| Melanotic encapsulation,a specifically anopheline defense mechanism against malaria,is probably initiated by binding of soluble pattern-recognition receptors to the parasite surface factors and activation of a serine protease cascade that ultimately leads to proteolytic activation of prophenoloxidase. Active phenoloxidase then cross-links and melanizes other proteins associated with the invading parasites. It is unclear how PPO is transported into the midgut tissues,activated by a protease cascade,and engaged in the melanization process of Plasmodium oocysts in the midguts of female Anopheles mosquitoes. Four stages will be involved in Plasmodium development within or outside the mosquito midgut,ie,gametes,zygotes,ookinetes,and oocysts of Plasmodium. After oocysts development is matured,sporozoites start to release and reach the salivary gland to become infective stage. In this article,we have used a model of inducible P.yoelii oocysts melanization in An.stephensi after administration of NA to explore the melanization and signals-modulating mechanisms based on the analysis for the level of differentially expressed melanization-engaging genes or proteins in hemocytes or midguts. Firstly,melanotic granulae within the P.yoelii oocysts were observed by light microscopy and transection electron microscopy. Abnormal metabolism and ultrastructure in the midgut were shown. Widen cellular gap was shown in some midgut cells. Secondly,differential protein concentration in the hemolymph and midgut of An.stephensi adult females fed on different meals was examined with Bradford method. By statistical analysis,NA treatment leads to a significantly decreased hemolymph protein concentration. No marked difference of midgut protein concentration between NA and IB groups. Thirdly,one-dimensional SDS-PAGE showed tens of differentially expressed hemolymph protein binds in NA-,IB-,NB or S-fed adult females. Especially expressed difference at the binds with molecular weight of 160kDa and 66kDa are significantly shown. Increased expression of the MW of 66kDa proteins was resulted in NB-,or,IB-fed group after 1st day. But after 6th ～8th day of infection,decreased expression was induced. Following 1st and 2nd day's NA treatment,66kDa proteins showed up-regulation.Up to 3rd day,the bind had a decreased expressed trend. Otherwise,the bind with molecular weight of 160kDa showed up-regulated expression in NB-,or IB-fed group at 1 st day'blood-borne. Then down-regulated expression was manifested after that.Fourthly,hemolymph and midgut proteins from NA-,or IB-treated mosquitoes were separated by two-dimensional gel electrophoresis and stained with Coomassie blue. From fifteen differential spots in the hemolymph,we seemly have found out serine protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot analysis using antibodies against serine protease 22D or PPO revealed the proteins in hemolymph and midgut extracts of NA- and IB-treated mosquitoes. SP22D and PPO became more abundant in the hemolymph of An.stephensi upon challenge with P.yoelii. Their levels reached the peak at 6th day after Plasmodium infection and then gradually decreased after NA-induced melanization. But increased content of PPO in the midgut was seen after the melanization. 2-DE followed by Western blotting revealed that both PPO and SP22D were constitutively expressed into the hemolymph of adult females and PPO is existed as a heterodimer in vivo. Alternately,SP22D may be normally expressed as heterodimer. Our results show PPO and SP22D in the hemolymph of An.stephensi is up-regulated expression after P.yoelii infection. But after melanization,lower level of the two proteases in hemolymph and increased level of PPO in the midgut are simultaneously shown. After NA-administration,some decreased expressed proteins related with carbohydrate metabolism,structure,or cellular communication were resulted by peptide mass fingerprints maps followed two-dimensional gel electrophoresis.Fifthly,using degenerate primers,PPO1,S...
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