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

Abstract

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.

Olfactory conditioning of the sting extension reflex in honeybees: Memory dependence on trial number, interstimulus interval, intertrial interval, and protein synthesis

“…1A) reflexively extend their sting (sting extension response, or SER) upon delivery of an electric shock to the thorax (Nú ñez et al 1997;Balderrama et al 2002), thus showing a typical defensive response to potentially noxious stimuli (Breed et al 2004). After successful conditioning, the odorant elicits SER, a conditioned response that can be retrieved 1 h after conditioning (Vergoz et al 2007). This form of conditioning is indeed aversive as shown by the fact that bees trained in this way and transferred to the operant context of a Y-maze, where they can freely walk and choose between the shock-associated odor and a non-shock-associated odor, explicitly avoid the punished odor and choose the non-shock-associated odor 1 h after conditioning ).…”

“…Furthermore, although olfactory SER conditioning leads to an aversive memory that can be retrieved either in a Pavlovian (Vergoz et al 2007) or in an operant framework ) 1 h after conditioning, it is unknown whether it leads to the formation of robust LTMs, retrievable some days after conditioning. In the honeybee, one pairing of an odorant with sucrose (i.e., one conditioning trial) leads to an early long-term memory (e-LTM) that can be retrieved 24 to 48 h after conditioning.…”

“…Despite the important progress made in understanding the behavioral, cellular, and molecular bases of this appetitive learning (Menzel 1999;Giurfa 2007), until recently it has been impossible to study aversive learning in bees in such a way that behavioral records would be accompanied by access to the nervous system. This gap has been filled by a novel conditioning protocol in which individually harnessed bees learn to associate an initially neutral odorant (CS) with a mild electric shock (US) (Vergoz et al 2007). Bees fixed individually on a metallic holder (Fig.…”

“…Three conditioning trials, on the other hand, lead to a stable late long-term memory (l-LTM) that can be retrieved 72 h or more after conditioning. Although the presence of short-term memories (STM) and medium-term memories (MTM)-retrievable just after conditioning or 1 h after it, respectively-have been shown in olfactory SER conditioning (Vergoz et al 2007;Carcaud et al 2009), no evidence thus far supports the presence of LTM after this form of learning. Here we performed a series of experiments aimed at characterizing the effect of variables such as ISI and ITI on learning and retention performance in olfactory SER conditioning.…”

Queen mandibular pheromone: questions that remain to be resolved

“…Evidence that aversive learning in insects involves DA signalling is compelling, particularly in fruit flies where DA-releasing neurons have been shown to convey the negative reinforcing properties of punishment signals (Riemensperger et al 2005;Schroll et al 2006;Schwaerzel et al 2003). Consistent with this model, inhibition of DA signalling with DA receptor antagonists has been shown to selectively impair aversive learning in bees (Vergoz et al 2007a).…”