The overall aim of the following experiment is to test the innate and circadian photo behavior of drosophila lava by means of a light dark preference test. This is achieved by first rearing the experimental larvae under defined light dark conditions to establish an initial circadian clock in the larvae. Next, the larvae is switched to constant darkness, which allows the larval circadian clock to free run.
Once at the L three feeding stage, the lava is sorted into groups to test their light preference at different circadian time points. Results are obtained that show innate and circadian light preference based on calculating a preference index and comparing between time points and experimental groups. Though this method can provide insight into the innate and circadian regulation of light sensing and the processing of photo behavior, it can also be applied to other systems such as olfaction, cassation, chemo, thermo taxis.
Likewise, it can be modified for learning and memory tests. Demonstrating the procedure will be Alina and BMA To graduate students from my laboratory To collect eggs, allow mating adult flies to lay for 12 hours on fresh files and transfer them to new vials. Keep transferring the adults every 12 hours for a 12 hour light, 12 hour dark cycle.
Keep the 12 hour egg collections in the incubator. For the experiment, raise the animals the early L three larva stage. These are feeding lae available 84 to 108 hours from egg.
Laying Correct staging of LAE is critical for the success of the experiment. Since late L three, LAE changed the photo behavior To test for circadian components of visual behavior. 48 hours before testing and just before the larvae subjective, dawn, put the collected larvae into a cardboard box.
Then transfer the box to a constant darkness incubator for 48 hours until the experiment, these larva must remain undisturbed in their dedicated space. Prepare the lid of a nine centimeter Petri dish using a 20 centimeter square divided in four quadrants. As a guide.
Glue 10 centimeter squares of aluminum foil on opposite quadrants and cover them in black tape. Be sure to cover the border of the lid. Next glue, A 20 centimeter square divided in four quadrants under the light source on the table.
Mark the circumference of a test dish on the sheet to act as a guide for consistent displacement. Install an LED lamp above the Petri dish using an iron support stand. Adjust the height in a way that the whole test plate is homogenously illuminated.
Adjust light intensity at a test site to between 350 and 760 luxe by either further adjusting the lamp height or by using an adjustable power supply. The experiment room needs red light illumination and must have the same temperature as where the animals are reared from. The vials reared two days under constant darkness.
Collect the lava in their food with a spatula from the top five millimeters of the food. Spread the food on the outer side of a Petri dish lid. Add some water to the food and gently mix it with the spatula.
Add a drop of tap water to a new dish using a paintbrush. Transfer the larvae to the drop of water to wash them. Then use a wet paintbrush to collect the larvae using the wet paintbrush.
Select only the early feeding L three larvae and avoid late wandering L three larvae. Transfer the selected larvae to the center of a test plate. Feeding L three larvae have anterior sphericals that are open and protruded to the outside.
In a finger-like form. The posterior sphericals have three openings each and four groups of large branched hairs. The saliva glands extend to the second abdominal segment.
Once 30 larvae have been placed at the center of the test plate, cover it with the prepared lid. Turn on the LED lamp. Start a timer and let the larvae move freely on the plate for five minutes.
Then quickly remove the lid and count the number of larvae in the dark and in the light quadrants. Marking the position of each lava can ease the counting. A quick photograph can also help larvae showing unclear light preference like larvae crawling on the walls or borrowing into agar should be counted as neutral preference and added to the total number of larvae when the light preference index is calculated.
After counting the larvae, discard the plate and use a new test plate for the next experiment. After performing 10 to 15 trials per genotype, proceed with analysis following the protocol. The light dark preference of Canton ES lava was compared at two different circadian times.
CT zero and CT 12, subjectively, dawn and dusk larvae or groan for 48 hours under standard conditions and then switch to constant darkness for three days. Light dark testing under 350 luxe revealed that light photosensitivity was higher in larvae tested at CT zero compared to larvae tested at CT 12. Using this formula, the darkness preference of CT zero lava was 0.52 compared to 0.36.
The CT 12 larvae comparing the indexes using a wilcoxon test produces significant P value of 0.023. After watching this video, you should have a good understanding of how to test the light preference of troph larva at different circuit and time points to this goal. You should be able to entrain on stage F three oph larva, test their photophobic behavior and calculate the light preference index for statistical comparison.
