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Bacteriophage integrase-directed insertion of transgenic constructs into specific genomic loci has been widely used by Drosophila community. The attP40 landing site located on the second chromosome gained popularity because of its high inducible transgene expression levels. Here, unexpectedly, we found that homozygous attP40 chromosome disrupts normal glomerular organization of Or47b olfactory receptor neuron (ORN) class in Drosophila. This effect is not likely to be caused by the loss of function of Msp300, where the attP40 docking site is inserted. Moreover, the attP40 background seems to genetically interact with the second chromosome Or47b-GAL4 driver, which results in a similar glomerular defect. Whether the ORN phenotype is caused by the neighbouring genes around Msp300 locus in the presence of attP40-based insertions or a second unknown mutation in the attP40 background remains elusive. Our findings tell a cautionary tale about using this popular transgenic landing site, highlighting the importance of rigorous controls to rule out the attP40 landing site-associated background effects., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Genetics Society of America.)

In Drosophila, 50 classes of olfactory receptor neurons (ORNs) connect to 50 class-specific and uniquely positioned glomeruli in the antennal lobe. Despite the identification of cell surface receptors regulating axon guidance, how ORN axons sort to form 50 stereotypical glomeruli remains unclear. Here we show that the heterophilic cell adhesion proteins, DIPs and Dprs, are expressed in ORNs during glomerular formation. Many ORN classes express a unique combination of DIPs/dprs, with neurons of the same class expressing interacting partners, suggesting a role in class-specific self-adhesion between ORN axons. Analysis of DIP/Dpr expression revealed that ORNs that target neighboring glomeruli have different combinations, and ORNs with very similar DIP/Dpr combinations can project to distant glomeruli in the antennal lobe. DIP/Dpr profiles are dynamic during development and correlate with sensilla type lineage for some ORN classes. Perturbations of DIP/dpr gene function result in local projection defects of ORN axons and glomerular positioning, without altering correct matching of ORNs with their target neurons. Our results suggest that context-dependent differential adhesion through DIP/Dpr combinations regulate self-adhesion and sort ORN axons into uniquely positioned glomeruli., Competing Interests: The authors have declared that no competing interests exist.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Critical to evolutionary fitness, animals regulate social behaviors by integrating signals from both their external environments and internal states. Here, we find that population density modulates the courtship behavior of male Drosophila melanogaster in an age-dependent manner. In a competitive mating assay, males reared in a social environment have a marked advantage in courting females when pitted against males reared in isolation. Group housing promotes courtship in mature (7-day) but not immature (2-day) males; this behavioral plasticity requires the Or47b pheromone receptor. Using single-sensillum recordings, we find that group housing increases the response of Or47b olfactory receptor neurons (ORNs) only in mature males. The effect of group housing on olfactory response and behavior can be mimicked by chronically exposing single-housed males to an Or47b ligand. At the molecular level, group housing elevates Ca2+ levels in Or47b ORNs, likely leading to CaMKI-mediated activation of the histone-acetyl transferase CBP. This signaling event in turn enhances the efficacy of juvenile hormone, an age-related regulator of reproductive maturation in flies. Furthermore, the male-specific Fruitless isoform (FruM) is required for the sensory plasticity, suggesting that FruM functions as a downstream genomic coincidence detector in Or47b ORNs-integrating reproductive maturity, signaled by juvenile hormone, and population density, signaled by CBP. In all, we identify a neural substrate and activity-dependent mechanism by which social context can directly influence pheromone sensitivity, thereby modulating social behavior according to animals' life-history stage.