Ecdysone

Flies were reared on standard cornmeal-molasses food at 25°C unless otherwise indicated. The following Gal4 lines from the Janelia Farm collection [38 (link)] were used to label follicle cells and corpus luteum cells: R47A04 (Oamb), R49E12 (5-HT2A), R10E05 (AstC-R2), and R42A05 (kay). To knockdown mmp1 or mmp2 or overexpress Timp in adult flies, actGal4/Cyo; tubGal80^ts virgin females were crossed to the following lines at 18°C and shifted to 29°C immediately after adult eclosion: UAS-mmp1^RNAi (Bloomington Drosophila stock center, B31489), UAS-mmp1^RNAi2 [39 (link)], UAS-mmp1^RNAi3 (Vienna Drosophila RNAi Center, V108894), UAS-mmp1^E225A (a dominant negative form of Mmp1) [40 (link)], UAS-mmp2^RNAi [39 (link)], UAS-mmp2^RNAi2 (VDRC, V107888), UAS-mmp2^RNAi3 (BDSC, B31371), UAS-Timp [41 (link)]. To knock down mmp1 or mmp2 or overexpress Timp in follicle cells of mature egg chambers, UAS-dcr2; R47A04 virgin females were crossed to the RNAi lines described above at 29°C. To knock down ecdysone synthesis genes, UAS-dcr2; R47A04 virgin females were crossed shd^RNAi (VDRC, V17203), dib^RNAi (VDRC, V101117), or phm^RNAi (VDRC, V108359). To overexpress mmp1 or mmp2 in mature follicle cells, R47A04 virgin females were crossed to UAS-mmp1 or UAS-mmp2 [41 (link)] at 21°C. Control flies were derived from specific Gal4 driver crossed to wild-type Oregon-R. Mmp2::GFP fusion genes were generated through recombinase mediated cassette exchange of MiMIC insertion (MI02914) in the third coding intron of mmp2 (S4 Fig.) [42 (link)]. Mmp2-Gal4 line is from an Gal4 enhancer trap [43 (link)], and UAS-RedStinger (BDSC, B8547) and UASpGFP-act79B; UAS-mCD8-GFP were used as reporters. sqh-EYFP-Mito (BDSC, B7194) and sqh-EYFP-ER (BDSC, B7195) were used for tracking mitochondria and endoplasmic reticulum, respectively.

Mitophagy was examined using a FACS-based analysis of mitochondrially targeted mKeima (a gift from A. Miyawaki, Laboratory for Cell Function and Dynamics, Brain Science Institute, RIKEN, Japan). U2OS cells stably expressing an ecdysone-inducible mt-Keima were induced with 10 μM ponasterone A, transfected with GFP-Parkin for 18 h and treated with 20 μM CCCP for 4 h. For flow cytometry analysis, cells were trypsinized, washed and resuspended in PBS prior to their analysis on a LSR Fortessa (BD Bioscience) equipped with 405 and 561 nm lasers and 610/20 filters (Department of Microbiology and Immunology Flow Cytometry Facility, McGill University). Measurement of lysosomal mitochondrially targeted mKeima was made using a dual-excitation ratiometric pH measurement where pH 7 was detected through the excitation at 405 nm and pH 4 at 561 nm. For each sample, 100,000 events were collected and single, GFP-Parkin-positive cells were subsequently gated for mt-Keima. Data were analysed using FlowJo v10.1 (Tree Star). For statistical analysis, a one-way ANOVA with Tukey's post test was performed on data from two independent experiments. *P<0.05; **P<0.001; ***P<0.0001; n.s., nonsignificant.

The ES [76 ] derives from a 7-year study where standardised methodology was used in a strategy [43 ] to screen the seeds/spores of 4155 species of vascular plants for the presence of ecdysteroid agonists and antagonists. Samples were micro-extracted with methanol (in which ecdysteroids are highly soluble) and the extracts were partially purified (to remove non-polar lipids and pigments) before analysis with 2 or 3 ecdysteroid-specific radioimmunoassays (using the black, white and DBL-1 antisera to quantify compounds structurally similar to ecdysone and 20-hydroxyecdysone; Refs. [43 ,77 (link)]) and the Drosophila melanogaster BII microplate bioassay for ecdysteroid agonists (detecting the presence of compounds with the biological activity of 20-hydroxyecdysone; Ref. [78 (link)]). The threshold for the detection of ecdysteroids was 150 ng ecdysone equivalents/g for the black and white antisera, 75 ng ecdysone equivalents/g for the DBL-1 antiserum and 7.2 μg 20-hydroxyecdysone equivalents/g for the BII bioassay. The methods used are described in detail on the website [76 ], and the data are presented in tabular form. The samples were either randomly selected (N-Series; 2500 species) or selected according to potential interest (S-Series; 2000 species). The criterion for being deemed positive was a linear response (pg ecdysone equivalents vs. extract aliquot size) in at least one RIA or a positive response in the agonist version of the BII bioassay.

The MEME motif-based algorithm [52 (link)] was used to analyze a 2500 bp sequence upstream of the 5’ transcription start site of the HPx1 gene (Aedes aegypti genome, version AaegL3.4). The Fimo tool [53 (link)] was used to specifically search for cis-regulatory elements associated with ecdysone molecular signaling, as previously described [54 (link)].

Ecdysone was purchased in powder form and was dissolved in ethanol. Then it was mixed with 2% agarose containing 5% sucrose to make the different doses of ecdysone tubes, and the same amount of ethanol as in the ecdysone solution was used to make the vehicle control tubes. Only 2% agarose and ecdysone were used to make ecdysone starvation tubes. Five- to seven-day-old flies were loaded into locomotor tubes for ecdysone treatment and behavior recording to verify that ecdysone promotes sleep in ecdysone-treated flies. For ecdysone feeding/starvation assay, all flies were kept in normal tube on days 0, 1, and 2. Flies were then transferred to either starvation tubes, or ecdysone starvation tubes between ZT23 and ZT0 on day 2. Flies were kept in these ecdysone/starvation tubes for 1 day and transferred back to normal tubes again between ZT23 and ZT0 on day 3 and recorded for another day. Data from days 2 to 4 were used for sleep analysis. For gaboxadol hydrochloride treatment, it was dissolved in water and diluted to 0.1 mg/ml as final concentration for locomotor tube experiments. Sleep was measurement from days 2 to 4, after flies were loaded into gaboxadol tubes at day 0. For TARGET system experiments, flies were raised at permissive temperature 18°C, and 5- to 7-day-old adult flies were used to loaded and were kept at 18°C from days 0 to 2, 31°C from days 3 to 4, and 18°C for day 5. Data from days 2 to 5 were collected for sleep analysis.
BODIPY493 was used for brain LD staining. Flies were loaded into locomotor tubes and subjected to 0.5 mM ecdysone treatment for 12 or 36 hr. Both control and ecdysone-treated flies were then dissected in the phosphate-buffered saline (PBS), fixed in 4% Paraformaldehyde (PFA) solution for 20 min, and washed three times with PBS + 0.3% Triton (PBT). Then brains were left in PBT at 4 degrees overnight and transferred to 1 µg/ml BODIPY493 in PBT for 20 min. Later they were mounted for imaging.
Brains were imaged with the oil-immersion ×40 lens of a confocal microscope at a resolution of 1024 × 1024. Raw images were processed with FIJI ImageJ. The first step was to remove non-lipid trash and exclude artificial signals or signals outside the brain area. The second step was to quantify the LD count, area, and total brain tissue area. Later, the LDs count will be normalized by the brain tissue area, and LD size calculated by LD area divided by total count. Quantifications were conducted by using ImageJ Macro.