CONSTITUTIVE AND BLOOD MEAL-INDUCED TRYPSIN GENES IN ANOPHELES-GAMBIAE

Trypsin genes in Anopheles gambiae are arranged as a tightly clustered gene family consisting of seven related coding sequences, devoid of i ntrons. The two blood meal-inducible members of this family, Antryp1 a nd Antryp2, were shown to play a crucial role in the breakdown of the blood meal constituents. The role of Antry3,4,5,6, and Antryp7 in the process of blood meal digestion remains to be elucidated. We have exam ined the localization and the expression patterns of these trypsins as well as the functional interactions in blood meal digestion between t rypsins and other gut-specific proteases. Northern blot and RT-PCR ana lysis indicated that the genes Antryp3,4,5,6, and Antryp7 are all cons titutively expressed in unfed female mosquitoes. Soon after blood feed ing the mRNA of these trypsin genes became undetectable and appeared a gain at the end of the gonotrophic cycle. The blood meal-inducible try psin Antrypl was also constitutively expressed at low level in the gut of adult female mosquitoes. This trypsin was the only member of this gene family to be expressed in the gut of male and female pupae. By us ing antisera that specifically recognized recombinant Antryp4 we were able to show that the corresponding protein in Anopheles is synthesize d and stored in the gut epithelium of unfed females as zymogen. Secret ion and activation of this trypsin was shown to occur in the midgut lu men immediately after fluid ingestion and independently of the protein content of the meal, Recombinant trypsins expressed in Escherichia co li, with the exception of Antryp5 and Antryp6, were able to activate i n vitro recombinant A. gambiae chymotrypsinogen, thus suggesting that blood meal ingestion is able to trigger a cascade of events leading to the activation of several proteases. (C) 1995 Academic Press, Inc.