Visual Classical Conditioning in Wood Ants

Common advantage of this technique is that visual memories can be studied in a controlled manner in fixed rather than free-moving wood ants. This method has a potential to be used for investigating the neural basis of visual learning in a very well-established model for insect navigation. To select the ants that are motivated to eat, place a slide with the drop of sucrose inside a small box with fluon covering the walls.

Then place each selected ant in the box and wait to see if it feeds on the sucrose drop. If the ant feeds on the sucrose drop immediately transfer it to an empty box. After this, transfer each ant to a separate tube and briefly place the tube in the freezer to immobilize the ants.

Place the immobilized ant in the holder through the cut in the cardboard, grasping it by the joint between its head and the thorax. Then use insect pins to ensure the antennae remain on the side of the head. Using a custom-made heating element, melt wax to adhere the tip of an insect pin to the ant's head, parallel to the cardboard.

After the wax dries, remove the insect pins from the antennae, and carefully remove the ant from the holder. Next, fix the insect pin holding the ant in a modeling clay cylinder, and fix a custom-made plastic holder beneath it. Ensure the ant maintains a typical standing posture and that the whole body, except the head, can move freely.

First, position a camera with a macro lens on top of the white acrylic box directly above the ant holder, and place the ant in the holder inside the box. Then, attach a needle and sucrose-filled syringe, the unconditional stimulus, to a bright blue cardboard rectangle, the conditional stimulus, for use as a visual cue. For paired training, start recording the ant 10 seconds before presenting the conditional stimulus.

If the ant performs the Maxilla Labium Extension Reflex, or MaLER, during this time, postpone the trial for a few seconds. Move the syringe with the conditional stimulus in front of the ant for 10 seconds. Then, exude a single drop of sucrose from the syringe and feed the ant for five seconds.

Repeat the paired training procedure 10 times with five minutes between each trial. For unpaired training, start the video recording 10 seconds before the trial. Then, present the conditional stimulus as previously described.

After 2.5 minutes, deliver the unconditional sucrose stimulus directly to the ant's mouthparts with a syringe without the blue cardboard. After the training trials are complete, begin the camera recording 10 seconds prior to the testing phase. Then, present the conditional stimulus to the ant for 10 seconds.

After the test is over, deliver the sucrose unconditional stimulus to the ant to confirm the ant is still motivated to feed. Using the recording made during training and testing, score the ant's responses during the 10 seconds of conditional stimulus presentation. Finally, separate the ant's responses during the conditional stimulus presentation into three categories, full extension with movement as if feeding, full extension without movement, or partial extension of the maxilla labium or maxillary palps.

In this protocol, ants that underwent paired training performed increasingly more MaLER in response to the conditional stimulus. On the contrary, unpaired ants showed no significant increase in MaLER during the training. To examine their short and mid-term memory, ants were tested either 10 minutes or one hour after the last training trial.

For both tests, the proportion of ants performing MaLER in response to the conditional stimulus was higher when they had undergone paired training. MaLER responses were divided into three categories, FEM, FE, and PE.Typically, ants performed FEM or PE more often than FE.However, only a few ants consistently demonstrated the same type of response. While attempting this procedure, it's important to remember to handle ants carefully to reduce stress levels and any potential damage, particularly during harnessing.

By adapting this procedure, different visual stimuli can be used in order to investigate the properties wood ants use to form visual memories. After its development, this technique paves the way for researchers in the field of insect neuroscience and navigation to explore visual associative memories with wood ants for microfa.