Materials and Reagents

1. Petri dishes 10 cm Ø (SARSTEDT, catalog number: 82.1473 )
   
2. Fly stocks
   Chromosome, Bloomington stock center No.:
   w¹¹¹⁸ (X, BL 6326)
   w*; ppk-Gal4 (X, 3^rd, BL 32079)
   w*;UAS-TNT^E (X, 3^rd, BL 28997)
   
3. Agar Kobe I (Carl Roth, catalog number: 5210.4 )
   
4. Agar plates (see Recipes)
   
5. Fly food (see Recipes)
   1. Agar (strings) (Gewürzmühle Brecht, Eggenstein, Germany, catalog number: 00262 )
      
   2. Corn flour (Davert, Newstartcenter, catalog number: 17080 )
      
   3. Soy flour (Davert, Newstartcenter, catalog number: 46985 )
      
   4. Brewer’s yeast (ground) (Gewürzmühle Brecht, Eggenstein, Germany, catalog number: 03462 )
      
   5. Malt syrup (MeisterMarken – Ulmer Spatz, Bingen am Rhein, Germany, catalog number: 728985 )
      
   6. Treacle (molasses) (Grafschafter Krautfabrik, Meckenheim, catalog number: 01939 )
      
   7. Nipagin (Methyl 4-hydroxybenzoate) (Sigma-Aldrich, catalog number: 54752-1KG-F )
      
   8. Propionic acid (Carl Roth , catalog number: 6026.3 )
      

Equipment

1. Brush (Size 1, Boesner, model: Da Vinci Nova Serie 1570 , catalog number: D15701)
   
2. Forceps Dumont #3 (Fine Science Tools, catalog number: 11231-30 )
   
3. Camera Basler ace acA2040-25gc (Basler, catalog number: 105716 )
   
4. Stereoscope ZEISS Stemi-2000C (Pulch und Lorenz, catalog number: 455053-0000-000)
   Manufacturer: ZEISS, model: Stemi-2000C .
   
5. External light souce LED Schott KL 1500 LCD (Pulch und Lorenz, catalog number: 150.200)
   Manufacturer: SCHOTT, model: KL 1500 LCD .
   
6. Hot probe, custom made (see below, Figure 1):
   1. Thermometer Greisinger electronic GTH1170 (Conrad, catalog number: 100599-62 )
      
   2. Electrolube polyurethane (Electrolube, catalog number: UR5634RP250G )
      
   3. Electronic parts (see supplementary material)
   Temperature control device and hot probe design:
   In principle, a commercially available and precise temperature-controlled soldering iron as used in several other studies (Neely et al., 2011; Tracey et al., 2003; Zhong et al., 2012) should provide sufficient accuracy to perform this assay. Here, we designed a temperature control device to keep the temperature very precisely at a constant value of 46 °C (Figure 1). One problem is the high temperature loss when the probe dips into the water film on the agar plate, which results in lower and variable temperatures on the larva. To prevent this caveat, the device has to regulate the temperature very fast to keep the previously set values. One such design has been previously developed and employed (Babcock et al., 2009) by using a custom-built temperature controller.
   
   
   Figure 1. Hot probe setup and design. A-C. Custom built controller for setting the temperature with connected probe. D. The temperature-controlled hot probe consists of a diode (Z-diode or regular diode) with one wire shortened and shaped as a probe tip (facing left). The second wire is connected to the electronic controller and the diode is sealed and insulated with a polyurethane resin to prevent excessive temperature leakage. An external temperature sensor (thermocouple, e.g., Greisinger electronic GTH1170) can be optionally included by soldering the sensor to the probe for external temperature monitoring.
   
   For our hot probe design, our in-house workshop employed a universal diode (1N4001) or alternatively, a Z-diode as a heating element. The diode is heated by a constant and precisely controlled direct current. The forward voltage is independent of the current (which is kept constant), but only depends on the temperature of the barrier layer of the diode. This allows using the occurring voltage for precise measurement and regulation of the barrier layer temperature.
      One of the two conducting diode wires functions as the hot probe and should be shaped as follows: The protruding wire is shortened to 4-5 mm and 1.6 by 0.9 mm width, similarly to the previously described size and shape (Tracey et al., 2003). The tip of the hot probe should be spatula shaped and should not have any sharp edges. When the temperature of the tip is below 40 °C, touching the larva should not cause rolling behavior. It should also be as short as possible (4-5 mm) to prevent unnecessary heat loss and large temperature differences between the tip and the diode barrier layer, where the temperature is measured.
      Voltage measurement and temperature regulation are achieved using an appropriate electronic system (built in-house and custom programmed) connected to the second diode wire. It displays the current temperature and adjusts the power at the diode tip to maintain the desired constant temperature. The precise circuit and block diagram, electronic parts and custom programming are provided as supplementary material.
   Calibration of hot probe diode:
   The insulated diode is placed in a 37 °C water reservoir (~1 L bottle with a magnetic stirrer, the temperature measured with a high precision reference thermometer). A very short impulse (2-10 msec only to prevent significant heating of the diode) is used to measure the voltage of the diode barrier layer at 37 °C. The same forward voltage measurement is performed at 55 °C. The forward voltage change between the two calibration points linearly correlates with the temperature and thus allows using the diode voltage for temperature measurement and control within this temperature range.
   

Software

1. Video software
   1. Stream Pix6 (Norpix Inc., Montreal, Quebec, ordered from Rauscher GmbH, Germany, art. no. Streampix STP-6-S-STD)
      
   2. Windows VLC Media Player (www.videolan.org)
      

Procedure