Approximately 10 cups of larval diet were used to get the necessary flies for the preliminary thermal limit testing as well as enough excess to start a new culture with which to do hardening and thermal tolerance testing (Experiment 2). The temperatures planned for the test ranged from 28 to 52°C, increasing in increments of 4°C, for upper lethal temperature (ULT), and 14 to -10°C, decreasing in increments of 4°C, for lower lethal temperature (LLT). The actual test temperatures were averaged from thermocouple readings and are presented in Figure 1. Flies were tested at peak emergence (1-2 days after first adult emergence) and allowed to become accustomed to the vials for 30 minutes at 25°C before testing.
Each containing a group of 10 flies (five vials for males and five vials for females), were used to test each temperature using a programmable water bath (Model: K25-cc-NR, huber, Germany) with a 1:1 propylene glycol-water mix. Each vial represents a replicate. The different vial sets were exposed to Spain phone number list each of the test temperatures for 2 hours. They were then checked at 1-, 2- and 24-hour periods after temperature exposure to determine the best recovery time for future use. Vials took roughly 10 minutes to reach test temperature in the water bath (Figure 1). Due to the numbers needed, multiple different egg groups were collected a day or two apart so that there would be enough male and female flies of the same age to run roughly two temp tests a day using the one water bath.
Temperature test order was randomized. Survival was defined as normal behavior (locomotion or sustained movement) or reaction to mild external stimuli such as rolling the test vial in a hand or prodding with a pencil. The observations were recorded for analysis to calculate fly survival %, and to determine lethal (temperature causing only 50% survival) and lower and upper sublethal temperatures (most extreme temperatures where survival was still 100%). The calculated upper sublethal temperature was found to be within 1°C of the calculated ULT making its use impractical in Experiment 2. A sublethal temperature of 35°C was used instead due to its use in a study looking at two other.