High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila provides an excellent model to study the genetic underpinnings of alcohol sensitivity because you can quantify alcohol sensitivity precisely under controlled genetic backgrounds and environmental conditions. Current methods to measure alcohol sensitivity in Drosophila test groups of flies. We present a simple, low-cost, high-throughput method to assess alcohol sensitivity in large numbers of single flies.

Variation in alcohol sensitivity in Drosophila can be translated to similar phenotypes in humans because flies share about 75%of their genes with human orthologs. The assay we developed can also be applied to measure the effects of acute toxicity of volatiles on other insects, including other fly species or disease vectors. Begin by creating a cardboard template of a 24-well cell culture plate.

Trace around the plate, and cut out the designated area. Then use the cardboard template to create a piece of small insect screen mesh the size of the plate. Place a small line of hot glue around the perimeter of a 24-well culture plate, and affix the screen mesh on top of the wells.

Then use the hot glue gun to secure a wooden craft stick to three sides of the culture plate. Prepare as many plates as can fit in the filming chamber. To create the filming chamber, cut a hole the size of the video camera lens on one side of a polystyrene box and an additional slit on the size of the illumination pad on the opposite side.

Insert the illumination pad into the slit, and position the camera in the lens hole above the illumination pad. Place all testing materials in a controlled environment, preferably a behavioral chamber with approximately 30%humidity, 25 degrees Celsius temperature, uniform airflow, and noise levels less than 65 decibels. Use the previously made cardboard template to cut two pieces of cheesecloth.

Then pipette one milliliter of 100%ethanol through the screen mesh into each well. Dry the mesh, and place the two pieces of cheesecloth on top. Cut a small piece of thin, flexible, plastic cutting board by using the cardboard template as a guide for expanding the area by one to two centimeters on one of the short sides.

After cutting, ensure that the plastic still fits between the three wooden craft sticks on the testing apparatus but hangs off one end. Prepare an aspirator according to manuscript directions, and use it to transfer one fly per well into a 24-well cell culture plate, covering any wells with previously aspirated flies with the flexible plastic. Record the well position and any relevant genotype or phenotype information of each fly.

Hold the flexible plastic flush with the top of the cell culture plate containing the flies, and invert the plate onto the top of the modified cell culture plate with the ethanol. Use the craft sticks to align the inverted cell culture plate, making sure that each well with ethanol aligns with each well containing a fly. Ensure that the illumination pad is lit at full brightness, and start recording with the video camera.

Expose the flies to the ethanol by carefully removing the plastic between the two plates, making sure to not dislodge the cheesecloth. When it is suspected that all flies have lost postural control, tap firmly in the center of the plate. If there is movement, continue to record, tapping every one to two minutes until no movement occurs.

Terminate the recording once there is no movement. To recover the flies, remove the top plate from the testing apparatus, and aspirate the individual flies into chosen containers. Replace the ethanol in the modified cell culture plates at least once per hour to maintain consistent ethanol exposure throughout the assay, and repeat the experiment for as many samples as desired.

Watch the video recording to determine fly sedation time, which is defined as the moment a fly loses postural control and locomotor ability. To ensure accuracy, watch the film in reverse, and record the time that the fly begins to move. This method can be used to generate data on a large number of flies within 10 minutes.

Two 24-well plates were used to simultaneously measure ethanol sedation times for 48 individual flies of two DGRP lines with different sensitivities to alcohol. Large sample sizes increase accuracy and reduce error to environmental variation. RAL_555 flies were less sensitive than RAL-177 flies.

Males and females of RAL_177 showed no sexually dimorphic effect, whereas the females of line RAL_555 were less sensitive to ethanol exposure than the males. When attempting this protocol, it is crucial to perform the aspiration of flies and the inversion of the cell culture plate with precision to achieve the desired result. Since the ability to assess single flies avoids confounding effects due to group interactions, this assay enhances the utility of the Drosophila model for high-throughput studies on acute effects of alcohol exposure.