BIOL 332 - Practical - Measuring Sensory Modalities in Drosophila.

Aim of the practical.

To understand the genetic and neural circuitry underlying male courtship behaviour in the fly.

Context of practical.

1. Have previously generated two mutant fly stock of unknown genotype.
2. Want to measure the behaviours of each of these mutant genotypes.

Assays used in this practical.

1. Courtship assay.
2. Negative geotaxis assay.
3. Olfactory avoidance assay.
4. Feeding assay.

Courtship assay step 1.

Add object female to chamber and start primary timer.

Courtship assay step 2.

When the male begins courting, note the time and start the courtship timer.

Courtship assay step 3.

If the male stops courtship, pause the courtship timer, then resume when he starts again.

Courtship assay step 4.

If successful copulation occurs, note the time this happens from the primary timer and stop the assay.

Courtship assay step 5.

In the absence of copulation, continue the assay for 10 minutes.

Courtship assay step 6.

Calculate courtship index (CI) by:
CI = total courtship time/observation time.

Courtship assay step 7.

Repeat process for other mutant genotype.

Negative geotaxis assay step 1.

Obtain tube with 10 3-5 day old male flies.

Negative geotaxis assay step 2.

Bang tube on the bench so flies are at the bottom, then place upright and start timer.

Negative geotaxis assay step 3.

After 45 secs, count the number of flies above a line towards the top of the tube.

Negative geotaxis assay step 4.

Repeat twice for each tube, and repeat process for each genotype.

Negative geotaxis assay step 5.

Calculate performance index:
PI = 1/2(total+top-bottom)/total.

Olfactory avoidance assay step 1.

Prepare vial of 5 male flies which have been without food for 2 hours.

Olfactory avoidance assay step 2.

Divide each vial into two sections.

Olfactory avoidance assay step 3.

Dip cotton swab in benzaldehyde solution and place into the top of the vial, then start timer.

Olfactory avoidance assay step 4.

After 15 secs count the number of flies in each compartment. Count every 5 secs up to 60 secs.

Olfactory avoidance assay step 5.

Calculate avoidance score for the vial.

Olfactory avoidance assay step 6.

Repeat with remaining vials and genotypes.

Feeding assay step 1.

Prepare normal samples of food, low-quality blue dyed food, and high quality blue-dyed food.

Feeding assay step 3.

Allow flies to feed for 30 mins.

Feeding assay step 4.

Make standard curve of blue food dye, by serial 1:1 dilutions.

Feeding assay step 5.

Place flies in ice bucket for 5 mins until they are unconscious.

Feeding assay step 6.

Fill Eppendorf tubes with 250 uL of PBS.

Feeding assay step 7.

Add flies to Eppendorf tubes containing PBS.

Feeding assay step 8.

Homogenise flies then centrifuge for 25 mins.

Feeding assay step 9.

Transfer supernatant to fresh Eppendorf tube, then centrifuge again for 10 mins.

Feeding assay step 10.

Transfer 100 uL of each sample and standard curve sample to a 96 well plate.

Feeding assay step 11.

Read absorbance in plate reader at 625 nm.