Multiple reversal olfactory learning in honeybees

Abstract

In multiple reversal learning, animals trained to discriminate a reinforced from a non-reinforced stimulus are subjected to various, successive reversals of stimulus contingencies (e.g. A+ vs. B-, A- vs. B+, A+ vs. B-). This protocol is useful to determine whether or not animals "learn to learn" and solve successive discriminations faster (or with fewer errors) with increasing reversal experience. Here we used the olfactory conditioning of proboscis extension reflex to study how honeybees Apis mellifera perform in a multiple reversal task. Our experiment contemplated four consecutive differential conditioning phases involving the same odors (A+ vs. B- to A- vs. B+ to A+ vs. B- to A- vs. B+). We show that bees in which the weight of reinforced or non-reinforced stimuli was similar mastered the multiple olfactory reversals. Bees which failed the task exhibited asymmetric responses to reinforced and non-reinforced stimuli, thus being unable to rapidly reverse stimulus contingencies. Efficient reversers did not improve their successive discriminations but rather tended to generalize their choice to both odors at the end of conditioning. As a consequence, both discrimination and reversal efficiency decreased along experimental phases. This result invalidates a learning-to-learn effect and indicates that bees do not only respond to the actual stimulus contingencies but rather combine these with an average of past experiences with the same stimuli.

Keywords: honeybee; learning; multiple reversal; olfaction.

Figure 1

Conditioned responses during multiple reversal learning in honeybees. Proboscis extension responses (% PER) to odors A and B during four consecutive differential conditioning phases. Bees experienced two contingency inversions between phases: A+ → A- and B- → B+ from the first to the second phase, A- → A+ and B+ → B- from the second to the third phase, and A+ → A- and B- → B+ from the third to the fourth phase. n = 111 bees.

Figure 2

Average excitatory (Δ_e) and inhibitory (Δ_i) reversal learning scores (+S. E.) computed for three consecutive reversal phases (2nd, 3rd, and 4th conditioning phases). Δ_e was calculated as the difference in responses to the CS+ between the fifth and the first trial of a reversal phase (Δ_e = CS+_trial5 – CS+_trial1); Δ_i was the difference in responses to the CS− between the first and the fifth trial of a reversal phase (Δ_i = CS−_trial1 – CS−_trial5). Statistical comparisons of excitatory scores between phases are indicated by letters (e.g. a, b). Comparisons of inhibitory scores between phases are indicated by letters with prime (e.g. a′, b′). Asterisks indicate significant difference between excitatory and inhibitory scores within a phase. n = 111 bees.

Figure 3

Average differentiation index (Di) obtained in the four consecutive differential conditioning phases (+ S. E.). Di was calculated as the difference between the responses to the CS+ minus the responses to the CS− in the last conditioning trial of each phase (Di = CS+_trial5 – CS−_trial5). The difference between the Dis of the 3rd and 4th phase was marginally non-significant (P = 0.055). n = 111 bees.

Figure 4

Conditioned responses during multiple reversal learning in three categories of honeybees. Proboscis extension responses (% PER) to odors A and B during four consecutive differential conditioning phases. Categories were defined by determining individual success in solving the 1st and the 2nd conditioning phases. The criterion used to define success in solving each phase was the presence of a dual correct response in the last (fifth) trial, i.e., PER to the CS+ and absence of PER to the CS−. (A) First category (n = 35 bees) included individuals that were not able to solve the very first discrimination of the 1st phase (A+ vs. B−). (B) Second category (n = 42 bees) included individuals that mastered the very first discrimination, but were unable to solve the subsequent reversal discrimination of the 2nd phase (A− vs. B+). (C) Third category (n = 34 bees) included individuals that solved the discriminations of the 1st and the 2nd phase, for which, therefore, the question of success in further reversal learning (3rd and 4th phases) was pertinent.

Figure 5

Average excitatory (Δ_e) and inhibitory (Δ_i) reversal learning scores (+ S. E.) computed for the three categories of bees, for the three reversal phases (2nd, 3rd, and 4th conditioning phases). (A) First category (n = 35 bees) included individuals that were not able to solve the very first discrimination of the 1st phase (A+ vs. B−). (B) Second category (n = 42 bees) included individuals that mastered the very first discrimination, but were unable to solve the subsequent reversal discrimination of the 2nd phase (A− vs. B+). (C) Third category (n = 34 bees) included individuals that solved the discriminations of the 1st and the 2nd phase, for which, therefore, the question of success in further reversal learning (3rd and 4th phases) was pertinent. Statistical comparisons of excitatory scores between phases are indicated by letters (e.g., a, b). Comparisons of inhibitory scores between phases are indicated by letters with prime (e.g. a′, b′). Asterisks indicate significant difference between excitatory and inhibitory scores within a phase.