Molecular cloning of a second prophenoloxidase cDNA from the mosquito Armigeres subalbatus: prophenoloxidase expression in blood-fed and microfilariae-inoculated mosquitoes

Melanization constitutes an important component in various aspects of insec t life, including cuticular sclerotization, egg-shell tanning, melanization of parasites and wound healing. Recently, a cDNA encoding prophenoloxidase (pro-PO), a key enzyme in the biosynthesis of melanotic material in insect s, was cloned from microfilariae (mf)-inoculated mosquitoes, Armigeres suba lbatus. However, results of Northern blot analyses indicated that two pro-P Os might be present in Ar. subalbatus and these pro-POs might be responsibl e for two distinct physiological functions, egg-shell tanning and melanizat ion of parasites. Subsequently, the second pro-PO cDNA (As-pro-PO II) was c loned from blood-fed Ar. subalbatus by rapid amplification of cDNA ends pol ymerase chain reaction. The 2210 bp As-pro-PO II cDNA contains a 41 bp 5'-n on-coding region, a 2064 bp open reading frame and a 105 bp 3'-non-coding r egion. A hydrophobic signal peptide for endoplasmic reticulum targeting is not found in the N-terminal region. The deduced amino acid sequence of As-p ro-PO II shares a high degree of identity (81.5%) with that of the As-pro-P O I obtained from mf-inoculated Ar. subalbatus, Both Northern blot and reve rse transcription polymerase chain reaction analysis demonstrated that thes e two mosquito pro-POs are persistently expressed in mosquito haemocytes an d not in fat body, midgut, or ovaries. The expression of As-pro-PO I and As -pro-PO II in mosquitoes is associated with melanization of mf and blood fe eding, respectively.