Documentation

Introduction

Locust Y Maze is a classical conditioning paradigm used to assess associative olfactory learning responses in the desert locust Schistocerca gregaria. A Y-maze is a modified form of T maze similar to rodents Y-maze. Locust Y-maze arena is an acrylic Y shaped apparatus occupied with a Y shaped rod. It consists of two arms separated from each other at an angle of 90o. The top is covered with a transparent lid, providing a clear view of the subject throughout the test. 

Locust Y-maze is usually used to evaluate olfactory learning behavior in desert locusts. In the experiment, locusts secured in an acrylic box are starved for 24 hours and then subjected to trials involving an unconditioned stimulus (US) paired with a conditioned stimulus (CS). Later on, the animals are allowed to pass through the test arena in the presence of stable airflow (directed towards their antennae) and a light source. Stable airflow is provided by an air pump and extraction lamp, whereas LED is used as a light source. The conditioned olfactory responses of locusts are monitored and recorded with the help of a video camera such as Noldus Ethovision XT.

Another form of Y-maze is a long arm Y-maze used to assess rodent’s inherent ability to explore new places. 

Apparatus and Equipment

Locust Y-maze arena consists of a main arm (24cm long) and two decision arms 15cm each. Two arms of the maze are separated by an angle of 90o. The acrylic maze is 22cm high and 10cm wide. It is occupied with a Y shaped rod in the center. This rod is elevated 11cm above the floor of the maze and has a circular cross-section of 2.5cm diameter. The main arm of the rod is 21cm long, and the length of two decision arms is 13cm each. Gregarious animals are allowed to walk only along the rod occupying the central area of the maze. A lid provides access to the inside of the maze on the top. The lid is made of acrylic glass to enable a clear view of subjects throughout the test. The end walls of decision arms have a hole (1cm in diameter) in the center to facilitate the passage of odors into the maze. These odor entrances are connected to odor containers.

An air pump is used along with an extraction fan to maintain stable airflow inside Y-maze. The airflow is directed from decision arms towards the long arm. A 100W heat lamp is used as a light source above the junctions of the arms of the maze. 

Training Protocol

Pre-training

Before subjecting to the Y-maze test arena, locusts are exposed to olfactory conditioning. The pre-training involves the following steps:

1. Starve gregarious animals of either sex for 24 hours and secure them in an acrylic box. Let the locusts rest for 5 minutes before the training begins.
2. Put CS (for example, 25ml of pure lemon extract) on 1cm2 filter paper and place it at the end of a tube. Connect the tube to an air pump and extraction fan. Place the tube in such a way as it is 5cm away and directed towards the subjects’ antennae. 
3. Place the locusts in front of the tubing 20 seconds before trial. Multiple trials are needed for associative training. The training consists of 10-15 trials, and the inter-trial interval (ITI) should be 5 minutes.
4. In each trial, first, expose the locusts to CS for 5 seconds and then simultaneously to CS/US (conditioned stimulus and unconditioned stimulus) for 20 seconds.  
5. Discard the locusts who fail the training and return the rest to the habituation tank. 

Procedure

After pre-training, locusts are subjected to the Y-maze test arena. Perform a single odor preference test in the Y-maze conditioning paradigm at the following intervals after training: 10min, 1hour, and 24hours. The test should be conducted as follows:

1. Before each test, place the experimental locust inside a cylindrical holding tube. The tube is attached to the rod of the maze. Keep the end of the tube facing the maze open so that it allows locusts to exit. Cover the other end with mesh to facilitate the airflow. 
2. Connect the vial containing 1ml of odor to the hole in the walls of decision arms. The vial must be attached to the air pump and extraction fan to enable stable airflow. Attach one decision arm to CS and the other to a novel odor.
3. Place the locust inside the holder and leave undisturbed for a few seconds.
4. Turn on the airflow and place the holding tube on the rod’s main arm at a position closest to the extraction fan. 
5. Give 5 minutes to each locust to exit the holding tube. Allow them to make a choice between arms. Let them touch the end of either of the decision arms with their legs or antennae. Record their choices. 

Note: The locusts who fail to choose or fall of the rod are discarded.

6. After each test, remove the locust and wipe the rod with 70% alcohol to get rid of possible traces of pheromones, and then let it dry. 

Literature Review

Assaying Visual Memory in the Desert Locust

Dillen et al., 2015 performed an experiment to determine whether the foraging behavior in desert locusts is visually oriented or not. Furthermore, feeding-dependent regulation of Schgr-sNPF transcript levels was studied. The insects were subjected to the training arena consisting of a rectangular cage. This cage had two smaller cages at the far end, and these smaller cages were lined with green or yellow laminated paper on the inside. Later on, the subjects were exposed to the Y-maze test. The effect of short neuropeptide F on visual memory of locusts could not be demonstrated. However, they succeeded in establishing an experimental assay to demonstrate the efficiency of learning and memory in Schistocerca gregaria.

A long-latency aversive learning mechanism enables locusts to avoid odors associated with the consequences of ingesting toxic food

Simões et al., 2012 studied how post-ingestive aversive memories enable desert locusts to make appropriate olfactory decisions in a Y-maze. They observed the effect on associative memory of these insects when subjected to odor and toxin-free food accompanied by toxin nicotine hydrogen tartrate (NHT) injections. It was concluded that locusts could form specific long-lasting aversive olfactory associations which they use to abstain from toxic food when they are foraging. 

Data Analysis

The following behaviors and parameters can be recorded using the Locust Y maze

• Time spent in the main arm  
• Time spent in the decision arms
• Number of visits to decision arms
• Latency to choose the decision arm 

Strengths and Limitations

Strengths

• Y-maze test presents a robust behavioral measure of appetitive conditioned associative memory
• Locust Y-maze arena is easy to use.
• It yields easily reproducible results because of its simplicity.

Limitations

• The reflexes observed and recorded in the locust Y-maze arena is not an exact representation of the behavioral responses of the subject in its natural habitat.
• Y-maze has a higher rate of probability success. It is a simple two-choice maze. Thus, the probability success by default is 50 percent. 

Summary

• Locust Y Maze is a conditioning paradigm used to assess associative olfactory learning responses in the desert locust Schistocerca gregaria.
• A Y-maze is a Y shaped arena with a long arm and two short arms. The short arms are separated from each other at an angle of 90o. The central area is occupied with a Y-shaped rod. The lid of the maze is made of acrylic glass to provide a clear view of animals inside the maze. 
• The results are recorded based on individual responses to the stimulus.

References

Dillen, S., Chen, Z., & Broeck J. V. (2015). Assaying Visual Memory in the Desert Locust. Insects, 6(2), 409-418. doi:10.3390/insects6020409

Simões, P. M., Ott, S. R., & Niven, J. E. (2012). A long-latency aversive learning mechanism enables locusts to avoid odours associated with the consequences of ingesting toxic food. Journal of Experimental Biology, 215(10), 1711-1719. doi: 10.1242/jeb.068106

Simões, P., Ott, S. R., & Niven, J. E. (2011). Associative olfactory learning in the desert locust, Schistocerca gregaria. Journal of Experimental Biology, 214(15), 2495-2503. doi:10.1242/jeb.055806