Arduino-Based Volatile Delivery System for Evaluating Honeybee Learning.

The proboscis extension reflex (PER) conditioning protocol is a widely used paradigm for studying learning and memory in honeybees. Accurate and consistent delivery of odor stimuli is essential for ensuring reproducible conditioning results, yet many existing systems require complex and expensive equipment. Here, we present a low-cost, fully documented Arduino-based system for automated volatile delivery during PER conditioning assays. The system integrates solenoid and servo valves, allowing controlled airflow between clean air and odorant channels, with stimulus timing managed by easily modifiable open-source code. Using this setup, we successfully conditioned Apis mellifera workers to associate 1-hexanol with a sucrose reward, achieving response rates comparable to those reported in previous studies. After three conditioning trials, 90% of honeybees exhibited conditioned responses, and 75% retained the association after five trials. This accessible and flexible system provides a reliable tool for researchers and educators aiming to implement classical conditioning protocols without requiring specialized technical expertise or infrastructure.