1.     Set up bottles of fly food to generate progeny

a.     Use 10 virgin females and 5 males in each bottle as parents to control for crowding

b.     Rear flies on standard cornmeal-dextrose agar food at 25 oC and 60% humidity

c.     After 4 days, clear parents

2.     For two days after eclosion, collect flies without using CO2

a.     Transfer offspring to new bottles, adding ~200 flies per bottle to prevent overcrowding

b.     Allow flies to mate for 24 hours

3.     Make experimental arenas

a.     Obtain 150 mm x 15 mm dishes with lids

b.     Evenly space five 35 x 10 mm dishes within the larger dish and adhere to the bottom of the larger dish using double-sided tape

c.     Remove and dispose of lids for the 35 x 10 mm dishes

d.     Create arenas with defined relative humidity:

       i.     0% RH produced by adding 25 mL Drierite around the smaller dishes

      ii.     70% RH produced by adding 25 mL saturated NaCl around the smaller dishes

e.     Equilibrate arenas in 25oC in the dark for ~10 hours

4.     Separate males and females using CO2

a.     Collect 20 sex-segregated flies in empty food vials

b.     When all flies are separated, place all vials on ice to anesthetize flies

c.     Swiftly knock flies into each small Petri dish

d.     Use 20-25 replicates per condition (4-5 arenas)

e.    Cover each small dish with mesh that has been autoclaved and cut into 50 x 50 mm squares

f.      Seal the mesh around each small dish using zipties

g.     Cover the arena using the lid, then seal with Parafilm

5.     Count, blind to genotype and humidity conditions, the number of dead flies each hour for 12 hours