The microplate feeder assay provides a simple, high-throughput, economical method to measure Drosophila feeding behavior, and it offers multiple advantages over other, more elaborate methods. Quantifying consumption by measuring absorbance with a plate reader eliminates manual measurements and obviates manual data entry. Data are also amenable to programmatic extraction and processing.
With this high-throughput assay we can quantify the consumption of water-soluble nutrients, drugs, pharmaceuticals, or toxins, and the system can be modified for applications to a variety of insect species. Begin by pouring the molten agarose into a reagent trough and dispense 80 microliters of molten agarose into each well of a 96-well microplate using a multi-channel pipette. Refrigerate the leftover agarose for up to a week in a sealed bag and remelt it for making additional plates.
If the barrier strips are too loose, coil them around the finger to give them curvature to hold them in the channels. Insert the barrier strips into the barrier strip channels to prepare the couplers. Affix the coupler to a starvation plate, making sure not to use the coupler to manipulate the plate as the coupler may slip off.
Ensure that the angled corner of the coupler matches the angled corner of the microplate to maintain correct orientation. Under CO2 anesthesia, sort three-to five-day-old flies. Load individual flies by column into the starvation plate.
Close each column as it fills by adjusting its barrier strip to the closed position. Carefully record the sample layout within the microplate. Once the starvation plate is filled, allow the flies to recover spontaneously after removing the CO2, and starve them for six hours starting from their initial anesthetization time.
Prepare 10 milliliters of liquid food in a 15 milliliter conical tube by dissolving 0.4 grams of sucrose and 0.1 gram of yeast extract in 10 milliliters of distilled water. Vortex the tube until the solids fully dissolve. Add 40 microliters of dye stock solution and transfer the liquid food into a 10 milliliter syringe tipped with a 0.45 micrometer filter.
Filter approximately 1.5 milliliters of the solution at a time into a 1.7 milliliter microcentrifuge tube. Set the syringe containing the solution aside and filter the additional solution as needed during feeder plate preparation. Prepare a feeder plate by sealing the bottom of a 1536-well microplate with a sealing film.
Use a sealing paddle to adhere to the film thoroughly. Then, trim excess film off the left and right edges with a razor blade. Dispense 10 microliters of the filtered liquid food column-wise into the upper left-hand well for each cluster of four wells of the 1536-well microplate.
Once all the wells are filled, apply a sealing film to the top of the plate, following the same steps used to seal the bottom of the microplate. Repeat for the desired number of plates. Centrifuge the plates at 200 times G for 10 seconds to settle the fluid.
Do not allow the plate to be chilled since this can cause condensation to build up in the wells, obscuring absorbance readings. Perforate the wells on the top surface of the plate with the needle probe tool equipped with a 0.25 millimeter diameter needle, using the same order to perforate as was used when dispensing the solutions. Wipe off the needle between solutions to prevent cross-contamination.
Flip the plate and perforate the wells on the bottom. Read the plate's absorbance at 630 nanometers without a lid. Place an internal lid on the top sealing film to ensure that the condensation rings encircle the perforated wells, then place the external lid on the plate.
Place the feeder plate face up on the coupler such that the guides align the appropriate holes of the feeder plate and starvation plate. Ensure the coupler and plates are correctly oriented. Once all the feeder plates are loaded onto the couplers, open the wells for the plates by adjusting the barrier strips on the coupler.
Place the coupler and plate assemblies in the secondary container. Place the lower half of a pipette box containing soaked paper towels into each secondary container to provide humidity. Close the lid of the secondary container and transfer it to a controlled environment.
Allow the flies to consume for 22 hours. After the 22 hours of exposure, check each plate for dead flies and update the plate layout accordingly. After all the plates are checked, anesthetize the flies en masse by pumping CO2 inside the secondary container.
After approximately 60 seconds, ensure that all the flies are immobilized. Gently tap the flies into the starvation plate and replace the plastic barrier strips. Remove the feeder plates for reading.
Reread the plate's absorbance at 630 nanometers. Repeat the process until all plates have been read. Evaporation was quantified for every well and was found to determine whether any correlations exist among the wells of individual plates.
Pearson correlation coefficients for evaporation versus rows and evaporation versus columns were calculated to evaluate trends between evaporation and well locations. Consumption for three-to five-day-old Canton-SB flies was quantified to establish the validity of the protocol. Flies were given a choice between a 4%sucrose solution with 1%yeast extract and a 4%sucrose solution supplemented with 15%ethanol and 1%yeast extract.
Both males and females showed an overwhelming preference for the solution with ethanol and yeast extract. It is essential to maintain consistency when constructing the feeder plates, ensuring that each fly is presented with an identical consumption scenario in regard to food volume, evaporation, and access. This technique will allow researchers in the Drosophila field to perform high-throughput assays for consumption and preference behaviors with higher throughput and at lower cost compared to traditional methods.
