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Validation of the doubly labelled water technique for bumblebees Bombus terrestris (L.)

The doubly labelled water (DLW) technique was validated for the bumblebee Bombus terrestris (L.) using respirometry (RESP) from tethered roundabout flights. We injected small volumes (1 µl) of a mixture containing low concentrations of deuterium and 18O and withdrew 1-2 µl of haemolymph to determine initial 18O concentration. The injected isotopes were equilibrated with the body water pool after 10 min, and high material turnover allowed the analysis of final blood isotope concentrations after 5-7 h. On average (n=16), values measured using the DLW technique exceeded values measured using RESP by 3.1±9.9 %, a difference that was not statistically significant at the 99 % confidence level. The absolute error was 7.4±7.1 % (mean ± s.d.). Isotope dilution spaces of both deuterium and 18O were almost identical with the body water pool. We corrected for isotope fractionation, using a slightly higher value than usual for one of the fractionation factors. The single most important variable to influence DLW results, which could not be measured with the desired accuracy, was the volume of the final body water pool N. An overestimate of final N possibly resulted in the DLW overestimate.