Supporting information S1 Supplementary assay protocols

To measure antioxidant defence, ROS and oxidative damage, individual larvae were homogenized using a pestle, diluted 15 times (v/w) in phosphate buffer saline (PBS, 100mM, pH 7.4) and centrifuged for 5 minutes (16,100 g; 4°C). The resulting supernatant was used in the assays.

For quantification of the superoxide anion concentration we used a modified version of the protocol by ElstnerHeupel [1]. The supernatant (40 µl) was first incubated at 20°C for 30 minutes in the presence of 4 µl hydroxylamine hydrochloride (10mM). Afterwards, 40 µl 17 mM sulfanilamide - 7 mM 2-naphtylamine was added and the mixture was incubated at room temperature for 30 minutes. To individual wells of a 384-well microtiterplate we added 35 µl of the mixture and measured absorbance at 450 nm. A calibration curve was established using sodium nitrite. Superoxide anion concentrations were quantified in duplicate per larva (intra-assay coefficient of variation: 2.21 % ) and both readings were averaged and expressed in M for the statistical analyses.

We measured the activity of two key antioxidant enzymes in insects, superoxidedismutase (SOD) and catalase (CAT) [2]. For the SOD activity we used the protocol of De Block Stoks [3] based on the SOD assay kit WST (Fluka, Buchs, Austria). This measures the formation of a formazan dye upon reduction of the tetrazolium salt WST-1 with the superoxide anion. In each well of a 96-well microtiter plate we mixed 200 µl of WST working solution, 20 µl body supernatant and 20 µl enzyme working solution. After an acclimatization period of 20 minutes at 37 °C, absorbance was measuredat 450 nm. The more SOD activity, the less formazan production. One SOD unit is the amount of enzyme needed to cause 50 % inhibition of the rate of the colorimetric reaction per µg protein. Protein content was measured using the Bradford method [4].

To measure CAT activity we used the protocol of De Block Stoks [3] (based on [5]). The body supernatant was further diluted 16 times with PBS. We filled each well of a 96-well microtiter plate (suited for the UV-spectrum) with 80 µl PBS (100mM, pH 7.4), 20 µl diluted supernatant and 100 µl 20 mM hydrogen peroxide. CAT activity was measured in duplicate (intra-assay coefficient of variation: 1.82 %)as the degradation of H2O2 with absorbance measurements at 240 nm every 30 seconds during 2 minutes. CAT activity was calculated based on the slope of the linear part of the reaction plot. One CAT unit is the amount of enzyme needed to decompose 1 µmol H2O2/min per µg protein.

We measured oxidative damage to lipids by measuring an often used biomarker of lipid peroxidation, the formation of malondialdehyde (MDA)[6]. We followed the preferred HPLC based technique to measure MDA [6]. Note that MDA may be present in ingested food and thereby altering background MDA levels [6]. This is unlikely to cause bias in our results as we have shown previously [7] that predation risk did not change food intake in the study species. Sample preparation was based on the protocol described in Miyamoto et al. [8]. First, 100 µl supernatant and 100 µl TBA 0.4% (40 mg TBA in 10 ml 0.2 M HCl) were mixed.This mixture was incubated at 90 °C for 60 minutes and cooled on ice. Afterwards, we added 330 µl n-butanol, mixed and centrifuged the mixture for 3 minutes (4 °C; 1,000 g). Finally, 10 µl of the supernatant (in duplicate; intra-assay coefficient of variation 3.12 %) was injected in an HPLC/UV-Vis system on a C 18 column (250 x 4.6 x 5 µm) (see[9]). The mobile phase was 30 mM KH2PO4-methanol (65 + 35, v/v %, pH 4); the flow rate was isocratic, 1 ml/min. Chromatograms were monitored at 535 nm and the retention time of MDA was 3.88 min. A calibration curve was established using 1,1,3,3-tetraethoxypropane (TEP, malonaldehyde, bisdiethylacetal). MDA concentrations were expressed in nmol/mg fat.The fat content was measured based on the protocol of Bligh & Dyer [10].

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