Electronic Supplementary Material s34

Electronic Supplementary Material

S1 – HPLC

After microdissection, brain tissue punches were ejected into 100 µl sodium acetate buffer (3g sodium acetate, 4.3 ml glacial acetic acid, 16 NaOH pellets and 1000 ml deionized H2O, pH=5), to which an internal standard (α-methyl dopamine, 94.2 ng/ml) was added. Samples were frozen at -80˚ C to facilitate cell lysis, thawed on ice and centrifuged at 17,000 rpm for 5 min. Supernatant (90 µl) was injected into the chromatographic system (Waters 717+ autosampler) and analyzed with an electrochemical LC-4B potentiostat (Bioanalytical Systems). Mobile phase (pH=2.75, flow rate 1.8 ml min-1) consisted of 900ml of deionized H2O, 250mg NaOH, 18.63mg Na2EDTA, 21g citric acid, 0.85ml TEA, 105mg sodium octane sulfonic acid (SOS) and 25ml acetonitrile. The electrode potential was set at +0.6 V with respect to an Ag/AgCl reference electrode.

S2 – Plasma cortisol analysis

Plasma (10 µl) was added to 290 µl of diluent, of which 100 µl was added to an assay plate with standard curve samples. 100 µl of hormone conjugate was added to each well. The plate was incubated, washed and 200 µl of TMB (3, 3’, 5, 5’ tetramethylbenzidine) substrate was added to each well. The samples and standards were then incubated for a second time before stop solution was added. We determined hormone levels by reading the absorbance at 405 nm (Bio-Tek EL800) and using the standard curve to calculate the concentration. The cortisol ELISA kit we used has an intra-assay variance of 6.6 to 10 percent coefficient of variance, which is dependent on cortisol levels.

S3 - Cortisol GLM

S4 – Mean monoaminergic activity for uninfected and experimentally infected fish

Mean monoaminergic activity measured as monoamine metabolite/monoamine ±95% C.I. Back-transformed means presented for dopamine data in the striatum and serotonin data in the hippocampus and hypothalamus. Brain region codes as defined in Methods. U=uninfected; EI=experimentally infected; DA=dopaminergic activity; 5-HT=serotonergic activity. Dopaminergic activity was not measured in the hippocampus.