Members of a trypsin gene family in Anopheles gambiae are induced in the gut by blood meal.

Abstract

Serine proteases are among the enzymes that play a crucial role during the digestion of the blood meal in the gut of mosquitoes. The identification of the corresponding genes would have important implications for 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 for 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 Anopheles gambiae, the mosquito vector of malaria in Africa. Mosquito trypsin‐related sequences were amplified by PCR using as template cDNA derived from RNA of blood fed mosquitoes. Cloning of the PCR product revealed 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 acid level. The deduced amino acid sequences clearly identify them as trypsins. Five additional trypsin sequences were found in overlapping genomic 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 detected among the proteins of a midgut lysate of blood fed mosquitoes using antisera against recombinant Antryp1. In addition, the recombinant polypeptides derived from Antryp1 and Antryp2 expressed in Escherichia coli showed a strong proteolytic activity against different sets of blood proteins. We conclude that the products of Antryp1 and Antryp2 play an important role in the breakdown of the proteins during the digestion of the blood meal in the mosquito gut.

Differential peptidomics highlights adipokinetic hormone as key player in regulating digestion in anautogenous flesh fly, Sarcophaga crassipalpis

“…In 57 carnivorous Diptera, either hematofagous or cadaver recyclers, the major protease involved is trypsin 58 (Borovsky et al, 1996). In accordance with the specific feeding behavior of anautogenous insects, in 59 these species, trypsin occurs in two differentially regulated forms: early trypsin that is constantly 60 present at a relatively low level even in sugar fed insects and late trypsin that appears after the intake of 61 a protein meal (Lu et al, 2006;Muller et al, 1993;Borovsky et al, 1996). In anautogenous mosquitoes, 62 late trypsin is a specific midgut protease, characteristic for adult blood feeding females only (Noriega & 63 Wells, 1999 (Ma et al, 2000).…”

Gene Expression Studies in Mosquitoes

“…A number of trypsins, peptidases, other enzymes, and transport proteins are recognized as being involved in this developmental phase. There are early and late phases of digestion that vary among the different mosquito species in the specific timing and genes involved (Barillas-Mury et al, 1995;Müller et al, 1993). The best characterized genes are those that encode carboxypeptidases (CPs, Table 2.3).…”

Induction of actin gene expression in the mosquito midgut by blood ingestion correlates with striking changes of cell shape

“…In anophelines, there is evidence to suggest that PM components are stored in secretory vesicles and released into the lumen upon blood ingestion (Devenport et al, 2004). Blood uptake also triggers a complex program of gene expression that includes the activation and/or up-regulation of genes involved in blood digestion and nutrient uptake (Muller et al, 1993;Lemos et al, 1996;Noriega and Wells, 1999;Holt et al, 2002;Dana et al, 2005;Marinotti et al, 2006).…”