Research Plan 2 - Rough
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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 calorie-rich 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.
Transcript of Research Plan 2 - Rough
PurposeObserve the feeding behavior of Drosophilia melanogaster mutant in their
taste receptor genes following a period of prolonged food deprivation.
HypothesisThe flies mutant for sugar receptors will still prefer the calorie-rich food.
Materials and methodsFeeding assaysStandard cornmeal medium, AnesthesiaVials MilliQ waterKim-wipe tissue60-well microtiter plates
Glycogen Measurement1% agar Blue and red tasteless food dyesAll tested sugars: sucrose ???Silicon plate Insect pins Dissecting microscope Fine tweezersDigital camera.
Sleep Analysis MaterialAgar containing 100mM sucrose (5% sucrose)Agar containing 0.3 mM sucralose Regular fly foodExcel-based macro.
Procedure1) 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 assayAgar 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 DissectionsA 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 generated10)Gycogen measurement.
