Aversive Learning in Honeybees Revealed by the Olfactory Conditioning of the Sting Extension Reflex

Invertebrates have contributed greatly to our understanding of associative learning because they allow learning protocols to be combined with experimental access to the nervous system. The honeybee Apis mellifera constitutes a standard model for the study of appetitive learning and memory since it was shown, almost a century ago, that bees learn to associate different sensory cues with a reward of sugar solution. However, up to now, no study has explored aversive learning in bees in such a way that simultaneous access to its neural bases is granted. Using odorants paired with electric shocks, we conditioned the sting extension reflex, which is exhibited by harnessed bees when subjected to a noxious stimulation. We show that this response can be conditioned so that bees learn to extend their sting in response to the odorant previously punished. Bees also learn to extend the proboscis to one odorant paired with sugar solution and the sting to a different odorant paired with electric shock, thus showing that they can master both appetitive and aversive associations simultaneously. Responding to the appropriate odorant with the appropriate response is possible because two different biogenic amines, octopamine and dopamine subserve appetitive and aversive reinforcement, respectively. While octopamine has been previously shown to substitute for appetitive reinforcement, we demonstrate that blocking of dopaminergic, but not octopaminergic, receptors suppresses aversive learning. Therefore, aversive learning in honeybees can now be accessed both at the behavioral and neural levels, thus opening new research avenues for understanding basic mechanisms of learning and memory

RNA-sequencing elucidates the regulation of behavioural transitions associated with mating in honey bee queens

This study was funded by a BBSRC ISIS grant BB/J019453/1, a Royal Holloway Research Strategy Fund Grant, and a Leverhulme Grant F/07537/AK to MJFB. BPO was supported by Australian Research Council Discovery grants DP150100151 and DP120101915. FM was supported by a Marie Curie International Incoming Fellowship FP7-PEOPLE-2013-IIF-625487 to MJFB. We would like to thank Dave Galbraight (Penn State) and Alberto Paccanaro (RHUL) for support with analysis of RNAseq data and four anonymous reviewers for providing thoughtful insights that helped to improve the manuscript.Peer reviewedPublisher PD

Leonard et al._OIKOS_2018

Sheet 1 (Absolute conditioning): Absolute PER conditioning protocol using 6 conditioning trials. Conditioned stimulus (predictor variable) included 8 volatiles (linalool, dipentene, myrcene, geranium, linalool and petrol pollution, dipentene and petrol pollution, myrcene and petrol pollution, geranium and petrol pollution). Response variable = proboscis extension (binary response following exposure to conditioned stimuli: 0 no proboscis extension, 1 proboscis extension). Sheet 2 (Recall tests): Proboscis extension following exposure to 1 of 8 conditioned stimuli (linalool, dipentene, myrcene, geranium, linalool and petrol pollution, dipentene and petrol pollution, myrcene and petrol pollution, geranium and petrol pollution), 1 hr, 24 hrs and 48 hrs post conditioning. Response to conditioned stimuli and conditioned stimuli with or without the addition of petrol exhaust pollution was recorded

Effects of natural mating and CO2 narcosis on biogenic amine receptor gene expression in the ovaries and brain of queen honey bees, Apis mellifera

A queen honey bee mates at ~6 days of age, storing the sperm in her spermatheca for life. Mating is associated with profound changes in the behaviour and physiology of the queen but the mechanisms underlying these changes are poorly understood. What is known is that the presence of semen in the oviducts and spermatheca is insufficient to initiate laying, and that copulation or CO2 narcosis is necessary for ovary activation. In this study we use real-time quantitative PCR to investigate the expression of biogenic amine receptor genes in the brain and ovarian tissue of queens in relation to their reproductive status. We show that dopamine, octopamine and serotonin receptor genes are expressed in the ovaries of queens, and that natural mating, CO2 narcosis, and the presence of semen in the spermatheca differentially affect their expression. We suggest that these changes may be central to the hormonal cascades that are necessary to initiate oogenesis

Figure 5

<p>The effect of octopaminergic and dopaminergic receptor antagonists on olfactory conditioning of the sting extension reflex (SER). Responses (SER) of bees trained to discriminate an odorant reinforced with an electric shock (black squares) and a non-reinforced odorant (white squares) during 12 acquisition trials (6 reinforced and 6 non-reinforced). A retention test was conducted 1 h after the last acquisition trial (black bar: odorant previously reinforced; white bar: odorant previously non-reinforced). a) Responses (SER) of control bees injected with Ringer into the brain (n = 40); b) Responses (SER) of bees injected with the octopaminergic antagonist mianserine 3.3 mM into the brain (n = 40); c) Responses (SER) of bees injected with the dopaminergic antagonist flupentixol 2 mM into the brain (n = 40). Ringer-and mianserine-injected bees learned to discriminate the reinforced from the non-reinforced odorant and remembered the difference one hour later. Flupentixol-injected bees did not learn to discriminate the reinforced from the non-reinforced odorant, nor did they respond appropriately in the retention tests. Similar results were obtained with other concentrations of octopaminergic and dopaminergic antagonists (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000288#pone-0000288-g006" target="_blank">Fig. 6</a>). These results show that dopamine, but not octopamine receptors are required for aversive olfactory learning in honeybees.</p

Figure 4

<p>The effect of electric shock on appetitive olfactory conditioning of the proboscis extension reflex (PER). Responses (PER) of bees trained to associate an odorant with sucrose solution along six conditioning trials and experiencing six electric shocks (‘shock group’; black dots and bars; n = 40) or six placements in the conditioning setup (‘placement group’; white dots and bars; n = 40) interspersed pseudorandomly. Bees in both groups learned to respond to the rewarded odorant irrespective of the presence or absence of shock. One hour later, both groups behaved similarly in the retention test (bars) and responded significantly more to the conditioned odorant than to the novel odorant. Thus, repetitive stimulation with the electric shock did neither affect appetitive olfactory learning nor olfactory memory one hour after conditioning. *: p<0.0001.</p

Figure 2

<p>Associative olfactory conditioning of the sting extension reflex (SER) in honeybees. a) Responses (SER) of bees trained with an odorant explicitly paired with an electric shock (black squares; n = 38) and with odorant and unpaired electric shock (white squares; n = 39) during 6 trials. Only the bees in the paired group learned the association and extended their sting as a response to the odorant. One hour after conditioning an olfactory aversive memory was present in the paired (black bar), but not in the unpaired, group (white bar). b) Responses (SER) of bees (n = 48) trained to discriminate an odorant reinforced with an electric shock (black squares) and a non-reinforced odorant (white squares) during 12 trials (6 reinforced and 6 non-reinforced). Bees learned to discriminate between odorants as a result of conditioning. *: p<0.0001.</p