Research Paper Volume 15, Issue 6 pp 1748—1767

Figure 1. Single-fly whole-organism respirometry protocol and sample randomization. (A–D) Represented is the flow chart of treatment, handling, and randomization procedures to which each cohort of n = 60 flies were subjected during four separate weeks (2 young cohorts and 2 aged cohorts) for a total of 240 flies. (A) During each round of whole organism respirometry, n = 60 Young (4–8 days-old) or n = 60 Aged (28–32 days old) male Oregon-R flies were separated into 3 treatment groups of n = 20 flies and subjected to Non-injected (Ni), Tris-injection, or FHV-injection treatment. After treatment, each fly was placed into a single vial and placed in a 25°C incubator (12 h light:12 h dark cycle). (B) Prior to respiration measurement, flies were taken out from the 25°C incubator and checked for viability. Next, they were transported in an isothermal container to prevent variations in temperature. At the Comparative Energetics Organismal Core facility, a 24-well Microplate (Loligo Systems) was used to measure the oxygen consumption rate (OCR) of individual flies. After measurement, flies were placed back into respective vials and returned to the 25°C incubator in an isothermal container. The sample transport cycle was repeated for the duration of the longitudinal OCR measurements. (C) On each microplate, a cohort of n = 20 randomly selected flies were measured alongside n = 4 blank wells (negative controls). (D) On each subsequent timepoint (24 h, 48 h, and 72 h post-treatment), well position was re-randomized for each microplate cohort resulting in the cohort being measured at the same time each day.