Table 1.

Troubleshooting Table

STEP	PROBLEM	POSSIBLE REASONS	SOLUTION
6	There is no multimeter reading	There is no power being supplied to the circuit	Make sure that the male plug is connected to the female mount, and that there is power supplied to the wall socket.
28 (OR) at any later point during the use of the device	The piezoelectric does not deflect	Loose/incorrect connections in the circuit	Ensure that the circuit follows the schematic shown in Fig 3a. Check to see whether any of the connections from the SPST relay or the voltage booster detached from the breadboard. If so, reconnect the wires into the appropriate position. Check the integrity of the other soldered connections, and resolder any loose connections.
		Arduino does not work	With the power supply connected into the wall, use a multimeter and check to make sure the Arduino micro controller is producing +5V power at the connector labelled +5V on the micro controller. If this is not displaying 5V, replace the Arduino.
		Piezoelectric is damaged	If the circuit connections are intact, the piezoelectric may need to be replaced.
28 (OR) at any later point during the use of the device	Display does not show any voltage reading when the device is plugged in	Display is damaged	If all the breadboard connections from the display are intact, plug in the power supply and measure the voltage level incoming to the display sensor using a multimeter. If this voltage input changes when the potentiometer is turned, replace the LCD display.
		Potentiometer is damaged/ loose connections	If the voltage measured on the multimeter does not change when turning the potentiometer, check the electrical connections for the potentiometer and if it is working (see next item in the Troubleshooting Table).
28 (OR) at any later point during the use of the device	Display does not change when potentiometer knob is turned	Potentiometer is damaged/ loose connections	Unplug the power supply. With the multimeter adjusted to measure resistance, place on tip on the middle post and a second on one of the outside posts of the potentiometer. Turn the knob on the potentiometer - if the resistance changes when turning, check the integrity of the wiring connections from the potentiometer to the buck converter. If the resistance does not change, replace the potentiometer.
38	Gap between head and piezoelectric is not uniform across all flies in the high-throughput dTBI device	The piezoelectric may be mounted at an angle	The piezoelectric can be adjusted by using the mounting screws to even out the gap.
44	dTBI mortality is high for 45% head compression	45% is too high for the genotype	Try reducing the head compression by 5%.
		Food is not fresh or not flipped often enough	dTBI flies need to be flipped on to fresh food every 2d.
47	dTBI mortality increased over time after calibration	Gap between the fly head and piezoelectric may have reduced because the piezoelectric was mishandled after calibration	Use the angled mirror to verify that the gap is similar to when the device was calibrated. Tightening or loosening the mounting screws of the piezoelectric can be used to change the gap.
		The positioning of piezoelectric may be too ventral and past the antennae, or over the proboscis	Consult Supplementary Video 4 and Fig 5d for appropriate positioning of the piezoelectric over the head. Remain consistent with the position that was used during calibration.
47	dTBI mortality decreased over time after calibration	Piezoelectric may be wearing out and losing efficiency	Recalibrate the device and assess whether the head compression is comparable to calibrated values.
		The positioning of piezoelectric may be too dorsal and behind the antennae	Consult Supplementary Video 4 and Fig 5d for appropriate positioning of the piezoelectric over the head. Remain consistent with the position that was used during calibration.
53	Sham mortality is high	Rough handling of flies	Avoid injuring the fly body when sliding in and out of the collar. Use blunt forceps for manipulations.
53	Too many flies are decapitated during the collaring process	Metal plates in the collar are too rough	Use sandpaper to file the edges of the metal plates to smoothen them. Regularly clean the metal plates with 100% ethanol to remove dried tissue and body fluids from previously decapitated flies.
		The forceps are pushing only against the body while sliding flies in and out of the collar	Make sure that the forceps are resting in the groove while pushing against the fly body.