High-Throughput Assays of Critical Thermal Limits in Insects

Thermal tolerance is a critical component of species distributions and responses to climate change. Here we provide methods for rapidly assessing heat and cold tolerance in small insects. These methods for measuring cold tolerance, via the critical thermal minimum and heat tolerance, via heat knocked down time are high throughput, semi-automated and require minimal equipment.

Demonstrating the procedure will be David Awde, a postdoc from my laboratory. To set up a critical thermal minimum assay, power on a temperature controlled fluid bath and press the play button to run a program, raising and maintaining the temperature of the bath to 25 degrees Celsius. Wait five to 10 minutes to allow the fluid bathroom column to reach 25 degrees Celsius, before replacing the plug at the top of the column with a 5.08 centimeter diameter filter.

Tap approximately 40 flies from their food vial into the column through the funnel and quickly replace the funnel with the plug, taking care not to let any flies escape. Then allow the flies to settle. Occasionally tapping the bottom plug to encourage the flies to climb.

After five minutes, press the start button on the fluid bath gain, to begin the critical thermal minima ramping program. Click to open the thermal couple recording software on the computer and click record to begin recording the temperature inside the column, every second for the duration of the assay. Ensure that each temperature recording includes the timestamp specific to the second, so that temperature data can later be merged with data from the Drosophila funnel monitor.

Add five milliliters of 90%ethanol to a 15 milliliter conical centrifuge tube and place the tube in a rack below the column. Occasionally tap the bottom plug of the column to entice any flies on the bottom to climb. Most of the flies will be on a perch or near the top of the column by 15 degrees Celsius.

At 15 degrees Celsius, place a 75 millimeter outer diameter glass funnel into the Drosophila funnel monitor. Remove the bottom plug and collect any flies still on the plug in the ethanol. Adjust the retort ring, funnel monitor and funnel, so that they are under the column.

Make sure that the lip of the funnel completely seals the bottom of the column and insert the bottom of the funnel into the 15 milliliter collection tube. Open the Drosophila funnel monitor software. The software will immediately start recording the time and date at which the flies reach their critical thermal minima.

Flies that reach their critical thermal minima, will lose their neuromuscular function and fall from their perches, through the Drosophila funnel monitor. To monitor whether all of the flies have reached their critical thermal minima, as the temperature decreases, check the top plug and perches to see if any flies are still perched. When all the flies, have reached their critical thermal minima, move the Drosophila funnel monitor and funnel away from the column opening.

On rare occasions, flies may reach their critical thermal minima, but remain stuck in the column. Open the top plug to remove these flies. To set up a high throughput heat knocked down assay, use an aspirator and septum lid, to load individual carbon dioxide sedated flies, into each well of a modified 96 well no bottom plate and seal the plate with a clear tight-fitting lid.

Then place the plate over a supply of food and allow the flies to acclimatize to the 96 well plate for at least 48 hours. On the day of the assay, set the incubator to 37.5 degrees Celsius, after 30 minutes place the fly culture plate into the incubator with the bottom of the plate against the white paper on the bottom of the tray. Note the orientation of the well column and row names on the tray and in the frame of the webcam and place colored tape along the sides and edges of the plate to help verify the orientation.

When the plate is in position, close the incubator door and click record in the video recording software. After two hours, check the recording to see whether all of the flies, have reached their final resting spot and stopped moving. When all of the flies have stopped moving, click stop and dispose of the flies.

Open the video file and record the knockdown time of each fly in each well. The most consistent measure of the heat knocked down downtime between the trials and observations is the time at which a fly reaches its final resting spot. Then track the video in reverse, focusing on a single well and noting the time at which the fly first moves off its final resting spot.

Determining the final resting time of each fly can be difficult. To ensure an accurate analysis, it is important to take your time and to observe each well individually. In this representative analysis, females from the Drosophila genetic reference panel line, had significantly lower mean thermal minima temperatures than females from the DGRP line 714.

In addition, the heat knocked down time at 37.5 degrees Celsius, differ significantly between females from the 73 and 461 DGRP lines. With minor modifications, this method for measuring the heat knocked down time, could be used to measure other thermal tolerance traits, such as chill-coma recovery time or critical thermal maximum. By significantly reducing the hands-on time for investigators, these methods have allowed the exploration of genetic variation and thermal tolerance traits at a scale that was not previously possible.