6.1.8. Thermolimit respirometry
Thermal limits of insects are often determined by visual or video observation (e.g. Hazell et al., 2008; Hazell and Bale, 2011; Terblanche et al., 2011; and citations therein). These protocols test the ability of coordinated movement of the insects. Besides passive observation, such analyses use the "righting response" of insects to regain normal body position after being pushed or nudged by the experimenter.
An alternative is thermolimit respirometry (Lighton and Turner, 2004; Lighton, 2008). With this method, the thermal limits of respiration can be determined. In A. m. carnica, chill coma temperature was determined by thermolimit respirometry to be in the range of 9-11°C by the observation that discontinuous respiration ceased (Lighton and Lovegrove, 1990; Kovac et al., 2007; compare Hetz and Bradley, 2005). At these temperatures, muscular and neural functions of bees come to a halt (Esch, 1988). Honey bees can survive chill coma temperatures (<10°C) for a longer time, but are incapable of coordinated movement (Esch, 1988).
Thermolimit respirometry is especially useful in determining the upper critical thermal limit (CTmax). By applying a temperature ramp of 0.25°C min-1 (Terblanche et al., 2011), the respiratory CTmax of A. m. carnica was determined as 48.9°C, which equalled the (voluntary) activity CTmax of 49.0°C (Käfer et al., 2012). The method uses the fact that cyclic patterns of respiration cease suddenly at the CTmax (Lighton and Turner, 2004; Lighton, 2008; Käfer et al., 2012).
In order to better identify the point of respiratory failure, the absolute difference sum (ADS) of the respiratory signal (rADS) can be calculated (Lighton and Turner, 2004). This is done by summing the difference between the absolute values (without considering sign) of successive data points of the respiratory signal. Further improvement can be achieved by calculating the rADS residuals around the estimated temperature of the CTmax. The rADS residuals are the difference between the ADS curve and a regression line calculated through the values for a section of, for example, 10 min before and after the estimated thermal limit. A usually sharp inflection point of the rADS residuals accurately indicates the respiratory CTmax (Lighton and Turner, 2004; Käfer et al., 2012; Stabentheiner et al., 2012). This method can also be applied for the analysis of other cyclic events like the readout of activity detectors (see section 6.8.2.).
One has to keep in mind, however, that thermolimit respirometry cannot replace conventional methods, which use behavioural cues like voluntary and forced activity to completely determine the CTmax and CTmin. Especially lethal temperatures have to be determined with appropriate methods of activity monitoring and proper tests of survival (e.g. Ono et al., 1995; Ken et al., 2005; Hazell et al., 2010; Terblanche et al., 2011). Nonetheless, thermolimit respirometry provides a standardized, fast and objective possibility to determine thermal limits because insect respiration depends on active control of spiracle and abdominal respiratory movements to achieve sufficient exchange of respiration gases. Consequently, respiration and muscular and neural activity are closely related. If respiration fails it is likely that other muscular and neural functions in the honey bee body are beyond their limits.