Isolation and characterization of a dopa decarboxylase cDNA and the induction of its expression by an insect cytokine, growth-blocking peptide in Pseudaletia separata

Abstract

Parasitization by the wasp, Cotesia kariyai, elevates the concentration of an insect cytokine, growth-blocking peptide (GBP), in hemolymph of last instar Pseudaletis separata larvae. The increase of epidermal and hemolymph dopamine level is associated with the GBP increase. Both GBP and dopamine disturb host development and metamorphosis ( Hayakawa, 1995). Dopa decarboxylase (DDC) converts Dopa to dopamine, and its cDNA was isolated from P. separata, and the deduced amino acid sequence showed that it was highly homologous to other lepidopteran DDCs, showing 96, 90 and 86% identity with those of Mamestra brassicae, Bombyx mori, and Manduca sexta, respectively. A 3.2 kb DDC mRNA transcript was constitutively expressed at low levels in the epidermis, brain-nerve cord and hemocytes, and the expression was enhanced by injection of GBP in these tissues. Detailed characterization of the DDC mRNA expression in the epidermis showed that its expression reached a plateau 3 hr after the injection. DDC activity and DDC protein (55 kDa) level mirrored the mRNA expression. Immunocytochemistry with anti-DDC antibody confirmed that the enhanced DDC expression was localized in the epidermal cells. Dopamine concentration in the epidermis gradually increased and reached maximum 6 hr after the injection. When the epidermis of Day 1 last instar larvae was cultured in vitro in the presence of GBP, DDC mRNA increased, indicating that GBP acted on the epidermal cells directly to induce expression of the DDC gene.