Mayfly rearing

The parthenogenetic mayfly N. triangulifer (WCC-2 clone isolated from White Clay Creek, (Patent US5665555)) were reared at the Stroud Water Research Center (SWRC; Avondale, PA). Newly hatched eggs of N. triangulifer were partitioned into rearing jars using natural stream water from White Clay Creek (WCC) and natural WCC periphyton as a food source as described elsewhere³⁹. Mayflies were reared at four constant temperature treatments (22°, 26°, 28°, and 30 °C ± 0.1 °C) and three variable temperature treatments with a 5 °C daily oscillation (0.5 °C/h) from 19.5 °C to 24.5 °C, mean = 22 °C (Regimen 1- diel fluctuation within the thermal acclimation zone) 23.5 °C to 28.5 °C, mean = 26 °C (Regimen2-diel fluctuation outside the thermal acclimation zone), and 25.5–30.5 °C, mean = 28 °C (Regimen 3 -diel fluctuation further outside the thermal acclimation zone). In each rearing condition, 5–11 replicate jars were used, and each jar contained 50 larvae. Temperature starts to rise at 6:30 am and reaches high soak at 4:30 pm for 2 h. Temperature starts to fall at 6:30 pm and reaches low soak at 4:30 am for 2 h. All temperature treatments were exposed 15:9 (L:D) photoperiod that began at 5:00 am and ended at 8:00 pm, simulating a light regime near the summer solstice at ~ 40°N). Survivorship, development time, and adult body size (fitness) were recorded for each replicate jar. Survivorship was the percentage of 1st-instar larvae surviving to the adult stage. These data were arcsine transformed for statistical analysis. Development time was the number of days from the start of the experiment (egg hatch) to the median day of adult emergence from a given replicate jar. Adult body size was mean individual biomass of emergent adults from a given replicate jar, dried at 60 °C for 48 h.