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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

  1. 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.
  2. After 2—3 days, remove the flies from the bottles by gentle tapping. Do not use CO2.
  3. 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

  1. 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.
  2. 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).
  3. 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.
  4. (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.
  5. 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

  1. 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.
  2. 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.
  3. 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.
  4. Establish the temperatures on each side of the test plates by placing the cover on the apparatus.
  5. To prevent drying, lightly spray H2O onto the agarose surface of the test plate.

The Behavioral test

  1. 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.
  2. 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

Fig 1

Associated Publications

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

Author information

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.

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