Inhibitory learning of phototaxis by honeybees in a passive-avoidance task

Abstract

Honeybees are a standard model for the study of appetitive learning and memory. Yet, fewer attempts have been performed to characterize aversive learning and memory in this insect and uncover its molecular underpinnings. Here, we took advantage of the positive phototactic behavior of bees kept away from the hive in a dark environment and established a passive-avoidance task in which they had to suppress positive phototaxis. Bees placed in a two-compartment box learned to inhibit spontaneous attraction to a compartment illuminated with blue light by associating and entering into that chamber with shock delivery. Inhibitory learning resulted in an avoidance memory that could be retrieved 24 h after training and that was specific to the punished blue light. The memory was mainly operant but involved a Pavlovian component linking the blue light and the shock. Coupling conditioning with transcriptional analyses in key areas of the brain showed that inhibitory learning of phototaxis leads to an up-regulation of the dopaminergic receptor gene Amdop1 in the calyces of the mushroom bodies, consistently with the role of dopamine signaling in different forms of aversive learning in insects. Our results thus introduce new perspectives for uncovering further cellular and molecular underpinnings of aversive learning and memory in bees. Overall, they represent an important step toward comparative learning studies between the appetitive and the aversive frameworks.

Figure 1.

ICARUS—a passive-avoidance setup for inhibitory conditioning of phototaxis in honeybees. (A) The ICARUS setup under red light. Red and black cables represent the electrodes connected to upper and lower metal grids by which the shock is delivered. (B–D) Top view images of the ICARUS setup taken with the camera used for video recording the experiments. (B) Background stimulation with red LEDs only. (C) Illumination of one compartment with blue LEDs. (D) Illumination of one compartment with green LEDs. (E) Spectral emittance (continuous lines; left ordinate) of the three types of LEDs used in the setup (blue, green, and red) and spectral sensitivity (dashed lines; right ordinate) of the three types of honeybee photoreceptors (S, M, and L, for short, mid, and long wavelengths, respectively) as a function of wavelength. Spectral analysis of quantum catches—the proportion of incident photons that are captured by the photo-pigments—showed that red LEDs induced negligible activation of photoreceptors (Q_S = 0.6, Q_M = 0.64, Q_L = 1.84) while green LEDs activated mainly the L photoreceptors (Q_S = 0.84, Q_M = 3.25, Q_L = 44.22) and blue LEDs activated both L and M photoreceptors (Q_S = 0.36, Q_M = 21.42, Q_L = 23.19). Quantum-catch values depend on the spectrum of the stimulating light and the spectral sensitivity of the photoreceptor considered; they are used to infer the signal generated at the photoreceptor level.

Figure 2.

Multiple conditioning trials in a passive-avoidance task induce strong phototactic inhibitory learning and a memory retrievable 24 h after conditioning. (A) Schematic representation of the experimental protocol. After a familiarization period of 5 min in the setup under red light, three groups of bees (paired, unpaired, and no-chock) were subjected to three conditioning protocols in which the latency to enter a blue-lit compartment was measured as a proxy of learning and memory. The paired group received eight conditioning trials in which the action of entering the blue-lit compartment was paired with a mild electric shock. The unpaired group received eight trials consisting of stimulations with attractive blue light and mild electric shock separated by 30 sec. The no-shock group received eight trials consisting in which only the stimulation with the attractive blue light was present. In all groups, trials were separated by an intertrial interval of 1 min. Memory retention was tested 24 h after conditioning. The test session consisted of a familiarization period, and two tests separated by 1 min. In the first test, one of the compartments was illuminated with blue light; in the second test, one compartment was illuminated with green light. No-shock was delivered during tests. (B) Learning curves represented in terms of the latency (s) to enter the blue-lit compartment during conditioning trials for the three experimental groups. (C) Memory scores represented in terms of the latency (s) to enter the blue-lit and green-lit compartments. They are displayed in a logarithmic scale for better visualization. Each box extends from the 25th to 75th percentiles; the line in the middle of the box shows the median. Tukey's method was used for plotting whiskers and outliers. (D) Mean walking speed (cm/s) of bees of the three experimental groups during conditioning trials and intertrial intervals. Blue bars represent stimulations with blue light. #: significant difference between paired and unpaired. *: significant difference between unpaired and no-shock. ¤: significant difference between paired and no-shock. (E) Mean walking speed of bees (cm/sec) of each experimental group during the test session including familiarization, test trials, and intertest interval. Blue and green bars represent stimulation with blue and green lights, respectively. Error bars correspond to SEM. ns: nonsignificant, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.

Figure 3.

Inhibitory learning of phototaxis is mainly operant but involves a Pavlovian component. (A) Schematic representation of the experimental protocol. After a familiarization period of 5 min under red light in the setup, two groups of bees (master and yoked) were subjected to two conditioning protocols in which the latency to enter a blue-lit compartment was measured as a proxy of learning and memory. The master group received eight conditioning trials during which the action of entering the blue-lit compartment was paired with a mild electric shock. Each bee in the yoked group received eight trials consisting of stimulations with the attractive blue light and the mild electric shock following the exact same temporal sequence as that of its corresponding master bee. In both groups, trials were separated by intervals of 1 min. Memory retention was tested 24 h after conditioning. The test session consisted of a familiarization period, and two tests separated by 1 min. In the first test, one of the compartments was illuminated with blue light; in the second test, one compartment was illuminated with green light. No-shock was delivered during tests. (B) Learning curves are represented in terms of the latency (s) to enter the blue-lit compartment during conditioning trials for the two experimental groups. (C) Memory scores are represented in terms of the latency (s) to enter the blue-lit and green-lit compartments. They are displayed in a logarithmic scale for better visualization. Each box extends from the 25th to 75th percentiles; the line in the middle of the box shows the median. Tukey's method was used for plotting whiskers and outliers. (D) Mean walking speed (cm/s) of bees of each experimental group along with conditioning trials and intertrial intervals. Blue bars represent stimulations with blue light. (E) Mean walking speed of bees (cm/s) of each experimental group during the test session including familiarization, test trials, and intertest interval. Blue and green bars represent stimulation with blue and green lights, respectively. Error bars correspond to SEM. ns: nonsignificant, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.

Figure 4.

Inhibitory learning of phototaxis up-regulates Amdop1 receptor genes in the calyces of the mushroom bodies. (A) Learning curves represented in terms of the latency (s) to enter the blue-lit compartment during conditioning trials for the two experimental groups, paired and unpaired. Error bars correspond to SEM. (B) Schematic representation of brain dissections. Brains were separated in four sections enriched in mushroom bodies calyces (MBc, red), optical lobes (OL, yellow), antennal lobes (AL, dark blue) and central brain (CB, light blue), respectively. (C–F) Relative normalized expression of five genes of interest (Amdop1, Amdop2, Amdop3, AmoctαR1, and Am5-ht1a receptors) in each of the four dissected brain regions of paired and unpaired conditioned bees. The expression of each gene of interest was normalized to the expression of three genes of reference (Ef1a, Rps8, Rp49) for each bee. Expression distribution is represented as boxplots. Each box extends from the 25th to 75th percentiles; the line in the middle of the box shows the median. Tukey method was used for plotting whiskers and outliers. ns: nonsignificant, (*) P < 0.05, (***) P < 0.001.