1
Supporting information S1 Supplementary assay protocols
2
To measure antioxidant defence, ROS and oxidative damage, individual larvae were
3
homogenized using a pestle, diluted 15 times (v/w) in phosphate buffer saline (PBS, 100mM,
4
pH 7.4) and centrifuged for 5 minutes (16,100 g; 4°C). The resulting supernatant was used in
5
the assays.
6
For quantification of the superoxide anion concentration we used a modified version of the
7
protocol by Elstner & Heupel [1]. The supernatant (40 µl) was first incubated at 20°C for 30
8
minutes in the presence of 4 µl hydroxylamine hydrochloride (10mM). Afterwards, 40 µl 17
9
mM sulfanilamide - 7 mM 2-naphtylamine was added and the mixture was incubated at room
10
temperature for 30 minutes. To individual wells of a 384-well microtiter plate we added 35 µl
11
of the mixture and measured absorbance at 450 nm. A calibration curve was established using
12
sodium nitrite. Superoxide anion concentrations were quantified in duplicate per larva (intra-
13
assay coefficient of variation: 2.21 % ) and both readings were averaged and expressed in M
14
for the statistical analyses.
15
We measured the activity of two key antioxidant enzymes in insects,
16
superoxidedismutase (SOD) and catalase (CAT) [2]. For the SOD activity we used the
17
protocol of De Block & Stoks [3] based on the SOD assay kit WST (Fluka, Buchs, Austria).
18
This measures the formation of a formazan dye upon reduction of the tetrazolium salt WST-1
19
with the superoxide anion. In each well of a 96-well microtiter plate we mixed 200 µl of WST
20
working solution, 20 µl body supernatant and 20 µl enzyme working solution. After an
21
acclimatization period of 20 minutes at 37 °C, absorbance was measured at 450 nm. The more
22
SOD activity, the less formazan production. One SOD unit is the amount of enzyme needed to
23
cause 50 % inhibition of the rate of the colorimetric reaction per µg protein. Protein content
24
was measured using the Bradford method [4].
1
25
To measure CAT activity we used the protocol of De Block & Stoks [3] (based on [5]).
26
The body supernatant was further diluted 16 times with PBS. We filled each well of a 96-well
27
microtiter plate (suited for the UV-spectrum) with 80 µl PBS (100mM, pH 7.4), 20 µl diluted
28
supernatant and 100 µl 20 mM hydrogen peroxide. CAT activity was measured in duplicate
29
(intra-assay coefficient of variation: 1.82 %) as the degradation of H2O2 with absorbance
30
measurements at 240 nm every 30 seconds during 2 minutes. CAT activity was calculated
31
based on the slope of the linear part of the reaction plot. One CAT unit is the amount of
32
enzyme needed to decompose 1 µmol H2O2/min per µg protein.
33
We measured oxidative damage to lipids by measuring an often used biomarker of lipid
34
peroxidation, the formation of malondialdehyde (MDA) [6]. We followed the preferred HPLC
35
based technique to measure MDA [6]. Note that MDA may be present in ingested food and
36
thereby altering background MDA levels [6]. This is unlikely to cause bias in our results as
37
we have shown previously [7] that predation risk did not change food intake in the study
38
species. Sample preparation was based on the protocol described in Miyamoto et al. [8]. First,
39
100 µl supernatant and 100 µl TBA 0.4% (40 mg TBA in 10 ml 0.2 M HCl) were mixed. This
40
mixture was incubated at 90 °C for 60 minutes and cooled on ice. Afterwards, we added 330
41
µl n-butanol, mixed and centrifuged the mixture for 3 minutes (4 °C; 1,000 g). Finally, 10 µl
42
of the supernatant (in duplicate; intra-assay coefficient of variation 3.12 %) was injected in an
43
HPLC/UV-Vis system on a C 18 column (250 x 4.6 x 5 µm) (see [9]). The mobile phase was
44
30 mM KH2PO4-methanol (65 + 35, v/v %, pH 4); the flow rate was isocratic, 1 ml/min.
45
Chromatograms were monitored at 535 nm and the retention time of MDA was 3.88 min. A
46
calibration curve was established using 1,1,3,3-tetraethoxypropane (TEP, malonaldehyde,
47
bisdiethylacetal). MDA concentrations were expressed in nmol/mg fat. The fat content was
48
measured based on the protocol of Bligh & Dyer [10].
49
2
50
51
REFERENCES
52
1. Elstner, E.F., Heupel, A. 1976 Inhibition of nitrite formation from
53
hydroxylammoniumchloride: a simple assay for superoxide dismutase. Anal. Biochem. 70,
54
616-620.
55
56
57
58
59
2. Korsloot, A., van Gestel, C.A.M., van Straalen, N.M. 2004 Environmental stress and
cellular reponse in arthropods. Boca Raton: CRC.
3. De Block, M., Stoks, R. 2008 Compensatory growth and oxidative stress in a damselfly.
Proc. R. Soc. B 275, 781-785.
4. Bradford, M.M. 1976 A rapid and sensitive method for the quantification of microgram
60
quantities of protein, utilizing the principle of protein-dye landing. Anal. Biochem. 72, 248-
61
254.
62
5. Aebi, H. 1984 Catalase in vitro. Meth. Enzymol. 105, 121-126.
63
6. Monaghan, P., Metcalfe, N.B., Torres, R. 2009 Oxidative stress as a mediator of life history
64
65
trade-offs: mechanisms, measurements and interpretation. Ecol. Lett. 12, 75-92.
7. Janssens, L., Stoks, R. 2013. Synergistic effects between pesticide stress and predator cues:
66
conflicting results from life history and physiology in the damselfly Enallagma
67
cyathigerum. Aquat. Toxicol., 132-133, 92-99.
68
8. Miyamoto, S., Alves de Almeida, E., Nogueira, L., Gennari de Medeiros, M.H., Di Mascio,
69
P. 2011 Evaluation of malondialdehyde levels. In: Oxidative stress in aquatic ecosystems.
70
Eds. Abele, J., Vazquez-Medina, J.P., Zenteno-Savin, T., Wiley Blackwell.
71
72
9. Karatas, F., Karatepe, M., Baysar, A. 2002 Determination of free malondialdehyde in
human serum by high-performance liquid chromatography. Anal. Biochem. 311, 76-79.
3
73
74
10. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification.
Can. J. Biochem. Physiol., 37, 911-917.
4