The Roles of Dopa Decarboxylase and Dopachrome Conversion Enzyme in the Melanization Reactions of Mosquitoes

• Main Authors: Chiung-Yin Huang, 黃瓊瑩
• Other Authors: Cheng-Chen Chen
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/51069339664729518652

博士 === 國立陽明大學 === 熱帶醫學研究所 === 93 === The biosynthesis of melanin and melanotic material constitutes an important component in various aspects of insect life, i.e. cuticular sclerotization, egg tanning, wound healing and melanotic encapsulation. In mosquitoes, the biosynthesis of melanin is initiated by the hydroxylation of tyrosine to DOPA by phenoloxidase (PO). DOPA is a branch point of the melanization reaction; it may be oxidized to dopaquinone by PO or be decarboxylated to dopamine by dopa decarboxylase (DDC). Further oxidation of dopaquinone by PO produces dopachrome. Dopachrome is then converted to dihydroxyindole by dopachrome conversion enzyme (DCE) to produce melanin. In addition, dopamine, which is an important substrate of sclerotin, may also be oxidized to produce melanin. In order to clarify the melanization reaction of parasite and cuticle sclerotization in mosquitoes, cDNAs encoding Armigeres subalbatus DDC and DCE were cloned. Gene knockdown using RNA interference was used to elucidate their roles in the melanization reaction of parasites and cuticle sclerotization in Ar. subalbatus. Real-time PCR analysis revealed the significant increase of both DDC and DCE in immune-activated mosquitoes, which demonstrated their possible roles in mosquito immune reactions. A double subgenomic Sindbis virus carrying antisense fragment of Ar. subalbatus DDC was used to knock down DDC. The degree of melanization (DOM) of mf in DDC-knockdown mosquitoes was significantly decreased at 48 hr post mf challenge then gradually recovered at 72 and 96 hr, suggesting that DDC may participate the melanization reaction of parasites in mosquitoes. To further investigate the competitive roles of DDC and DCE, gene knockdown by double-stranded RNA (dsRNA) was used to elucidate their roles in the melanization reaction of parasites in Ar. subalbatus. Compared with controls, the DOM of mf in both DCE- and DDC-knockdown mosquitoes were significantly decreased, but the DOM of parasites in DDC-knockdown mosquitoes was significantly higher than those in DCE-knockdown mosquitoes. The results of the present study suggested that DOPA decarboxylation pathway and DOPA oxidation pathway are both required for melanization immune responses, but DOPA oxidation pathway may be the major route. For further elucidation of DDC and DCE in the cuticle sclerotization of mosquito pupae, dsRNAs of DDC and DCE were also inoculated into mosquito white pupae to study the roles of DDC and DCE in the cuticle sclerotization of mosquito pupae. As compared with water- inoculated controls, the moratlity rate of mosquito papae inoculated with DDC dsRNA progressively increased over time, and only a few DDC dsRNA-inoculated papae emerged to deformed adults. In contrast, the mortality rate of mosquito pupae inoculated with DCE dsRNA showed no significant change with time and most of them emergerd to normal adults. Our results suggested that DOPA decarboxylation pathway is the major route of cuticle sclerotization of mosquito pupae, and DOPA oxidation pathway may play no role in the cuticle sclerotization of mosquito pupae.