Purpose

Observe the feeding behavior of Drosophilia melanogaster

mutant in their taste receptor genes following a period of prolonged
food deprivation.
Hypothesis
The flies mutant for sugar receptors will still prefer the calorierich food.
Materials and methods
Feeding assays
Standard cornmeal medium,
Anesthesia
Vials
MilliQ water
Kim-wipe tissue
60-well microtiter plates
Glycogen Measurement
1% agar
Blue and red tasteless food dyes
All tested sugars: sucrose ???
Silicon plate
Insect pins
Dissecting microscope
Fine tweezers
Digital camera.
Sleep Analysis Material
Agar containing 100mM sucrose (5% sucrose)
Agar containing 0.3 mM sucralose
Regular fly food
Excel-based macro.
Procedure
1) Anesthetize and collect 50 straight winged flies. Allow them to
recover in cornmeal for 2 days before experiment.
2) After 4-6 days, starve flies for 5,10,15,2 hours in vials containing
2 mL of milliQ water soaked in Kim-wipes.
3) Cold-anesthetize flies and transfer to 60 well microtiter plates for
two choice preference assays.
Prepare a two choice assay
Agar substrate containing sugars was made 1% agar and
0.5-0.6%
blue. Color-label 1% agar without sugar red.
4) Allow to feed for 120 minutes.

5) Observe color of abdomen. Flies subjected to the two-choice

assay with sucrose versus plain agar were sorted into two groups
sucrose eating and no eating. Each group was dissected.
Crop and Gut Dissections
A fly is immobilized on a silicon plate, pinned, legs and
wings removed under a dissecting microscope. Cuticle of
thorax and abdomen are then peeled off in PBS using fine
tweezers to expose crop and gut.
Take images.
PER assay
Flies starved for 22 hours (in the presence of water) were
tested with water before the experiment and only flies that
did not respond to water were used. The taste bristles on
the labellum or the legs were stimulated by a Kim-wipe
thread soaked in tastant solution. Per response were
scored as follows: no extension = 0, half extension = 0.5,
and full extension = 1.

6) Hemolymph glucose and trahalose concentrations were

measured. 10 flies
starved on agar (**) for 5,10,15, or 22 hours were decapitated
and their hemolymph was drawn with a capillary pipette.
7) Mix 0.5 uL of hemolymph with 100uL of the Glucose Assay Kit
adjusted to a
pH of 6.8.
8) 10uL of pig kidney trehalase per 5mL of glucose reafent was
added to the mixture, incubated at 37 degrees C for 16 hours
and measured with a fluorescent plate reader using quantitative
NADH fluorescence (**).
9) Standard curves are generated
10)
Gycogen measurement.