The role of dopamine and serotonin in conditioned food aversion learning in the honeybee

Abstract

For most animals, eating entails the risk of being poisoned. Learning how to identify foods with toxins is an important mechanism that reduces the risk of poisoning. While conditioned food aversions have been studied in vertebrates for over 50 years, the neural circuits underlying this form of learning have been difficult to elucidate because of their complexity. Insects, such as fruit flies and honeybees, are important models for the study of the neural mechanisms of learning and memory, but conditioned food aversions have not yet been reported from either species. My collaborators and I recently established that the honeybee has the ability to learn to avoid odours associated with toxins in food using two independent neural pathways. In these experiments, we found that honeybees can learn to associate scents with toxins that they can pre-ingestively detect using their proboscis. This form of learning is primarily mediated by the neurotransmitter, dopamine. We also found a second mechanism: bees can learn to avoid odours associated with the malaise caused by ingesting toxins. This form of learning is mediated by serotonin. Our data are the first to show that two different mechanisms account for conditioned food aversions in insects. 

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Learning to avoid eating toxic food is as important as learning to find and obtain nutrition. Central to this form of learning is the ability to integrate sensory information with changes in state caused by intoxication. Although conditioned food aversions have been studied for the past 50 years in vertebrates, the molecular mechanisms of the neural circuits underpinning this form of learning have proven difficult to pinpoint.1 Several neuromodulators such as dopamine (DA) and serotonin (5HT), as well as glutamate and endocannabinoids, participate in the acquisition and consolidation of a conditioned food aversion1–3 but their exact roles as pre- or post-ingestive signaling mechanisms in these neural circuits remain unclear. Determining whether these pathways are functionally independent and mediated by different neuromodulators is fundamental to our understanding of the way that the brain evaluates the quality of ingested food.

Invertebrates such as nematodes, sea slugs, fruit flies and honeybees have simpler brains—using them as model systems has made it possible to dissect the way that neuromodulators function during learning.4–7 In flies and bees, octopamine (OA) is the primary neuromodulator of circuits involved in appetitive learning,8,9 whereas learned avoidance of odors signaling electric shock is mediated by DA.9–11 A recent study of food aversion learning in the nematode C. elegans demonstrated that nematodes without 5HT receptors failed to learn to avoid odors associated with the ingestion of pathogenic bacteria.12 Considerably less is known about the role of 5HT in appetitive and aversive learning in insects, however. Although conditioned food aversions have been demonstrated in slugs,13 cuttlefish,14 mantids15 and grasshoppers,16 they have not yet been reported in important insect models such as fruit flies or honeybees.

The honeybee, an olfactory-learner par excellence, is a mutualist with its food source and as such, rarely encounters food containing toxins. In fact, the honeybee possesses only 10 genes encoding gustatory receptors, perhaps indicating that it is limited in its ability to use bitter taste to pre-ingestively detect toxins.17 Free-flying honeybees will, however, avoid consuming sucrose solutions containing caffeine, nicotine and amygdalin.18,19 How they perform this, and whether or not they can learn to avoid cues associated with the presence of toxins, has not yet been tested.

Using a well-characterized assay for olfactory conditioning of the proboscis extension reflex (PER) in the honeybee,20 we found that honeybees can detect bitter compounds such as quinine and learn to avoid odors associated with its presence in sucrose solution.21 Our data are the first to establish that the bee has gustatory neurons that respond to toxins such as quinine and amygdalin on the proboscis. Furthermore, by mixing these compounds with sucrose, we found that the detection of toxins requires the activity of several classes of neurons, including those that respond most to sucrose alone. This suggests that the ability to detect toxins, at least in insects, is not a labeled line code in specific ‘bitter’ sensing neurons. Instead, our data support the hypothesis that the perception of bitter requires input from several classes of neurons simultaneously.

By presenting two toxins—the strongly bitter toxin, quinine and the difficult-todetect toxin, amygdalin—in a rewarding sucrose solution during olfactory conditioning, we established that learned olfactory avoidances that depend on bitter gustatory input occur rapidly whereas learning that relies on the malaise that occurs after toxin ingestion occurs on a slower time course. Learning to avoid odors associated with bitter substances develops within 5 min of the first learning trial and relies on input gustatory neurons located on the proboscis. This is in contrast to learned olfactory aversions arising after toxins are ingested: in this case, the post-ingestive pathway for learned inhibition of PER is expressed 20–40 min after initial toxin ingestion. The slow nature of this learned avoidance presumably arises because of physiological stress caused by toxin ingestion that could be signaled by the gut. Alternatively, if the toxin itself passed via the midgut into the hemolymph, it could be detected by neural tissues elsewhere in the body. This occurs in vertebrates, where lithium chloride, a salt often used to produce conditioned malaise by injection, activates the vagus nerve.22 Whether malaise is signaled by afferents projecting from the gut to the brain in insects is unknown.

These data clearly show that at least two pathways for learned suppression of PER exist in the honeybee: a pre-ingestive pathway that relies on gustatory input and a post-ingestive one arising after food ingestion. Importantly, our work also establishes that these two pathways are mediated by different neurotransmitters. Recent studies in crickets and in larval fruit flies have demonstrated that DA mediates learned avoidances of visual or olfactory cues associated with salt or quinine applied to the mouthparts.23–25 To see if this was also the case in the honeybee, we blocked DA, OA and 5HT receptors during differential conditioning with quinine and also observed that learned avoidances of bitter substances are primarily modulated by DA. In contrast, learning to associate odors with the malaise caused by ingesting amygdalin is mediated by 5HT. Our data are the first to identify a role for 5HT during food avoidance learning in an insect. We speculate that 5HT modulates the circuit that controls conditioned proboscis extension in honeybees. Identifying the mechanism that links the gut and brain during malaise learning remains to be determined.

Pollinators, such as honeybees, provide a vital service to plants by transporting pollen between the flowers of conspecifics. Plants generally produce toxins as a means of preventing herbivory; it is, therefore, counterintuitive that nectar and pollen contain toxins as they are the rewards offered to pollinators for visiting. However, many species do have toxins in their nectar such as the almond tree which produces nectar containing amygdalin.18,26,27 Our data show that the honeybee's ability to pre-ingestively detect toxins in sucrose solution depends on the chemical nature of the toxin, as some toxins are much more difficult to detect. Toxins in nectar could afford plants a selective advantage if they repelled nectar thieves but not pollinators. Even if pollinators post-ingestively learned that they had been poisoned, the time course of this form of learning is slower. A pollinator could, therefore, transfer pollen between plants before it was aware that it had been poisoned and so facilitate selection for this trait in a population of plants.

Abbreviations

OA	=	octopamine
DA	=	dopamine
5HT	=	serotonin
PER	=	proboscis extension reflex

Acknowledgements

The work was funded in part by small grants from Association for the Study of Animal Behavior and the Wellcome Trust.

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