This method can be used to address a key question in the field of somatosensation, the mechanism by which an animal, such as drosophila melanogaster selects its preferred environmental temperature. The main advantage of this technique is that it can establish the preferred temperature of the group of larvae in a single assay when confronted with a continuous range of temperatures. In addition to providing insight into temperature preferences of drosophila larvae, impress plate can also be adapted for use with other model organisms such as the worm, C elegans.
First, make yeast paste to prepare vials for a cross. Mix yeast granules in distilled water and grind them into a paste with a pestle. Then, add a dab of yeast paste close to the inner walls of standard drosophila vials just above the surface of the food.
To produce the larvae, collect 12 to 35 females and up to half as many males, but not more than 10 males. Combine these groups in the yeast paste-containing vial. Load the prepared vials into a 100-vial tray.
Include 20 open vials loaded with water in the tray to provide humidity. Then, seal the tray in a clear plastic bag and incubate it at 25 degrees Celsius for 48 hours. After 48 hours of feeding and mating, transfer the flies into new food vials to collect eggs.
Tap them over. Do not use carbon dioxide. Then, allow the flies to lay eggs for three hours at 25 degrees Celsius.
Later, remove the adults and place the vials containing the eggs back into the tray with water vials and close the bag. Then, develop the eggs at 25 degrees Celsius to the desired larval stage. To prepare a single directional gradient, first create a human ambient environment for the assay.
Place two aluminum blocks connected to separate water baths on wet paper towels, 10 centimeters apart. The water baths need to be warmed up in advance. Next, make 100 milliliters of 1%agarose and add 25 milliliters to each assay plate on a level surface.
Once the agarose has solidified, gently rub the surface with a melamine sponge to make the surface slightly coarse. Then, to promote efficient temperature transfer, fill any gaps between the aluminum blocks in the assay plates with water delivered from a spray bottle. Now, place the assay plates on the aluminum blocks and make sure that the demarcations are two centimeters from either edge to exactly match the edges of the aluminum blocks.
Next, spray a film of water onto the surface of the plates so the gels do not dry out. Next, cover the system with a cardboard box to reduce evaporation and help stabilize the temperature of the gel surface. Wait for five to 10 minutes to allow the temperature to equilibrate.
Then, check the surface temperature on the plate in at least 12 locations to insure that there is an even temperature throughout, give or take two degrees Celsius. Then replace the box cover until the assay is performed. To isolate clean larvae, first add about 40 milliliters of 18%sucrose solution to a 50 milliliter test tube.
Then transfer larvae from the food piles into the solution, using a scoopula. Next, mix the larvae around in the solution thoroughly, using the scoopula. Now, wait for 30 to 60 seconds for the larvae to float to the top layer of the tube.
Then pour off the solution with larvae into another 50 milliliter tube and top it off with fresh 18%sucrose. Again, wait for the larvae to float up. The presence of food can influence the results.
Therefore, in order to obtain reproducible results, it is important to clean the larvae thoroughly to minimize contamination with food. Do so gently, to avoid damage to the larvae. Next, place a 300 micron cell strainer on top of a 50 milliliter tube, then remove any dead adult bodies and floating debris from the surface of the sucrose solution.
Now, pour the larvae through the strainer to collect them on the mesh screen. Then, run about 50 milliliters of water through the strainer twice to further clean the larvae. Then, onto an empty 35 millimeter dish, rinse off most of the larvae from the strainer, using water.
Use a paintbrush to transfer the remaining larvae on the mesh. Then, siphon off most of the water from the dish using a micro pipette. Then place the lid on the dish upside down so larvae cannot escape.
Now, allow the larvae to recover for 10 to 20 minutes before initiating the assay. First, remove the cardboard box over the gel and quickly check the gel surface temperature. If the surface is dry, spray a small amount of water on the surface.
If the gel is good, then load 100 to 200 larvae at the center of each plate, using a small paintbrush. Then, place a lid over each assay plate to trap the larvae and cover the set-up with a cardboard box to prevent light exposure. The assay is now in progress.
After 10 to 30 minutes, remove the cardboard box and the microplate lid. Then, photograph the plates from above to record the position of the larvae. Place the box above the camera to avoid reflection on the surface of the gel.
To clean up the plate, aspirate all the larvae wherever they may have scattered. Now, calculate the percentage distribution of larvae in each zone, using image analysis software. Make lines every two centimeters, based on the demarcations on the assay plate and count the number of larvae in each zone.
When counting, ignore larvae distributed in ridges 0.5 centimeter from each edge as gel thickness and temperature conditions are not uniform near the edge. In 18 degree Celsius to 28 degrees Celsius, single directional gradient was made using water baths set to 16.8 degrees Celsius and 31 degrees Celsius. The temperature distribution along the gradient was found to be nearly linear.
Control larvae were assayed at various ages. First, second and early-third instar larvae showed peak preferences for the 24 degrees Celsius zone. During mid-third instar, most larvae accumulated in the 18 degree Celsius zone.
Non-wandering late-third instar larvae were tightly clustered in the 18 degree Celsius zone. The proclivity to accumulate in the 18 degree Celsius zone in the control strain, which was White 1118, was not due to an edge effect, since the late-third instar larvae still accumulated in the 18 degree Celsius zone, using a bidirectional gradient. When we analyzed late-third instar larvae-harbored mutations required for discriminating temperature, the results changed.
Larvae with a null mutation in trpA1 distributed equally over the entire 18 degree Celsius to 28 degree Celsius gradient. Larvae missing just the A and B isoforms or the C and D isoforms of trpA1 also showed severe impairments. Larvae with a mutation in one of two genes encoding phospholipase c beta, norpA, were also disrupted.
However, larvae with a mutation in a gene encoding the other phospholipase c beta, plc21c, behaved normally. After watching this video, you should have a clear understanding about how to obtain reproducible results using a continuous thermal gradient, which will allow you to compare the temperature preferences of wild type and mutant drosophila larvae. Once mastered, this technique can be done in 30 minutes for a single assay.
While attempting this procedure, it is important to pay attention to the condition of the fly vials and not use the larvae if they are dried out. This technique simplifies the discovery of new genes required by drosophila larvae for selecting their favorite temperature in the comfortable range.
