Identification of salivary gland-specific regulatory regions from the mosquito Anopheles gambiae.

The application of the powerful tools of genetics and molecular biology to studies on mosquito vectors and malaria parasites has contributed to a significant advancement in the understanding of the complex interactions between Anopheles and Plasmodium. Moreover, the development of genetic manipulation techniques and the progress of genome sequencing projects raised great expectations for the development of novel malaria control strategies. In this context the availability of salivary gland-specific promoters, which may allow the expression of a given foreign gene in the salivary glands of transgenic mosquitoes, may prove a useful tool both for studies on vector-parasite interactions and, potentially, for the development of novel strategies for vector control. We report here the promoter analysis of the Anopheles gambiae AgApy and D7-related genes in the fruitfly Drosophila melanogaster and in the mosquito Anopheles stephensi. We initially focused our attention on the analysis of AgApy. This was the first salivary gland-specific gene isolated from An. gambiae and it encodes the platelet inhibitor apyrase. We have already shown that a 800 bp fragment located immediately upstream of AgApy is at least partly recognized in D. melanogaster, where it is able to drive the expression of a reporter gene in adult salivary glands (Lombardo F et al, 2000 J Biol Chem, 275: 23861-23868). Because in the meantime a genetic transformation technique of An. stephensi was developed (Catteruccia F et al, 2000 Nature, 405: 959-962) we decided to test in this mosquito the activity of the same promoter fragment. Therefore, the AgApy-LacZ-SV40t cassette (which contains the AgApy promoter, the E. coli LacZ reporter and the SV40 terminator) was transferred into a Minos-based vector carrying the Green Fluorescent Protein (EGFP) as transformation marker. 351 embryos were injected and the 65 G0 fertile adults obtained (18,5%) were pooled in five breeding groups and outcrossed to wild type mosquitoes. Screening for GFP expression of G1 larvae showed that only two groups, A and B, yielded fluorescent larvae. Based on the different intensity of fluorescence six bona fide homozygous lines (AW1, AS1, BW1, BW2, BI1 and BS1) were further analyzed for the presence and the expression of the transgene. Southern blot analysis indicated the presence of single insertions (AW1, AS1) and respectively of two (BW1), three (BW2, BI1) and four copies (BS1) of the transgene. With the only exception of BW2 the LacZ gene is transcribed in adult males and females of all transgenic lines as shown by RT-PCR expression analysis. In contrast, histochemical stainings revealed β-galactosidase activity only in the salivary glands of BS1 females, which contain four copies of the transgene. A weak staining was visible, after 10-15 hrs of incubations, in groups of cells in the distal-lateral lobes whereas a more intense blue color was detectable in the proximal portion of the medial lobe. This pattern only partially overlaps the endogenous expression profile of AgApy in An. gambiae and suggests that the promoter fragment that we used may lack of some tissue-specific enhancer or other essential regulatory regions. In the meanwhile we had proceeded with the characterization of the D7-related (D7r) genes, which are expressed in female salivary glands and are closely clustered in a 6 Kb region on chromosome arm 3R (Arcà B et al, 2002 Insect Mol Biol, 11:47-55). The intergenic spacers of the D7r genes appeared good promoter candidates and, therefore, we tested the ~1 Kb regions located immediately upstream of the D7r2 and D7r4 genes by D. melanogaster transformation. Different transgenic lines were established and histochemically analyzed for the presence of β-galactosidase activity. Intense blue colour was detectable after 2-3 hours of incubation in the adult salivary glands of both male and female fruitflies. Moreover, the staining was tissue-specific and showed very similar pattern in different individuals and different lines. These results indicate that the D7r2 and D7r4 promoter regions drive strong and tissue-specific expression of the LacZ reporter gene into the salivary glands of the fruitfly and are therefore very promising as salivary gland promoter candidates. The activity of the D7r4 promoter is currently being tested in the Asian malaria mosquito An. stephensi.