Petri dish hygrotaxis arena

1.     Set up bottles of fly food to generate progeny

a.     Use 7 virgin females and 2 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

c.   Transfer flies to new bottles after 3 days

d.   Let flies mature for 3 additional days (7 day old flies)

3.     Make experimental arenas

a.     Obtain 100 mm x 15 mm dishes with lids

b.     Center one 35 x 10 mm dish 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:

1. Inner chamber: 70% RH produced by adding 10 mL saturated NaCl in the smaller dish OR 96% RH produced by adding 10 mL ddH₂O in the smaller dish
2. Outer chamber: 20% RH produced by adding 20 mL saturated LiCl around the smaller dish OR 70% RH produced by adding 20 mL saturated NaCl around the smaller dish

e.   Cover the arena using the 100 mm x 15 mm lid

f.    Equilibrate arenas in 25^oC and 70% humidity in the dark for ~2 hours

4.     Separate males and females using ice

a.     Collect sets of 5 female and 5 male flies in empty 29 mm food vials with a cotton plug

b.   Use 20-25 replicates per condition

c.     When all flies are separated, place vials in sealed 1000 mL Nalgene jars containing 200 mL desiccant

d.   Desiccate flies at 25^oC in the dark for ~5 hours

e.   Remove vials from desiccant and place on ice to anesthetize flies

f.      Swiftly knock flies into empty 100 mm x 15 mm dishes

g.    Cover each dish containing flies with 66-68 nylon mesh that has been autoclaved and cut into 125 x 125 mm squares

h.      Seal the mesh around each dish containing flies, using zipties

5.   Record hygrotaxis behavior 

        a.  Use infrared lighting and camera to track movement

        b. Remove lids from the equilibrated arenas

        c.  Invert dishes containing flies atop corresponding experimental arenas

        d.  Record for 300 seconds

6.   Count, blind to genotype and humidity conditions, the number of flies in the inner and outer chambers, every 5 seconds