Genotypic Influence on Aversive Conditioning in Honeybees, Using a Novel Thermal Reinforcement Procedure

Abstract

In Pavlovian conditioning, animals learn to associate initially neutral stimuli with positive or negative outcomes, leading to appetitive and aversive learning respectively. The honeybee (Apis mellifera) is a prominent invertebrate model for studying both versions of olfactory learning and for unraveling the influence of genotype. As a queen bee mates with about 15 males, her worker offspring belong to as many, genetically-different patrilines. While the genetic dependency of appetitive learning is well established in bees, it is not the case for aversive learning, as a robust protocol was only developed recently. In the original conditioning of the sting extension response (SER), bees learn to associate an odor (conditioned stimulus - CS) with an electric shock (unconditioned stimulus - US). This US is however not a natural stimulus for bees, which may represent a potential caveat for dissecting the genetics underlying aversive learning. We thus first tested heat as a potential new US for SER conditioning. We show that thermal stimulation of several sensory structures on the bee’s body triggers the SER, in a temperature-dependent manner. Moreover, heat applied to the antennae, mouthparts or legs is an efficient US for SER conditioning. Then, using microsatellite analysis, we analyzed heat sensitivity and aversive learning performances in ten worker patrilines issued from a naturally inseminated queen. We demonstrate a strong influence of genotype on aversive learning, possibly indicating the existence of a genetic determinism of this capacity. Such determinism could be instrumental for efficient task partitioning within the hive.

Appetitive but not aversive olfactory conditioning modifies antennal movements in honeybees

“…During our experiments, no deleterious effects on the bees' vitality or their behavioral responses were observed as a result of this marking. In particular, marked bees showed olfactory learning performances that are fully consistent with standard performances, both for appetitive (Bitterman et al 1983;Giurfa and Sandoz 2012) and aversive learning (Vergoz et al 2007;Junca et al 2014). Two previous studies in which both appetitive and aversive conditioning Oscillation frequency changed significantly for the CS+ but not for the CS2.…”

“…Another important classical conditioning procedure, the olfactory conditioning of the sting extension response (SER) was developed only recently (Vergoz et al 2007). In this procedure, the odor CS is associated with an aversive US (electric shock: Vergoz et al 2007; thermal shock : Junca et al 2014). Once the association has been made, bees extend their sting to the aversively reinforced odor alone.…”

“…Lastly, we used two standard protocols for conditioning bees appetitively (Bitterman et al 1983) or aversively (Junca et al 2014). However, it is important to bear in mind that there are differences concerning the application of the US, between the two protocols.…”

“…In PER conditioning, the US was a compound applied to the antennae and then to the proboscis (Bitterman et al 1983). In SER conditioning, the US was a heated probe applied to the mouthparts (Junca et al 2014). The two protocols thus differ in the mode of delivery and their respective contact with the antennae.…”

The complexity of learning, memory and neural processes in an evolutionary ecological context

“…Indeed, a comparison of four different strains of N. vitripennis, including homozygous as well as genetically diverse populations [43], and a panel of different isofemale lines of D. melanogaster [44] showed considerable differences in memory formation. Likewise, variation in learning was found between a sexual and asexual population of the parasitic wasp Venturia canescens [45], between individuals of different bumblebee colonies [46,47], and between genetically different worker bees [48].…”