are able to evaluate sensory information based on prior experience, similarly to adult flies, other insect species, and vertebrates. Larvae and adult flies can be taught to associate odor stimuli with sugar reward, and prior work has implicated both the octopaminergic and the dopaminergic modulatory systems in reinforcement signaling. Here we use genetics to analyze the anatomy, up to the single-cell level, of the octopaminergic/tyraminergic system in the larval brain and subesophageal ganglion. Genetic ablation of subsets of these neurons allowed us to determine their necessity for appetitive olfactory learning. These experiments reveal that a small subset of about 39 largely morphologically distinguishable octopaminergic/tyraminergic neurons is involved in signaling reward in the Drosophila larval brain. In addition to prior work on larval locomotion, these data functionally separate the octopaminergic/tyraminergic system into two sets of about 40 neurons. Those situated in the thoracic/abdominal ganglion are involved in larval locomotion, whereas the others in the subesophageal ganglion and brain hemispheres mediate reward signaling.

Appetitive and aversive olfactory learning induce similar generalization rates in the honey bee

Appetitive and aversive learning drive an animal toward or away from stimuli predicting reinforcement, respectively. The specificity of these memories may vary due to differences in cost–benefit relationships associated with appetitive and aversive contexts. As a consequence, generalization performances may differ after appetitive and aversive training. Here, we determined whether honey bees show different rates of olfactory generalization following appetitive olfactory conditioning of the proboscis extension response, or aversive olfactory conditioning of the sting extension response. In both cases, we performed differential conditioning, which improves discrimination learning between a reinforced odor (CS?) and a non-reinforced odor (CS-) and evaluated generalization to two novel odors whose similarity to the CS? and the CS- was different. We show, given the same level of discriminatory performance, that rates of generalization are similar between the two conditioning protocols and discuss the possible causes for this phenomenon.

Sucrose responsiveness, learning success, and task specialization in ants

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.