From these plates, thirty eggs were transferred aseptically to the surface of non-nutritive agar (2% wt/vol agar in water) having 2 3 mg yeast grains distributed on the agar surface in a 35 mm Petri plate.
The samples were filtered through nitrocellulose membranes with 0.45-µm pore diameter, and the filters were placed over 1.5% non-nutritive agar plates, supplemented with Escherichia coli (6 ).
To determine when wild type larvae reach critical weight, we starved carefully staged larvae of defined age classes on non-nutritive agar and measured the time it takes for them to reach pupariation from the onset of starvation.
After three days the males were discarded and equal numbers of females from each reciprocal cross were placed together in a plastic chamber with a spoon containing non-nutritive agar with a generous smear of live yeast for egg collections.
Eggs for the experiment were collected over a six days period by placing Petri dishes containing non-nutritive agar with a generous smear of live yeast in the cages.
We then starved larvae of different age classes on non-nutritive agar media and measured the time it took for them to reach pupariation from the onset of starvation, checking for pupariation every 2 hr.
For ecdysone feeding experiments, we added 0.15 mg of 20E (SciTech Chemicals, Dejvice-Hanspaulka, Czech Republic) to 1 g of normal fly medium or starvation medium (1% non-nutritive agar).
Six to 12 typical colonies (yellow and 1 to 3 mm diameter) were transferred to nutritive soft agar (T1N1, 0.75% agar) and incubated for 24 h at 37°C.
