Quantifying Triglycerides in Insects

The TAG content quantification was performed using the Triglycerides Reagent (Thermo Scientific, 981786). The assay is based on the enzymatic cleavage of the fatty acid moieties from the glycerol backbone of mono-, di-, and triacylglycerols and the subsequent quantification of the released glycerol. A detailed description of the method can be found at^{23 (link)}. Monoacylglycerols are negligible in various insects^{24 (link)} and Drosophila^{23 (link),25 (link)} and only very little diacylglycerol and free glycerol amounts are present, which are negligible in comparison to the triacylglycerol amounts^{23 (link),25 (link)}. In this study, we actually also quantified free glycerol levels, however, a fluorometric assay was used given the extremely low levels of free glycerol (see below). In brief, the assay was performed by transferring an aliquot of the supernatant of the above mentioned homogenate (25 µL) to a 96 well plate (Sarstedt, 82.1581) followed by a dilution with 25 µL 0.05% Tween 20 (1:2). Samples and standard were incubated at 37 °C with the Infinity reagent and the absorbance was measured at 510 nm. The total amount of TAG was determined using a glycerol standard (Sigma Aldrich, G7793).