Characterization of a serine protease and hemolymph polypeptides from the human malaria vector, Anopheles gambiae

The interactions of malaria parasites with different mosquito tissues (e.g. midgut, hemolymph, and salivary gland) are being studied intensively to develop new ways to reduce or stop disease transmission at the level of the vector. Serine proteases and hemolymph components are important factors that can affect development at one or all stages of the parasite in the mosquito. This study focused on the molecular/immunological characterization of a mosquito serine protease, AgSp24D, and on the patterns of hemolymph polypeptides from Anopheles gambiae.; AgSp24D is a 271 amino acid protein containing the serine, histidine and aspartic acid residues found in serine proteases. Northern analysis revealed that transcription products occur mainly in adults and males. Plasmodium-refractory mosquitoes express higher levels of AgSp24D mRNA than susceptible mosquitoes. The thorax was the primary site for expression in the adults. Polyclonal antisera against an AgSp24D fusion protein recognizes two polypeptides of 50 kDa and 60 kDa in immunoblots of whole body homogenates of adult mosquitoes. The strongest signal was detected in thorax homogenates although signal was also detected in all tissues except hemolymph. The polyclonal antibody cross reacted to several species of mosquito, a fruit fly, a cricket, a tobacco hornworm, and bovine cardiac and skeletal muscle. AgSp24D protein levels did not change after infection of Armigeres subalbatus with microfilarial worms that migrate to and develop in the thorax.; The effects of various immune elicitors on the large hemolymph polypeptides of A. gambiae were investigated. Wounding induced at least six hemolymph polypeptides (79, 57, 44, 41, 32 and 25.7 kDa) which appeared in the hemolymph within 30 min and reached a maximum level after approximately 6 hours as determined by silver staining. Dose response experiments with Escherichia coli showed that two polypeptides (33 and 25 kDa) pre-existing in the hemolymph before challenge disappeared in an E. coli cell number-dependent manner. Time course studies demonstrated that the 33 and 25 kDa polypeptides started to decline at 12-18 hrs after injection of E. coli. LPS, laminarin, and zymosan A caused a reduction in the 25 kDa polypeptide only.