Materials and Methods
Drosophila melanogaster
The life cycle of the common fruit fly from embryo to birth is roughly 9-11 days. This had large implications for our research as parents needed to be removed from the vial before young had hatched to prevent breeding between parents and offspring, which can cause contamination of fly samples. Flies begin breeding at roughly 3 days of age. As such, there were less than two weeks after eggs were laid, where the parents had to be removed from the vial. Throughout the research, several vials had to be discarded due to contamination. This occurred largely during the week-long hiatus of the lab due to the university scheduled spring break, and partially due to misplaced or mislabeled vials.
To prevent incorrect data, it was determined that any chance parent/offspring breeding would disqualify that vial from being tested. This led to less data collection than preferred.
Stage 1: Embryo (1 day)
Stage 2: First Instar Larva (1 day)
Stage 3: Second Instar Larva (1 day)
Stage 4: Third Instar Larva (2.5-3 days)
Stage 5: Pupa (3.5-4.5 days)
Crosses
Continuing on with what we had last semester (see Code and Data Sets)
RNAi lines to knockdown expression of genes shown to be overexpressed
CASK (see slideshow below for more info)
Vha100
Shrb (not shown to be overexpressed or manipulated in our data)
GR-control
GR-control is the non-ALS expressing genotype.
GR-80
GR-80 indicates the ALS expressing genotype.
Locomotor Assay Trials
The method of data collection consisted of placing flies in a 250mL graduated cylinder, tapping them down to the bottom, and testing how many flies would climb past a certain point within a certain period of time.
The testing period was 2 minutes long. Within a 2 minute period, flies were recorded on a video camera, and then the number of flies that crossed the 190mL line was manually counted. A piece of tape was placed behind the vial to increase the visibility of the flies.
The image below is a frame from one of the videos taken during a Locomotor Assay test. In this frame, the fly above the 190mL line, with the lighter colored tape, would be counted as having crossed the line. The one below the 190mL line, with the red tape, would not be counted as having crossed the line.
