Flow through respirometry system

Routine metabolic rate was measured as the rate of CO₂ produced by groups of 20 larvae of the same instar and strain using a flow-through respirometry system (Sable Systems International, Henderson, NV) (Hoekstra et al. 2013 (link)). Groups of larvae were collected onto the cap of 1.7 ml tube containing 0.5 ml of fly medium and placed inside one of four respirometry chambers that were housed in a temperature-controlled cabinet (Tritech Research, Los Angeles, CA) maintained at 25°. Between 8 and 13 biological replicates for each strain and instar were randomized across chambers and respirometry runs, during which each group of larvae was sampled for CO₂ production for two 10-min periods. CO₂ that accumulated in the chambers as a result of larval metabolism was detected using an infrared CO₂ analyzer (Li-Cor 7000 CO₂/H₂O Analyzer; LI-COR, Lincoln, NE). $\dot{V}{\text{CO}}_2$ was calculated from the mean fractional increase in CO₂ at a constant air-flow rate of 100 ml/min over a 10-min time interval for each replicate after baseline-drift correction. The wet weight of the group of larvae was recorded using a Cubis microbalance (Sartorius AG, Göttingen, Germany) at the beginning of each respirometry run.