Authors: Young Kwon , Hye-Seok Shim , Xiaoyue Wang & Craig Montell
When given a choice between two temperatures, Drosophila larvae will select the preferred temperature. This protocol outlines a step-by-step procedure for performing a two-way choice test on Drosophila larvae, to identify the preferred temperature.
Rearing 3rd instar larvae
- Tap adult flies over to bottles or vials containing fresh food and yeast granules. Use healthy flies that had not been exposed to CO2 for ≥3 days, and do not expose the flies to CO2 during the transfer.
- After 2—3 days, remove the flies from the bottles by gentle tapping. Do not use CO2.
- Allow the larvae to grow for an additional 2—3 days. Add H2O as needed to ensure that the fly food remains moist.
Collection of larvae from the food
- Scoop out food containing the larvae (~3—6 ml) into 40 ml of a 15% sucrose solution in a 50 ml tube. The food will sink and the larvae will float.
- After a brief incubation (~30—60 seconds), transfer the larvae (using a cut off P1000 pipet tip; diameter opening 5—8 mm) to a fresh 50 ml tube and wash the larvae with 15% sucrose until all remaining food debris is removed (usually 1—3 times).
- Transfer the larvae to a new tube, wash them with H2O at least two additional times to remove the sucrose. Be sure that all pupae and dead flies are removed.
- (Optional) If the collected larvae include more than 5% of 1st and/or 2nd instar larvae, transfer the larvae to a bacterial plate with moisturized 2% agarose, and remove the early stage instar larvae by aspiration.
- Before initiating the behavioral assays, keep the collected larvae for 15—30 minutes at room temperature (~22 °C) in a 35×10 mm Petri dish or in the cap from a 50 ml tube under a dim light with adequate moisture to ensure that they do not become desiccated.
Set-up for thermotaxis assays
- The apparatus for performing the thermotaxis assay consists of two adjacent aluminum blocks containing temperature controlled circulating H2O (Thomas Scientific, 9106). The two blocks are separated by a thin insulator (X-ray film) and held together in a plexiglass tray.
- Monitor the temperatures on the test plates using microprobe thermometers (BAT-12, Physitemp Instruments) and flexible implantable probes (IT-21, Physitemp Instruments) placed at the center of each side
of the test plates. The temperatures deviate ±0.2 °C over the whole area on each side of the test plate.
- The test plates used to perform the thermotaxis assays are plexiglass covers from 6×12 mini trays (Nunc 136528) coated with 7 ml of 2% agarose.
- Establish the temperatures on each side of the test plates by placing the cover on the apparatus.
- To prevent drying, lightly spray H2O onto the agarose surface of the test plate.
The Behavioral test
- Transfer 40—100 larvae (≥95% 3rd instar) from the Petri dish or cap described above to the middle of the two sides of the thermotaxis plate, and conduct the experiment in complete darkness.
- After 15 min, photograph the test plate, tabulate the number of larvae on each side of the plate, and calculate the preference index (PI) according to the following formula:
PI = [(no. of larvae on the 18 ºC side) – (no. of larvae on the side with the variable temperature)]/(total no. of larvae on both sides of the test plate)
Figure 1: Set-up for thermotaxis assays
Control of thermotactic behavior via coupling of a TRP channel to a phospholipase C signaling cascade, Young Kwon, Hye-Seok Shim, Xiaoyue Wang, and Craig Montell, Nature Neuroscience 11 (8) 871 - 873 27/07/2008 doi:10.1038/nn.2170
Young Kwon, Hye-Seok Shim & Xiaoyue Wang, Johns Hopkins University School of Medicine
Craig Montell, Johns Hopkins University School of Medicine, Biological Chemistry , Baltimore, MD 21205, USA
Correspondence to: Craig Montell ([email protected])
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.127. Originally published online 28 July 2008.