Experiment 2: learning to avoid pheromone trails – quinine+shock punishment
This protocol is extracted from research article:
Hard limits to cognitive flexibility: ants can learn to ignore but not avoid pheromone trails
J Exp Biol, Jun 4, 2021; DOI: 10.1242/jeb.242454

During experiment 1, we noticed that ants very rapidly learned to carefully probe the droplet with their antennae before drinking. This enabled them to avoid punishment for an incorrect choice, thus greatly reducing the cost of errors. Such reduced error costs may not have been sufficient to promote learning. We thus designed a further experiment (experiment 2) in which we introduced an electric shock device (henceforth ‘shocker’; Fig. 1B) as an unavoidable punishment. Shockers were affixed 4 cm from the bifurcation on each of the Y-arms. They consisted of a 3D-printed PLA body (an STL file can be downloaded in modifiable form from https://www.tinkercad.com/things/dl5ce7taHaI-ant-zapper), which offered a tunnel narrowing from 1 cm (the width of the Y-maze arms) to a 2 mm gap (Fig. 1B). The floor of the gap was covered with two slightly disconnected copper plates (Fig. 1B, left), which were connected via wires to a button and a laboratory power supply. When the ant walked through the gap, thereby touching each copper plate with at least one of her legs, she closed the electric circuit (if the shocker was activated) and got shocked with 7.5 V (Roussel et al., 2012). The front and back of the shocker were equipped with a polytetrafluoroethylene (Fluon®) plastic plate, preventing ants from passing the barrier except via the tunnel. Apart from adding the shocker, experiment 2 was identical in design to experiment 1 above. A total of 31 ants were tested.

While the overall procedure of experiment 2 was identical to that of experiment 1 above, we added two methodological improvements. Firstly, we established two pre-training trials prior to the trials, ensuring a standard baseline experience across subjects. Secondly, we added two unrewarded learning tests after trial 20 and 25, to increase the chance of uncovering any cryptic learning that may have occurred (Bortot et al., 2019).

In the pre-training trials, we confronted the ant with one rewarded trial followed by one punished trial, both presented on a linear runway (21 cm long, 1 cm wide). In the first trial, paper overlays covered with a control solvent were placed on the runway, which was also equipped with an inactive shocker and a droplet of sucrose solution that was presented behind the device. After reaching the sucrose drop, the ant was marked with acrylic paint on its abdomen while drinking the sucrose solution. Afterwards, she was allowed back to the nest. After unloading the sugar, the ant was allowed back onto the linear runway for a second pre-training trial. In this second trial, the ant was confronted with a linear runway covered with a pheromone trail, an activated shocker, and a droplet of quinine solution behind the device. After the ant experienced both negative stimuli (shock and quinine), it was transferred to the Y-maze for the test. Within the pre-training trials, the punishment trial was always carried out after the reinforcement trial to ensure the ant's participation.

During unrewarded learning tests, the arms of the Y-maze were equipped with the respective paper layers (control solvent or pheromone trail), but both shockers were inactivated, and the sucrose and quinine droplets were replaced by neutral water droplets. After entering one arm of the Y-maze, the connection to the Y-maze stem was interrupted for 1 min, therefore only allowing the exploration of both Y-maze arms. We recorded the overall time the subject spent on the correct (positive) arm before replacing the water droplets with the respective liquids (sucrose or quinine solution), allowing the ant to drink from the sucrose, and to return freely to the nest for further testing.

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