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Supporting information
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Quantification of metallothionein concentrations
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Each larva was homogenized in 160 µl homogenization buffer (10 mM tris-HCl, 85 mM
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NaCl, pH 7.4, Sigma-Aldrich, Belgium). Subsequently, a 40 µl aliquot of the homogenate
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was separated and directly stored at -80°C for analysis of the internal zinc concentrations; the
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remaining 120 µl of the homogenate was centrifuged (Eppendorf Centrifuge 5804 R,
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Hamburg, Germany) for 20 min (4°C, 13,200 g). Afterwards, two times 50 µl supernatant
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was used for MT analyses. We used the cadmium thiomolybdate saturation assay, employing
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isotopically enriched cadmium 109Cd (Klein et al., 1994). The oxidized MTs were first
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converted into native MTs with 10 µl reducing agent 2-mercaptoethanol (140 mM Mercapto-
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ethanol, 10 mM tris-HCl, 85 mM NaCl, pH 7.4) and Zn2+ (300 mM ZnSO4.7H2O) as metal
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donor. High molecular weight Cd-binding complexes were denatured by treatment with
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acetonitrile and metals bound to MT were removed with ammonium tetrathiomolybdate. The
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excessive tetrathiomolybdate and its metal complexes were removed with
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dimethylaminoethyl-sephacel (66% (v/v) suspension in the homogenization buffer). This was
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followed by saturation with 10 µl radioactive 109Cd isotope. The excessive 109Cd was
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complexed and removed by 100 µl Chelex-100 (66% (v/v) suspension in the homogenization
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buffer), shaken for 15 min, followed by centrifugation for 5 min (4°C, 8,000 g). Subsequently
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500 µl of the supernatant was incubated with 500 µl of acetonitrile for 3 min. After
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centrifugation, 800 µl of the supernatant with 109Cd bound to MTs was measured with a
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Wizard 3 1480 automatic gamma counter (Perkin-Elmer, Zaventem, Belgium) with standard
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errors ranging from 1 to 2%. The MT concentrations were calculated by assuming one MT
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molecule is saturated by 7 molecules of 109Cd (Kito et al., 1982).
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Quantification of internal zinc concentrations
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In the same larvae in which the MT concentrations were measured, 40 µl aliquot of the
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homogenate was dried at 60°C for 48 h. Subsequently, samples were digested by 200 µl of
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highly pure acid nitric (HNO3: 70%), followed by 30 minutes incubation at 110°C in a
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heating block. After cooling down, 10 µl hydrogen peroxide (H2O2: 30%) was added to the
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sample. Again, the sample was incubated at 110°Cfor 30 minutes. All samples were then
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diluted with mQ-water to exact 7 ml sample solutions. Zinc concentrations in the damselfly
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larvae were analyzed using ICP-OES (Thermo scientific, ICAP 6300 Duo, Waltham, MA,
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USA). Certified mussel reference material (CRM 278) of the Community Bureau of
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Reference (European Union, Brussels, Belgium) and blanks were used to verify the accuracy
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of the analysis. Recoveries were within 10% of the certified values. The zinc concentrations
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of the homogenization buffer were below the detection limit (2.5 µg/l), indicating they did
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not bias the observed zinc concentrations in the animals.
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References
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Kito H., Ose Y., Mizuhira V., Sato T., Ishikawa T., Tazawa T. (1982) Separation and
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Purification of (Cd, Cu, Zn)-Metallothionein in Carp Hepato-Pancreas. Comparative
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Biochemistry and Physiology C-Pharmacology Toxicology & Endocrinology, 73, 121-
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127.
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Klein D., Sato S., Summer K. H. (1994) Quantification of oxidized metallothionein in
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biological-material by a Cd saturation method. Analytical Biochemistry, 221, 405-
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409.
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