Respirometry

The CO₂ production was determined using an open flow-through respirometry (Sable Systems International, Las Vegas, NV, USA). The outside air firstly passed through the Sub-Sampler Pump (SS-4), then into the Mass Flow Controller (MFC2), finally into the Carbon Dioxide Analyzer (CA-10A). Data were collected, analyzed using Universal Interface (UI-2) and ExpeData data acquisition software (Sable Systems International), which also controlled the eight-channel multiplexer. Data were recorded every 1 s. All measurements were conducted during the daytime, which was the resting phase of the nocturnal scorpions. Each individual was weighed to the nearest 0.01 g with an electronic balance (Sartorius BP3100S, Gottingen, Germany) following the flow-through respirometry measurements.

In order to avoid air CO₂ fluctuations, an additional ∼30 l dry and empty plastic container was supplied in front of SS-4. We set the flow rate at 60 ml min^–1. Four respirometry chambers attached to a multiplexer were placed in the incubator, with one acting as a baseline and the other three containing scorpion individuals. Glass chambers that had relatively small volume (∼10 ml) were used to reduce the possibility of the scorpions moving, which may have lead to a several-fold increase in CO₂ production.

A complete experimental run lasted for 120 min, consisting of ten cycles (repeats). Each cycle lasted 12 min, with a 1.5-min baseline each at the start and end of the cycle, and three 3-min measurement periods of scorpion-containing chambers. The 3-min measurement period consisted of three phases (Fig. 5): lag phase ∼0.5 min, buffering phase ∼1 min and stationary phase ∼1.5 min. This procedure was adopted because (i) a preliminary trial verified that the CO₂ release in our experimental condition was continuous, which indicated a continuous pattern of gas exchange in M. martensii here; (ii) we have also extended the stationary phase to 10 min and similar result was obtained. Scorpion activity was determined when a sharp increase of CO₂ release was detected at lag or stationary phase. For each experimental run, the initial 30 min were not recorded and the first one to two cycles were usually abandoned as well, because scorpion activity was sometimes detected during these times. Median value of the remaining eight to nine cycles was calculated as the final result for each individual.

The change curve of carbon dioxide volume (VCO₂) during metabolic rate measurement. I, Lag phase, ∼0.5 min. II, Buffering phase, ∼1 min. III, Stationary phase, ∼1.5 min.