The Black cells phenotype is caused by a point mutation in the Drosophila pro-phenoloxidase 1 gene that triggers melanization and hematopoietic defects1

• Black cells is caused by a point mutation in pro-phenoloxidase 1 (PPO1).
• The amino acid change, A480V, has a distance effect on the conformation of the pro-domain cleavage site of PPO1.
• PPO1A480V causes spontaneous melanization inside crystal cells.
• Melanized crystal cells are unable to supply hemolymph pro-phenoloxidases 1 and 2.
• Crystal cell melanization affects larval hematopoiesis.

Melanization contributes to arthropod-specific innate immunity through deposition of melanin at wound sites or around parasites, with concomitant release of microbicidal reactive oxygen species. Melanization requires sequential activation of proteolytic enzymes in the hemolymph, including the final enzyme pro-phenoloxidase. Black cells (Bc) is a mutation causing spontaneous melanization of Drosophila crystal cells, a hemocyte cell type producing phenoloxidases. Bc individuals exhibit circulating black spots but fail to melanize upon injury. Although Bc is widely used as a loss-of-function mutant of phenoloxidases, the mutation causing Bc remained unknown. Here, we identified a single point mutation in the pro-phenoloxidase 1 (PPO1) gene of Bc flies causing an Alanine to Valine change in the C-terminal domain of PPO1, predicted to affect the conformation of the N-terminal pro-domain cleavage site at a distance and causing uncontrolled catalytic activity. Genomic insertion of a PPO1A480V transgene phenocopies Black cells, proving that A480V is indeed the causal mutation of the historical Bc phenotype.

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