Infestation of palm trees by the red palm weevil (RPW) Rhynchophorus ferrugineus in the tropics, Middle East, and North Africa represents a serious economic threat and has reached a critical level due to significant spread. The inability of early detection of this pest is the main obstacle against infestation management. Here, we investigated the potential use of the honeybees' olfaction for early detection of the odorant signature of the RPW's aggregation pheromone. We conditioned the Proboscis Extension Response (PER) of harnessed honeybee foragers using the RPW's aggregation pheromone as a conditioned stimulus in an appetitive Pavlovian learning protocol using either the synthetic pheromone or the natural pheromone released by groups of RPWs. To test the sensitivity of pheromone detection by bees, five groups of RPWs (25, 20, 15, 10, and 5 individuals each) were used to condition the bees. Medium- and long term- memory retention was evaluated after (4, 24, 48, and 72 hrs post-conditioning). The honeybees learned the odor of the RPW's pheromone both in its synthetic and its natural form. In the latter case, learning success increased with the number of weevils used as a pheromone source. The percentage of learners after six conditioning trials was 89%, 92%, 87%, 80%, 68%, and 49% for the control, 25, 20, 15, 10, and 5 RPW group sizes, respectively, and was positively correlated with RPW group size. Memory was retained for at least 24 h without a significant decay in all groups trained with different numbers of RPWs. Memory retention decreased significantly after 48 h. In conclusion, PER conditioning using RPW pheromone as a conditioned stimulus for subsequent detection of RPW infestations was valid up to a period shorter than 48 h for the 15 RPW group (27% of bees) and to 72 h for the 20 RPW group (25% of bees). The results show that honeybees could be used as promising bio-detectors for the red palm weevil. However, further research and field trials are needed to establish them as proven biological sensors.
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