Quantitative analysis of phenol oxidase activity in insect hemolymph

We describe a simple, inexpensive, and robust protocol for the quantification of phenol oxidase activity in insect hemolymph. Discrete volumes of hemolymph from Drosophila melanogaster larvae are applied to pieces of filter paper soaked in an L-3, 4-dihydroxyphenylalanine (L-DOPA) solution. Phenol oxidase present in the samples catalyzes melanin synthesis from the L-DOPA precursor, resulting in the appearance of a roughly circular melanized spot on the filter paper. The filter paper is then scanned and analyzed with image-processing software. Each pixel in an image is assigned a grayscale value. The mean of the grayscale values for a circular region of pixels at the center of the image of each spot is used to compute a melanization index (MI) value, the computation is based on a comparison to an external standard (India ink). Numerical MI values for control and experimental larvae can then be pooled and subjected to statistical analysis. This protocol was used to evaluate phenol oxidase activity in larvae of different backgrounds: wild-type, lozenge, hopscotch(Tumorous-lethal) (which induces the formation of large melanotic tumors), and body-color mutations ebony and yellow. Our results demonstrate that this assay is sensitive enough for use in genetic screens with D. melanogaster and could conceivably be used for evaluation of MI from hemolymph of other insects.