Bacterial‐injection‐induced syntheses of N‐β‐alanyldopamine and Dopa decarboxylase in the hemolymph of coleopteran insect, Tenebrio molitor larvae

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Abstract
Injection of Escherichia coli into larvae of the coleopteran Tenebrio molitor resulted in the appearance of a dopamine-like substance on the electrochemical detector. To characterize this dopamine-like substance, we purified it to homogeneity from the immunized hemolymph and determined its molecular structure to be N-β-alanyldopamine using the liquid chromatographic/tandem mass spectrometric method. Chemically synthesized N-β-alanyldopamine showed the same retention time on HPLC as the purified N-β-alanyldopamine from immunized larvae. To elucidate the molecular mechanism of N-β-alanyldopamine synthesis in vivo, we examined the enzyme activity of Dopa decarboxylase against E. coli-injected hemolymph of T. molitor larvae. The enzyme activity of Dopa decarboxylase increased dramatically ≈ 8 h after injection; Dopa decarboxylase activity of injected larvae being 10-times higher than naive larvae after 24 h. To evaluate the extent of quantitative changes of Dopa decarboxylase in response to bacterial challenge, Tenebrio Dopa decarboxylase was purified to homogeneity from the whole larvae and a cDNA clone for Tenebrio Dopa decarboxylase was isolated. RNA blot hybridization revealed that expression of the Dopa decarboxylase gene was activated transiently 3–8 h after E. coli challenge. Immunoprecipitation experiments showed that Tenebrio Dopa decarboxylase was detected from 8 to 24 h in E. coli-injected larval extract. Thus, bacterial injection into T. molitor larvae might induce transcriptional activation of a Dopa decarboxylase gene, and then synthesis of N-β-alanyldopamine. The synthesized N-β-alanyldopamine might be used as a substrate by phenoloxidase during melanin synthesis in the humoral defense response or the melanotic encapsulation reaction of the cellular defense response.