Respiratory quotient (RQ)

A mean winter respiratory quotient (RQ_Winter) was estimated as a weighted mean of measured lipid (RQ = 0.7), glycogen, and free carbohydrate (RQ = 1) changes between seasons (compare Erregger et al. 2017), by multiplying the RQ values by the mass of the respective energy stores, divided by the sum of masses. In addition, we included structure mass changes as a rough estimate of protein degradation (RQ = 0.8).

In order to allow a proper estimation of the time the wasps are able to survive with the energy reserves remaining in spring, we determined the respiratory quotient in summer individuals (workers) (RQ_Summer) of P. dominula AT and P. gallicus IT and, for comparison, in P. biglumis AT, according to the measurements in overwintering gynes by Kovac et al. (2022b). In short, seven wasps were placed individually in 2.23 ml respiration measurement chambers for about 2 h (to accumulate enough CO₂ and O₂-depleted air) and measured at 25 °C ambient temperature. Care was taken to have long enough measurement periods to include several respiratory cycles of resting individuals (see Käfer et al. 2015), which avoids unrealistically low RQ values during discontinuous respiration. An additional empty chamber served as a reference for control of instrument drift. A RM gas flow multiplexer (Sable Systems International, Las Vegas, USA) passed commercial dried air to a reference and a measurement channel (parallel mode) of an Uras 14 differential infrared gas analyzer (DIRGA; ABB, Zürich, Switzerland) followed by an Oxzilla 2 differential oxygen analyzer (Sable Systems). The air flow was regulated at 144 ml min⁻¹ by factory-calibrated Brooks 5850 S mass flow controllers. The multiplexer flushed the eight measurement chamber channels in sequential order. The air leaving the measurement chambers was dried with Drierite^® desiccant (Hammond Drierite Co. Ltd., Xenia, USA) before it entered the DIRGA and the Oxzilla. The Uras 14 CO₂ analyzer was calibrated against internal calibration cuvettes, and the Oxzilla 2 O₂ analyzer against air from outside the laboratory, before and after measurement (Stabentheiner et al. 2012). Any instrument drift and offset was compensated during evaluation according to the difference between the measurement channels and the reference channel. Data acquisition and evaluation was done with the DIRGA CO₂ gas analyzer system software (Centrol 5; Harnisch, Austria). The readout of dried air was integrated against time. In this way, the respiratory quotient was calculated as RQ = ∫CO₂/∫O₂.