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  • Flies were raised on standard cornmeal media at 25°C and 60% relative humidity under a 12-h light:12-h dark cycle. The white-eyed Canton-S [23], elav-GAL4, cry-GAL4, UAS-mCD8::GFP; UAS-mCD8::GFP, pdf-GAL4, UAS-TrpA1, pdf-GAL80, and VT4244-GAL4 lines were obtained from Dr. Ann-Shyn Chiang. The Oregon-R and red-eyed Canton-S strains were obtained from Dr. Li-Mei Pai. The cryb, crym, and UAS-cry lines were gifts from Dr. Patrick Emery. The UAS-cryRNAi (v7238 and v7239) flies were obtained from the Vienna Drosophila Resource Center (VDRC). Two Helmholtz coils were used to build the platform. Each coil was wound with 332 copper wires and was 20 cm in diameter. The distance between the two coil rings was 10 cm (Fig 1B). The platform generated a uniform magnetic field of different intensities (10 G, 20 G, 40 G, 60 G, and 80 G) by using a DC power supply to input different currents and voltages. The intensity of the magnetic field was measured using a Gaussmeter (Sypris Solutions, Inc., Model #7030, California, USA), with a hall probe manipulator, to evaluate the intensity of the magnetic field in the courtship chamber (Fig 1D). To evaluate courtship behavior, one male and one virgin female fly were placed in a chamber on the magnetic apparatus, and their courtship behaviors were recorded with a video camera. The courtship behavioral assay followed procedures established by a previous study [24]. Naïve males with no pre-test social experience were collected on the day of eclosion and kept individually in test tubes in a 25°C incubator with an L/D cycle. Target females were stored in groups (20 females per vial). The courtship assays were conducted between 2 and 6 h into the light cycle in the courtship chamber (1.2 cm diameter × 0.8 cm high), which contained a layer of yeast media. The flies were anesthetized with mild CO2, and both test males and target females (female Canton-S, 3 days after eclosion) were transferred to the behavior chamber, where the magnetic field was established. The courtship index is defined as the percentage of time that the tested male spent courting the target female during a 10-min recording period (e.g., tapping, following, vibrating wings, and attempting to copulate). For the TrpA1 studies, all of the flies were raised at 23°C before the experiments, and were placed in a 23°C or 30°C environment 10 min before and during the courtship behavioral assays. Drosophila brains were dissected in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 min at room temperature. After fixation, the brain samples were incubated in PBS containing 1% Triton X-100 and 10% normal goat serum (PBS-T) and degassed in a vacuum chamber to expel tracheal air with six cycles of depressurizing to 270 mm Hg followed by holding for 10 min). Next, the brain samples were blocked and penetrated in PBS-T at 25°C for 2 h and then incubated in PBS-T containing mouse 4F3 anti-discs large (DLG) monoclonal antibody (diluted 1:10, Developmental Studies Hybridoma Bank, University of Iowa) at 25°C for one day. After the samples were washed in PBS-T three times, the samples were incubated in a biotinylated goat anti-mouse antibody (diluted 1:200, Molecular Probes, Thermo Fisher Scientific) at 25°C for one day. Next, the brain samples were washed and incubated in Alexa Fluor 635-conjugated streptavidin (diluted 1:500, Molecular Probes) at 25°C for one day. After extensive washing, the brain samples were cleared and mounted in FocusClear (CelExplorer) for confocal imaging. Fly brain samples were imaged using a Zeiss LSM 700 confocal microscope with a 40X C-Apochromat water-immersion objective lens. To overcome the limited field of view, the brain samples were imaged twice, one for each hemisphere, with an overlap in between. We then combined the two parallel image stacks into a single dataset with ZEN image-processing software, using the overlapping region to align the two stacks. All data were analyzed parametrically with Prism 5 statistical software (GraphPad). Data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons tests or evaluated by paired t-tests. All data are presented as the mean + standard error of the mean (SEM).
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