MEMBERS OF A TRYPSIN GENE FAMILY IN ANOPHELES-GAMBIAE ARE INDUCED IN THE GUT BY BLOOD MEAL

Serine proteases are among the enzymes that play a crucial role during the digestion of the blood meal in the gut of mosquitoes. The identif ication of the corresponding genes would have important implications f or the control of mosquitoes and mosquito-borne diseases. Analysis of the genomic organization of these genes may lead to the isolation of a gut-specific, inducible promoter tor the expression of anti-parasitic agents in transgenic mosquitoes. Moreover, specific inhibitors could be designed on the basis of the structural properties of the enzymes. We report here on the identification of a trypsin gene family in Anoph eles gambiae, the mosquito vector of malaria in Africa. Mosquito tryps in-related sequences were amplified by PCR using as template cDNA deri ved from RNA of blood fed mosquitoes. Cloning of the PCR product revea led two distinct sequences. Corresponding full-length cDNA clones were obtained and sequenced. Antryp1 and Antryp2 code for proteins of 274 and 277 amino acids respectively, showing 75% homology at the amino ac id level. The deduced amino acid sequences clearly identify them as tr ypsins. Five additional trypsin sequences were found in overlapping ge nomic clones. The genes identified are tightly clustered within 11 kb and sequencing indicates that no introns are present. Northern and PCR analysis indicated that the transcription of both Antryp1 and Antryp2 is induced by blood feeding. Moreover, the Antryp1 protein was detect ed among the proteins of a midgut lysate of blood fed mosquitoes using antisera against recombinant Antryp1. In addition, the recombinant po lypeptides derived from Antryp1 and Antryp2 expressed in Escherichia c oli showed a strong proteolytic activity against different sets of blo od proteins. We conclude that the products of Antrp1 and Antryp2 play an important role in the breakdown of the proteins during the digestio n of the blood meal in the mosquito gut.