Possible Involvement of Manduca sexta Hemolymph Protease‐1 in Melanization

Abstract

Melanization is a complex innate immune response in insects, involving multiple proteases in the prophenoloxidase (proPO) activation cascade. Hemolymph protease‐1 (HP1), a clip‐domain serine protease expressed in hemocytes, is found constitutively in the Manduca sexta hemolymph, yet its function is still unknown. RNAi techniques have been unsuccessful as a means to study the protease in vivo. As a result, we created a model by using the Drosophila S2 cell system to study the protease. The HP1 cDNA was cloned and incorporated into the S2 cellular genome. The cells were selected, and a stable cell line was established. Western blot analysis indicated that the expression of proHP1 in the cell culture was highly efficient. When the stable cells were treated with dsRNA against HP1, the expression of HP1 was blocked. To test whether HP1 might influence the activation of hemolymph proPO, the stable HP1 S2 cells treated with or without dsRNA were cultured as a monolayer. After induction of HP1 gene expression of the stable cells, M. sexta plasma was added to the culture. We found that the culture media of the cells expressing the HP1 protein turned dark‐colored, with an increase of PO activity, whereas the culture media of the cells in which the HP1 gene was silenced by RNAi remained unchanged. This result suggests that HP1 may function in the melanization/PO activation cascade. (supported by NIH grant no. GM41247)