Ecdysone quantification from whole body samples of drosophila melanogaster larvae

[Abstract] Steroid hormones strictly control the timing of sexual maturation and final body size both in vertebrates and invertebrates. In insects, the steroid hormone ecdysone controls the timing of the molts between larval instars as well as the transition to metamorphosis. Growth during the final instar accounts for over 80% of the increase in final mass in insects, and the duration of this growth period is driven by a sequence of small ecdysone pulses that ultimately induce metamorphosis. Historically the biologically active form of ecdysone, 20-hydroxyecdysone (20E), was quantified using radio-immunoassays, bioassays, or chromatography assays. However, these assays are methodologically complicated and often time consuming. Furthermore, collecting samples for precise measurements of ecdysone concentrations using these assays is limited in small insects like Drosophila melanogaster. Here, we describe an accurate and sensitive method to collect carefully-staged third instar larvae suitable for preparing samples for ecdysone quantification using a commercially-available 20E enzyme immunoassay (EIA). Because we resynchronize larval development at the molt to the final instar, collect large samples, and weigh each sample, we are able to detect a small ecdysone peak early in the final instar known as the critical weight ecdysone peak. This method detects peaks as low as 6 pg 20E/mg larval sample, allowing us to quantify other small ecdysone peaks in flies – the necessary prerequisite for eventually determining their regulation and function.

In contrast, L2 larvae show 1) smaller mouth hooks, 2) "closed" anterior spiracles, 3) thinner tracheae, and yellow-color posterior spiracles. We mainly rely on the anterior spiracle morphology. B. Scheme of staging. At pre-staging, we collect only L2 larvae on new fly medium plates (pre-staging). Two hours later, we collect third instar larvae in food vials and put the rest of the L2 larvae in a new food plate for the next staging. If required, the larvae can be separated by sex, using either sex-specific genetic markers, such as paternally-inherited X or Y chromosome with a ubiquitous GFP marker that will only be expressed in males (for example This causes the larvae to float to the surface. We used the morphology of the anterior spiracles to distinguish between second and third instar larvae (Bodenstein, 1950, see also Figure 1A).
Remove all third instar larvae and transfer second instar larvae onto a new larval culture plate using either Dumont #3 forceps or entomology forceps. We usually remove all L3 larvae from sucrose solution and transfer L2 larvae by double-checking one by one. This is necessary for Therefore, the formula will be: 20E concentration (μg/μl) = (OD240) × (Dilution ratio; it will be 200 in this case) × 480.6/12,676.52 3. Dilute the stock solution to be 1,000 pg/μl, and make a dilution series: 1,000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 pg/μl for the standard curve using absolute ethanol.

Prepare standard solutions by taking 80 µl of the dilution series from
Step B3, such that you obtain 80,000, 40,000, 20,000, 10,000, 5,000, 2,500, 1,250, 625 pg/tube respectively. We prepare two replicate tubes for each concentration (see Figure 3). 6 www.bio-protocol.org/e3915  2. Wash collected larvae twice in double-distilled water to remove any fly medium residuals and wash out sucrose solution. Exclude larvae that appear noticeably smaller or larger for their cohort at this step, as it is likely that these larvae have not been correctly staged.
3. After drying larvae briefly on a small piece of paper towel for about a minute, weigh a group of larvae for one biological replicate together on an ultra-micro balance.
Usually, 20-30 mg of larvae (50-60 larvae for the onset of the L3 and 35-40 larvae for 24 h after the molt to the L3) is sufficient for accurate ecdysone quantification. 4. Place all weighed larvae in a 1.5 ml microcentrifuge tube, add three times the volume of absolute methanol (30 μl methanol for 10 mg larvae), and freeze them immediately on dry ice.

5.
Keep all samples at -80 °C until you are ready for ecdysone quantification (Parvy et al., 2005). 6. When you are ready to begin ecdysone quantification, place all tubes on dry ice and homogenize frozen larvae carefully using disposable pestles and a cordless hand-pestle motor (VWR International). 7. Centrifuge tubes at 4 °C at maximum speed for 5 min. 2. Set up a plate as outlined in the ecdysone EIA manufacturer's instructions (Porcheron et al., 1989; https://www.caymanchem.com/pdfs/501390.pdf).
3. Cover the plate with a piece of parafilm and incubate overnight at 4 °C with gentle agitation.
5. Empty the plate by turning it over. Rinse each well with 300 μl wash buffer. Repeat four more times.
6. After the 5th wash, blot the plate on a few layers of clean paper towel to ensure any liquid will be removed completely.
7. Add 200 μl of Ellman's reagent to each 96 well. Make sure the plate is sealed with a piece of parafilm and covered by a piece of aluminum foil, as the Ellman's reagent is light sensitive.
Incubate the plate at room temperature with gentle agitation. 8. Clean the bottom of the plate with 70% ethanol using paper towels. We then corrected the ecdysone concentration in the sample for sample weights obtained in Step C3 to obtain pg ecdysone/mg larval weight.

Notes
Detecting the smaller pulses of ecdysone in third instar larvae requires both accurately staging the larvae from the molt to the third instar and weighing the samples (Warren et al., 2006). 8 www.bio-protocol.org/e3915