Figure 4. Trainer-Specific Learning Is Associative.

Males were trained for 1 hr. Trainer condition is indicated above the panel, and tester type is indicated below each set of bars (mV, mature virgin; iV, immature virgin; im, immature male; -d, decapitated). Data are presented as means ± SEM.

(A) Learning requires a trainer-specific pheromone signal. Males were trained with an immature virgin courtship object in the presence of a pheromone-laced or control filter. Compared to males trained with a control filter, males that had been exposed to mature virgin pheromones in the context of an unsuccessful courtship suppressed subsequent courtship of mature virgins (*p < 0.1, **p < 0.05, ***p < 0.001). Filters that had been exposed to a mature virgin for 4 hr were more effective trainers than filters that had only been exposed for 1 hr (**p < 0.05). Pairing of the courtship object with an irrelevant pheromone (immature male) did not produce memory when tested with a mature virgin.

(B) Substitution of an immature male for the immature virgin courtship object produced the same results, indicating that the nature of the courtship object does not influence the specificity of the learning.

(C) Sequential presentation of cues does not support memory formation. Males were trained for 1 hr with an immature virgin, then exposed for 1 hr to a filter containing mature-virgin pheromone (left bar) or to the same cues in reversed order (middle bar). No memory was formed in comparison to time-matched control males who were trained for 1 hr with a mature virgin and tested after a 1.5 hr delay (right bar, **p < 0.05 for comparison with the null hypothesis).

(D) Copulation during training blocks learning. Males were exposed to a mobile mature virgin (left bar) for 1 hr and copulation was confirmed visually. Control males were trained for 1 hr with a decapitated mature virgin and did not copulate. Males were tested for memory 1 hr after training to allow copulated males to recover from fatigue. Courtship vigor of males that had copulated was unaffected after training and rest (p > 0.1), whereas males that did not copulate showed significant suppression of courtship (**p < 0.05 for comparison to the null hypothesis).