Behavioral research in Drosophila

Grooming assessment

For grooming experiment, 15 flies (both genders, aged 4–9 days before analysis) will be tested for each group. To record grooming behavior, each fly will be gently aspirated from its isolation tube and placed into a fresh well/arena 20 mm in diameter and 5 mm deep and allowed to acclimate for 1 min. The well must be quickly covered with standard perforated cover slide through which the flies could be recorded for at least 15 min. For the purpose of our experiment, up to six flies will be transferred into individual wells and recorded at the same time; loading typically took ~2 min. For adequate scoring, videos will be cropped to individual wells. Grooming will be defined as fly legs rubbing against each other or sweeping over the surface of the body and wings. Data will be collected for: 1. The percentage of time the fly spends grooming; and 2. The duration of individual grooming bouts. Grooming bouts will be recorded as ending when a fly either stops grooming and remains motionless for 2 s or stops grooming and walks at least 4 steps. Grooming bout will be scored as a discrete event and coded with start-time and duration, which is particularly helpful for the training of scorers and inter-rater reliability determinations. Grooming experiments will be performed under 25 °C and to avoid circadian rhythm confound, experiments will always be performed at the same time of day.

Climbing assessment

This protocol relies on the natural tendency of flies to climb, known as negative geotaxis, or The climbing assay.

For the climbing assay we will transfer 15 flies from a single vial into a 250 ml glass graduated cylinder. Marking the position of the cylinder will keep it constant every day. Experiments are going to be conducted in ambient light at temperature and humidity of 22 °C and 40% respectively. To avoid circadian rhythm confound, experiments are always performed at the same time of day. The top of the cylinder will be sealed with a parafilm to prevent the escape of any flies. Very lightly tapping the cylinder against a closed cell foam pad repeatedly with enough force will displace the flies to the inner bottom surface. Recordings will be taken for: 1. The time the first fly crosses the 150 ml line (cca. 17.5 cm from the bottom); 2. The percentage of trials in which the first fly will not cross the 17.5 cm line within 3 min; 3. The time for 50% of the fly population to cross the 17.5 cm line; 4. The percentage of trials in which 50% of the fly population will not cross the 17.5 cm line within 3 min; and 5. The percentage of flies that will cross the 17.5 cm line within 3 min. Trials will be performed for four times each group and their mean will be taken for a sample value. For data analysis we will exclude events in which a) no fly, and b) 50% of fly population haven’t been reached 17.5 cm within 3 min. Once the trial has been ended, we will dispose flies in 95% ethanol.

Social interaction assessment

For the social interaction assay, 15 flies (both genders, aged 4–9 days before analysis) will be tested for each treatment group. To minimize the disruption of standard environmental conditions, flies were reared in bottles and thus socially enriched kept as mixed genders to allow mating, and kept with standard food at all times prior to testing. Flies will be separated by gender the day prior to each experiment. The experiments are going to be performed at the same time of the day, in a range of 3-4 hours in the afternoon to reduce variation in performance linked to circadian rhythm, and under ambient light. All behavioral assays will be carried out in the same dedicated room at ~25°C. Social interaction will be observed using the social space. Flies will be collected from the bottles when ~3-4 days old, and sexed the day before the experiment, under cold anesthesia. Two hours prior each experiment, the vials will be habituated to the test room (25°C). Flies were collected from vials, using gentle suction with a mouth aspirator and introduced into the chamber through the opening between the bottom spacers. After the entrance was closed, the bottom of the chamber will be banged on a lab bench 3 times, to ensure that all flies will be at the same starting point. The data set will be used for most of the statistical analysis.

Social Space Index — the calculation of the Social Space Index (SSI) is based on the values obtained in the histogram representations of the social distance. SSI equals the percentage of flies in the first bin minus the percentage of flies in the second bin (SSI = first bin — second bin). SSI =<0 suggests a lack of social interactions. Kolmogorov-Smirnov tests will be used to statistically analyze histograms, and t- tests used for the SSIs.

All experiments are going to be run at least in triplicate.

Circadian rhythm assessment

For circadian rhythm experiment a 24-well plate is prepared, in the base of each well a preprepared mixture of agar and sugar is added. Drosophila young adults are then placed individually in each well, and an air permeable cover is put over the top of the well plate. The flies are then individually housed, have a humid environment and some available nutrition. Both genders (aged ~9 days before analysis) will be tested for each group. The well plate, loaded with flies, is then inserted into the chamber of the Zantiks MWP unit, and the script is set up to control the environment (temperature and lighting) and the measurement of the distance travelled by each individual fly. The script is set to last for 6 days: during the first three days the well plate is exposed to 12 hours Light, followed by 12 hours Dark, and then during the subsequent 3 days the plate is exposed to continuous dark. During all this time the activity (measured as distance travelled) is measured and written to a data file at frequency of 5 minute bin. Circadian rhythm experiments are performed under constant temperature and humidity conditions (~25 °C and 50–75 % relative humidity).

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