Anopheles gambiae salivary gland promoter analysis.

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 basic research in mosquito and parasite biology, and in the understanding of the complex interactions between Anopheles and Plasmodium. Moreover, the development of genetic manipulation techniques for An. gambiae and P. falciparum, and the availability of the complete sequences of human, mosquito and parasite genomes, 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. Recently, we described the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland-specific genes AgApy and D7r4 in transgenic An. stephensi (Lombardo et al. 2005). Specifically, an 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic An. stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal-lateral rather than in the distal-lateral lobes of female glands. We proposed some explanations to elucidate those data and to plan future work. First of all, the AgApy promoter employed in the An. stephensi transformation could be lacking in some of the sequence infomation needed for the correct and strong expression in the female salivary glands; moreover, the transcriptional machinery of An. stephensi could not perfectly recognize all the transcriptional information eventually enclosed in the An. gambiae promoter region. Taking advantage of the recent improvements in transformation techniques that made possible the genetic manipulation of An. gambiae mosquitoes, we established transgenic An. gambiae lines carrying a larger portion of the AgApy promoter (~ 2,4 kb) driving the LacZ reporter gene. A transformation vector based on the piggyBac transposon and marked with the dsRed gene under control of the 3xP3 promoter was used for the microinjection experiments. A number of different transgenic An. gambiae G1 founders were obtained: the progeny was analyzed by Southern blot revealing the presence of single as well as multiple copies of the transgene. Three independent lines containing one (E9), three (D6) and four copies (D4) of the transgene were selected and established for further analysis. RT-PCR expression analysis with LacZ-specific primers showed different expression profiles in the three transgenic lines suggesting that position effect and copy number affect reporter gene expression. In fact, LacZ mRNA was ubiquitously detectable in transgenic mosquitoes from the D4 line whereas individuals from the D6 line exhibited an expression profile restricted to female salivary glands and adult males. Finally, transgene expression in E9 mosquitoes appeared strong in larval and pupal stages, absent in female carcasses and rather weak in salivary glands and males. Hystochemical assays on whole glands and western blot analysis on salivary protein extracts failed so far to reveal β-galactosidase protein in transgenic mosquitoes of the different lines. Further analysis are therefore in progress to completely characterize the transactivation properties of this AgApy regulatory region in the An. gambiae transgenic lines.