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Appendix
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Supplementary methods enzyme assays
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All larvae were homogenized on ice in a mixture of 150 µl cacodylate buffer (0.01 mol/L
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Na-Coc, 0.005 mol/L CaCl2) and 150 µl proteinase inhibitor (Sigma®, P-2714). Two
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hundred µl of the supernatant was separated to measure PO and total PO activity. To the
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remaining pellet and supernatant PBS buffer (pH 7.4) was added (1:23 w:v dilution). The
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resulting supernatant was used to measure SOD and CAT activity. For each animal, we
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also assessed protein content using the Bradford method (Bradford 1976).
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The activity of PO and total PO was quantified following the protocol by Stoks et
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al. (2006). Shortly, 20 µl of the sample was added to microtiter plate wells. ProPO was
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activated using 5 µl chymotrypsin (5 mg/1 mL aqua dest) whereas 5 µl of aqua dest was
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added to wells in which PO was measured. The mixture was incubated for five minutes at
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room temperature. Afterwards, 25 µl PBS and 150 µl L-DOPA (10 mM in cacodylate
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buffer) was added. The reaction was allowed to proceed for 30 minutes at 30°C and
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readings were taken every 20 s on a plate reader (BioRad, Benchmark Plus). Enzyme
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activity was measured as Vmax (the slope of the reaction curve during the linear phase of
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the reaction that transforms L-DOPA into dopachrome under non-limiting substrate
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concentrations).
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Activity of the antioxidant enzymes was measured following the protocols by De
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Block and Stoks (2008). SOD activity was quantified closely following the protocol
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described in the SOD Assay Kit-WST (Fluka, Buchs, Austria), which measures the
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formation of a formazan dye upon reduction of the tetrazolium salt WST-1 with
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superoxide anions. We added 200 ml of WST working solution and 20 ml of enzyme
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working solution to 20 ml of the supernatant. This mixture was incubated for 20 min at
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37°C followed by reading the absorbance at 450 nm. The activity of CAT was quantified
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following the protocol of Aebi (1984). The supernatant was further diluted 16 times with
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PBS (pH 7.4). To 20 ml of the diluted supernatant, we added 80 ml of PBS and 100 ml of
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20 mM H2O2.
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One SOD unit was defined as the amount of enzyme necessary to decrease the
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colorimetric reaction with 50% at 37°C. One unit of CAT activity was defined as the
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amount that decomposes 1 µmol H2O2/min at 30°C and pH 7.4.
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Additional measurements
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To obtain additional information to better understand the observed downregulation of PO
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24h after autotomy we performed additional physiological measurements on a new set of
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L. viridis reared and manipulated in an identical way as the larvae of the first experiment.
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To evaluate the possibility of a transient upregulation of PO after wounding and to assess
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the duration of the observed downregulation of PO, we scored the activity of PO at
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additional time points after autotomy. We killed the larvae by flash freezing them 0h,
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0.5h, 1h, 2h and 48h after autotomy. We also assessed two other physiological variables
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24 after autotomy on an extra set of larvae. To determine whether overall protein
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synthesis rates were decreased 24 after autotomy we measured the RNA:DNA ratio. To
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evaluate the possibility that the observed downregulation of PO and SOD activity was
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due to hemolymph loss we measured the activity of another enzyme, trehalase, not related
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to immune function and antioxidant defence.
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Measurement of RNA/DNA ratio and trehalase
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RNA/DNA ratios were quantified following a modified protocol of Melzer et al. (2005).
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Nucleid acid concentrations were determined using a modified fluorescent dye-based
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method and ethidium bromide (EB, Sigma®, E1385) was used as a fluorophore. The
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trehalase activity was measured by estimating the glucose produced by hydrolysis of
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trehalose as described by Worland et al. (1998).
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First, larvae were homogenized on ice in 0.04 M Na/Na2 buffer pH 7.0 (1:1 w:v
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dilution) in RNA-se free eppendorf tubes. An aliquot of 50 µl of the supernatant was
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separated to measure trehalase activity. To the remaining pellet and supernatant 60 µl
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extraction buffer was added (50 mM Tris pH 7.5, 50 mM EDTA, and 0.05% SDS).
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Homogenates were placed on a shaking mill for 20 min at maximum power (Labinco
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L46), then sonicated three times for 10 s and again shaken for 5 min. The resulting
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supernatant was diluted (1:40) in TE buffer (50 mM Tris pH 7.5, 50 mM EDTA) and
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used in the assay. All homogenization and preparatory steps were performed on ice and
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by use of nuclease free materials.
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Then, samples were placed in wells of black 96-well microtiter plates (Greiner
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655209). One hundred µl of each sample was used for the determination of total
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fluorescence by adding 2 µl of EB (100 µg/ml). TE buffer was used as a control. After 15
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min of incubation in the dark, fluorescence was quantified by exciting the samples at 530
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nm and measuring their emission at 590 nm (Tecan Infinite M200). This gave an estimate
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of the combined amounts of DNA plus RNA. For the quantification of the amount of
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DNA, 100 µl of all samples were treated with 1 µl RNAse (20 mg/ml, Ribonuclease A
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from bovine pancreas, Fluka 83831) in order to assess the proportion of fluorescence
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caused by EB intercalating with DNA. Wells were incubated at room temperature for 1 h
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to promote digestion of RNA, where after 2 µl of EB was added. Fluorescence was
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recorded 15 min after EB incubation in the dark. Standard curves were established from
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RNA (R6625) and DNA (D4764). Samples were run in triplo and mean values for nucleic
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acid concentrations were taken.
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To measure trehalase activity, the reaction mixture contained 80 µl milliQ, 80 µl
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citrate buffer (50 mM sodium citrate, 0.1 M Na2HPO, pH 5.4), 15 µl sample and 20 µl
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trehalose (0.1 M). As a control, the reaction mixture was incubated with milliQ instead of
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trehalose. After incubation at 37°C for 30 min the reaction was stopped by boiling for 3
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min. The amount of glucose of the samples and their controls was measured using the
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glucose assay reagent (Sigma A7420). Each well contained 75 µl milliQ, 25 µl sample
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and 200 µl reagent. After 20 min incubation at 30°C absorbance was quantified at 340
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nm. Glucose concentrations were calculated by means of a glucose standard curve. All
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samples were measured in duplo. Trehalase activity is expressed as µg glucose/min under
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assay conditions.
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Statistical analyses
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We ran separate ANOVAs for each response variable (PO, RNA/DNA ratio, trehalase)
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with lamellae autotomy as the independent variable and lamellae autotomy as
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independent variable. For PO we compared the activity of PO at the different time points
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after autotomy with the control group (initial PO level at 0h) by Dunnett’s a posteriori
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tests.
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Results
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Compared to the initial PO levels (mean ± SE; 0h: 170.47 ± 9.45), there was no
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difference after 0.5 h (154.76 ± 8.32), 1h (186.83 ± 9.20) and 48h (182.19 ± 11.86) (all p
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> 0.640). Yet, we found a decreased activity of PO 2h after autotomy (123.69 ± 12.84)
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(F1,22 = 8.60; p = 0.009).
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There was no effect of autotomy on the RNA/DNA ratio (with lamellae: 2.311 ±
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0.098, without lamellae: 2.304 ± 0.098) (F1,32 = 0.003; p = 0.954) and the trehalase
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activity (with lamellae: 0.092 ± 0.014, without lamellae: 0.105 ± 0.014) (F1,32 = 0.46; p =
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0.505).
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References
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Aebi, H. 1984. Catalase in vitro. Methods Enzymol. 105, 121-126.
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Bradford, M.M. 1976. Rapid and sensitive method for quantitation of microgram
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quantities of protein utilizing principle of protein-dye binding. Anal. Biochem. 72,
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248-254.
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De Block, M. & Stoks, R. 2008. Compensatory growth and oxidative stress in a
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damselfly. Proc. R. Soc. B 275, 781-785. (doi:10.1098/rspb.2007.1515)
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Melzner, F., Forsythe, J.W., Lee, P.G., Wood, J.B., Piatkowski, U. & Clemmesen, C.
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2005. Estimating recent growth in the cuttlefish Sepia officinalis: are nucleic acid-
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Biol. Ecol. 317, 37-51. (doi:10.1016/j.jembe.2004.11.011)
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Stoks, R., De Block, M., Slos, S., Van Doorslaer, W. & Rolff, J. 2006. Time constraints
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mediate predator-induced plasticity in immune function, condition, and life
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Worland, M.R., Grubor-Lajsic, G. & Montiel, P.O. 1998. Partial desiccation induced by
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