Phenoloxidase assay raw data

Phenoloxidase levels in Drosophila tissues were measured using a simple procedure based on the transformation of L-dopa to dopachrome in the presence of phenoloxidase, measured as function of optical density, as described by Lario et al. (1993). Larvae were singly placed in freshly collected P. obesinymphae galls for 45 minutes, and subsequently removed with a small paintbrush. A control group of larvae of similar size was not exposed to the aphids and was labeled the “unattacked” group. In order to differentiate between phenoloxidase activation during mechanical wounding of the epithelia by aphid stylets, and circulating phenoloxidase activation in the hemolymph, Drosophila hemolymph was extracted from both the attacked larvae and the control group using fine needles. After hemolymph extraction, the larval midguts were removed using a pair of dissecting tweezers. The remaining cuticle was then placed in a 1.5 mL Eppendorf tube and ground by a pestle. The tubes were centrifuged briefly, and the liquid portion was removed by a pipette for use in the phenoloxidase assay, in addition to the extracted hemolymph. Immediately after extraction, the hemolymph was transferred to ice-chilled ELISA wells containing 50 μL of 20 mM TRIS buffer, pH 7.0, with 1% sodium citrate. The cuticle fluids were transferred by pipette to ELISA wells containing the same mixture. After the hemolymph and cuticle fluids were collected, 50 μL of 3,4-dihydroxyphenyl-alanine (L-dopa), 5 mg/mL in TRIS buffer, were added to each well. Mixtures were incubated at 32°C for 45 min, and the wells were subsequently read in an ELISA spectrophotometer at a wavelength of 490 nm. Blank wells containing 50 μL TRIS buffer and 50 μL L-dopa in TRIS but no hemolymph or cuticle fluids were used as controls.