A blog post highlighting the article by N. Tsvetkov, B. Madani, L. Krimus, S. E. MacDonald, and A. Zayed in Insectes Sociaux

By Armo Zayed

Assay for spatial learning and memory.

As central place foragers, honey bees have an amazing ability to fly several kilometers away from their colony to forage and then beeline back to their home without getting lost. Honeybee foragers can perceive and communicate spatial information via the famous waggle dance. But how are these traits encoded in the honey bee genome?

When Nadia Tsvetkov joined my lab in 2012, she was keenly interested in studying the genetics of spatial learning and memory in bees. She spent several months training bees to fly through mazes. The maze experiments were fun but took too long, and our sample sizes were far fewer than the hundreds of bees needed to tease out the likely subtle genetic effects on learning and memory. What we needed was an assay that was as fast and as easy to standardize as the proboscis extension reflex assay – the workhorse of insect olfactory learning and memory studies. We tried some different approaches (one involved a very beautiful but unwieldy maze constructed out of Christmas balls) until colleague Dr. Suzanne MacDonald, a vertebrate biologist at York University’s Department of Psychology, suggested that we try the food search task paradigm. The paradigm is commonly used to study spatial learning and memory in primates and rodents. A common protocol entails hiding toys or food in boxes within a testing arena that animals are allowed to explore. Over time, the animals learn and can recall the location of boxes that contain rewards.

So we set out to try a similar assay on bees. For prototyping, we used some common and inexpensive items; we made the testing arena of clear Tupperware containers and we employed several artificial flowers made out of Q-tips. After a few pilot assays, we decided that a small arena containing four flowers was the best compromise between complexity and length of the experiment. Nadia worked out the testing protocol that involved placing bees into the arena where one of the artificial flowers had a sucrose reward. Once a bee found and fed from the rewarding flower, it was removed and tested again for a total of three training trials. Finally, the bee entered the arena where none of the flowers had a sugar reward. This time, the bee had to rely solely on its memory to find the focal flower. Our data analysis showed that bees subjected to this test exhibited two telltale signs of learning and memory: they improved their ability to find the rewarding flower during training, and they were able to recall the location of the rewarding flower after training. The Food Search Box (FSB) was born.

We carried out two more experiments to test the utility of the FSB for studying spatial learning and memory in bees. We first compared the performance of nurses (young honeybee workers that nurse the brood) and foragers (older workers that forage outside the colony) in the FSB paradigm. While both nurses and foragers did equally well in the training trials, foragers did substantially better than nurses in the memory test. So, is it age (young vs. old) or behavioral state (nurse vs. forager) that is influencing spatial memory in the FSB? To answer this question, we carried out another study on same-aged workers. We treated these workers with either cGMP, which causes precocious foraging, or cAMP, which does not alter behavioral state. The cGMP-treated bees performed similarly to foragers in the FSB, while cAMP and the control bees performed like nurses in the assay. Taken together, the results of these two experiments indicate that behavioural state (nurse vs. foragers) is primarily associated with differences in spatial memory in the FSB.

We are very excited by the results of the FSB; we were able to test bees quickly without much attrition. It is feasible to screen hundreds of bees within a short period, opening up the door for genetic and genomic studies of spatial learning and memory in honeybees. While it is certainly possible to improve on the design to enhance automation (i.e., RFID readers or tactile sensors to passively record visits to artificial flowers), the low-tech version presented in the paper is very easy to set up and perform. We are looking forward to feedback from the community on the test, and we hope it will provide a useful tool for studying spatial learning and memory in honeybees and other insects.