Effects of cold immobilization and recovery period on honeybee learning, memory, and responsiveness to sucrose

In addition to human error and variation in laboratory conditions, there are numerous factors that can complicate comparisons among studies. Furthermore, differences in how experimental methods are executed can make it difficult to distinguish between effects of focal versus extraneous variables. Insect neural function is commonly evaluated using Pavlovian conditioning techniques; learning and memory in many species can be assessed using the proboscis extension reflex (PER). However, there are significant inconsistencies in methods used to immobilize insects prior to PER tests. We compared responses of honeybees immobilized in a refrigerator, on ice, and in a freezer, and evaluated influence of recovery interval before testing. Ice-chilling weakly decreased learning (response to an originally neutral odor) more so than refrigeration or freezing, but not 24-h recall of odor. We found no significant differences in responsiveness to sucrose relative to cooling method, but responsiveness was significantly lower among honeybees left to recover for only 0.75 h versus 1.5 or 3 h. Finally, we observed increased responsiveness to sucrose and geraniol between June and August. Our results suggest that inconsistencies in cold immobilization methods could confound interpretation and comparison of results from a large body of work on honeybee learning and memory.

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► Methods for evaluating honeybees’ proboscis extension reflex vary substantially.
► Three methods of cooling are used; differences in temperature affect time to immobilization.
► Cooling method affects learning (ice-chilling reduces) but not memory or responsiveness to sucrose.
► Longer recovery periods increase responsiveness to sucrose.