Reproductive behavior in Drosophila has both stereotyped and plastic components that are driven by age- and sex-specific chemical cues. Males who unsuccessfully court virgin females subsequently avoid females that are of the same age as the trainer. In contrast, males trained with mature mated females associate volatile appetitive and aversive pheromonal cues and learn to suppress courtship of all females. Here we show that the volatile aversive pheromone that leads to generalized learning with mated females is (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). cVA is a major component of the male cuticular hydrocarbon profile, but it is not found on virgin females. During copulation, cVA is transferred to the female in ejaculate along with sperm and peptides that decrease her sexual receptivity. When males sense cVA (either synthetic or from mated female or male extracts) in the context of female pheromone, they develop a generalized suppression of courtship. The effects of cVA on initial courtship of virgin females can be blocked by expression of tetanus toxin in Or65a, but not Or67d neurons, demonstrating that the aversive effects of this pheromone are mediated by a specific class of olfactory neuron. These findings suggest that transfer of cVA to females during mating may be part of the male's strategy to suppress reproduction by competing males.

Suppression of memory by new learning demonstrates that the dynamics of memory consolidation are subject to plasticity in Drosophila. This type of metaplasticity is essential for navigation of experience-rich natural environments.

Mechanisms for identifying appropriate mating partners are critical for species propagation. In many species, the male uses multiple sensory modalities to search for females and to subsequently determine if they are fit and receptive. Males can also use the information they acquire in this process to change their courtship behavior and reduce courtship of classes of targets that are inappropriate or unreceptive. In Drosophila, courtship plasticity, in the form of both nonassociative and associative learning, has been documented-the type of learning depending on the nature of the trainer. The conditions in which the male is presented with the training target can profoundly alter the cues that he finds salient and the longevity of the memory that he forms. With the exception of habituation and sensitization, these types of plasticity have an operant component in that the male must be courting to respond to the behavior-altering cues. Courtship plasticity is therefore a complex and rich range of behaviors rather than a single entity. Our understanding of these plastic behaviors has been enhanced by recent advances in our understanding of the circuitry underlying courtship itself and the identification of chemical cues that drive and modify the behavior. Courtship learning is providing a window into how animals can use a variety of sensory inputs to modulate a decision making process at many levels.

Genetic causes for disturbances of locomotor behavior can be due to muscle, peripheral neuron, or central nervous system pathologies. The Drosophila melanogaster homolog of human CASK (also known as caki or camguk) is a molecular scaffold that has been postulated to have roles in both locomotion and plasticity. These conclusions are based on studies using overlapping

Finding a mating partner is a critical task for many organisms. It is in the interest of males to employ multiple sensory modalities to search for females. In Drosophila melanogaster, vision is thought to be the most important courtship stimulating cue at long distance, while chemosensory cues are used at relatively short distance. In this report, we show that when visual cues are not available, sounds produced by the female allow the male to detect her presence in a large arena. When the target female was artificially immobilized, the male spent a prolonged time searching before starting courtship. This delay in courtship initiation was completely rescued by playing either white noise or recorded fly movement sounds to the male, indicating that the acoustic and/or seismic stimulus produced by movement stimulates courtship initiation, most likely by increasing the general arousal state of the male. Mutant males expressing tetanus toxin (TNT) under the control of Gr68a-GAL4 had a defect in finding active females and a delay in courtship initiation in a large arena, but not in a small arena. Gr68a-GAL4 was found to be expressed pleiotropically not only in putative gustatory pheromone receptor neurons but also in mechanosensory neurons, suggesting that Gr68a-positive mechanosensory neurons, not gustatory neurons, provide motion detection necessary for courtship initiation. TNT/Gr68a males were capable of discriminating the copulation status and age of target females in courtship conditioning, indicating that female discrimination and formation of olfactory courtship memory are independent of the Gr68a-expressing neurons that subserve gustation and mechanosensation. This study suggests for the first time that mechanical signals generated by a female fly have a prominent effect on males' courtship in the dark and leads the way to studying how multimodal sensory information and arousal are integrated in behavioral decision making.

The antennal lobe (AL) is the primary olfactory center in insect brains. It receives sensory input from the olfactory sensory neurons (OSNs) and sends, through its projection neurons (PNs), reformatted output to secondary olfactory centers, including the mushroom body (MB) calyx and the lateral horn (LH) in the protocerebrum. By injecting dye into the AL of wild-type Drosophila, we identified previously unknown direct pathways between the AL and the ventrolateral, superior medial, and posterior lateral protocerebra. We found that most of these areas in the protocerebrum are connected with the AL through multiple tracts, suggesting that these areas are sites of convergence for olfactory information. Furthermore, areas such as the superior medial protocerebrum now appear to receive olfactory output both directly from the AL and indirectly from lobes of the MB and the LH, suggesting a degree of functional interaction among these areas. We also analyzed the length and number of fibers in each tract. We compare our results obtained from wild-type flies with recent results from transgenic strains and discuss how information about odorants is distributed to multiple protocerebral areas.

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INTRODUCTIONIn Drosophila melanogaster, as in many other animals, courtship is a series of stereotypical behaviors carried out by a male responding to multimodal signals. Because different experimental conditions can engage distinct sensory modalities that affect male behavior, courtship experiments need to be carefully designed. There are several ways to manipulate sensory inputs to the test male. This protocol describes methods for designing and conducting experiments that measure the various parameters of courtship behavior.