Study On Proteins Involved In The Defenses Of Anopheles Dirus Against Plasmodium Yoelii

Malaria is a severe infectious parasitic disease with great damage to human health, which causes 1-3 millions deaths annually.Control of malaria encounters great challenge because of the emergence of drug and insecticide resistance in parasite and mosquito vector, respectively.It is difficult to control the transmission of the disease through traditional methods.Detailed understanding of the interplay between the parasite and mosquito genes that are major determinants of parasite development might permit the development of new concepts and tools for blocking transmission.The mosquito innate immune system is an obvious suspect for the mediation of parasite losses,but evidence for this hypothesis has only recently been obtained.Induction of antimicrobial peptides,phagocytosis and melanotic encapsulation are the main immune responses of mosquito against Plasmodium.Identification of the key molecules of the responses is very important.Melanotic encapsulation is the most important defense mechanism of Anopheles against Plasmodium,some key components of which such as serine protease exist in hemolymph.Most of current investigations on mosquito defense mechanism against Plasmodium have concentrated on Anopheles gambiae,but little was known about Anopheles dirus.Anopheles dirus is the main vector of Plasmodium falciparum and Plasmodium vivax in China and Southeast Asia.The development of oocysts of Plasmodium yoelii could be blocked by melanotic encapsulation.Hence, Anopheles dirus-Plasmodium yoelii is a suitable model for studies on the blocking strategy of malaria and immune responses of mosquito.In this study,we used this model to investigate polypeptides and genes against Plasmodium infection and relationship between serine protease and Plasmodium infection by different methods.This study includes three parts as following.Part 1:Polypeptides from hemolymph of Anopheles dirus infected and uninfected with Plasmodium yoelii were separated with two-dimensional polyacrylamide gel electrophoresis(2-DE) respectively.Then the gels were stained by Coomassie brilliant blue and analyzed by PDQuest 2D Elite analysis software.Mass spectrometry,amino acid sequencing and NCBInr database searching were carried out to analyze and forecast both the biological characters and functions in oocyst melanization of the differentially expressed proteins.As a result,37 differentially expressed polypeptide spots(DEPs) were found by comparing the two gels.Six of the DEPs,named DEP1-6,were examined by mass spectrometry.DEP1 was also surveyed by amino acid sequencing.Results of bioinformatic analysis showed that DEP1～6 were all unknown proteins and DEP1 might play a role in the defense of mosquito against Plasmodium.Part 2:AdSP3 was expressed in a bacterial expression system in order to assess the cross-reactivity between the production of expression and antiserum against Manduca sexta PAPs.And bioinformatic analysis was carried out to compare amino acid sequences of serine proteases from different insects,especially amino acid sequences of AdSP3 and Manduca sexta PAPs.At last hemolymph proteins of Anopheles dirus infected and uninfected with Plasmodium yoelii were separated respectively with electrophoresis and identified by antiserum against Manduca sexta PAPs.1.Pinpoint/AdSP3 expression vector was successfully constructed and determined by DNA sequencing.Then Anopheles dirus serine protease AdSP3 was expressed in Escherichia coli JM109.An expressed fusion protein with the molecular weight of 35kDa separated by SDS-PAGE,followed by Western blotting to determine the immnoreactivity of the expressed fusion protein with antiserum against Manduca sexta PAPs.Positive results were observed.Bioinformatic analysis also showed high similarity between serine proteases of Anopheles dirus and Manduca sexta PAPs.The results indicated that identification of serine protease of Anopheles dirus by antiserum against Manduca sexta PAPs was reasonable.2.Hemolymph proteins of Anopheles dirus infected and uninfected with Plasmodium yoelii were separated with electrophoresis respectively and identified by antiserum against Manduca sexta PAPs with Western blotting.Two proteins with different molecular weight were identified.The different results of the Western blotting between infected and uninfected mosquitoes showed that the positive proteins were related to Plasmodium infection. Part 3:Tissue location and quantification of serine protease of Anopheles dirus were investigated at both mRNA and protein levels.1.RT-PCR and Real-time PCR were carried out to invetigate tissue location and transcription level of AdSP3 at different time points after blood feeding.The results showed that AdSP3 was transcripted in both hemocyte and salivary gland.Whether AdSP3 was transcripted in midgut is still unclear because multiple bands of PCR product were amplified.We found different levels of transcription between the groups,which was related to the infection with Plasmodium yoelii.2.Immunocytochemical localization and confocal laser scanning microscopy were carried out to determine distributing of serine protease in mosquito and quantify the protease at protein level.Positive results were observed in hemocytes,salivary glands and midguts.Change of intensity of green fluorescence between different groups indicated that serine protease was related to melanotic capsulation of oocysts by Anopheles dirus.The purpose of our research is to find proteins of Anopheles dirus involved in defenses against Plasmodium yoelii infection.We also want to know whether serine protease is related to Plasmodium yoelii infection and its role in the defense against Plasmodium infection.The results demonstrate involvement of serine protease in defense against Plasmodium infection.This study will help us to understand the relationship between Plasmodium and mosquito vector better,and provide us with more experimental and theoretic grounds for research of melanization mechanism and new strategy to prevent malaria.