Post Genomic Functional Genetic Analysis of Parasite/Insect Interaction

The recent direction of biological research has focused on sequencing the total DNA content of organisms. This has led to the identification of potentially all the genes encoding for specific proteins present in particular species. What is needed now is to identify the function of these genes, their protein products and the tissues in which they are produced. The most common way to establish gene function is to isolate specific genes in the test tube, modify them and then return them to the organism. These modifications can either cause the organism to over-produce, or conversely, underproduce, this specific protein which in turn causes a specific physiological change that gives vital information on the gene's normal role. In the model organism, the fruit fly, there are numerous systems available to modify gene expression. However, to study important interactions that only occur between parasites and blood sucking insects requires similar methods to be developed for these important carriers of disease. This is particular relevant to mosquitoes which are biting nuisances worldwide and the most important vectors of many human diseases. The general complications of maintenance of blood-feeding insects make the development of efficient modification tools that maximize experimental flexibility a requisite. We will thus adopt the most successful method used in fruit fly research to the mosquito, Anopheles gambiae that is the primary transmitter of the malaria parasite in Africa. Different components of system will be initially tested in the simplified environment of isolated mosquito cells to rapidly identify those that function most efficiently. A selection of the most efficient tools will then be examined directly in mosquitoes to precisely define those that work in the more complex holistic environment of the whole organism. As a proof of principle that the system functions efficiently in mosquitoes we will over-produce and silence specific proteins thought to kill parasites in the insect. Using the flexibility of the system we will determine whether malaria parasite killing is more effective if the protein is produced by the mosquitoes' 'liver', blood cells or its stomach. Although the work is focused on Anopheles mosquitoes and the malaria parasite, the principles and methods should be transferable to other parasites transmitted by mosquitoes and blood sucking insects.

To study gene function in parasitized haematophagous insects requires the development of methodologies tailored to these non-model organisms. These approaches are urgently needed for mosquitoes, which are biting nuisances worldwide and the most important insect vectors of many human diseases. The labour intensive maintenance of blood-feeding insects makes the development of experimental flexibility in functional genetic characterisation an important requisite. The proposed aim is thus to adopt the binary Gal4/UAS system to A. gambiae mosquitoes, in order to create a panel of driver lines that initially target gene expression to the main tissues involved in parasite/vector interaction, namely the midgut, fat body and hemocytes. These driver lines will then become the references with which to examine parasite/vector interaction in the mosquito through tissue specific transgene expression and gene silencing. Initial experiments will screen alternative Gal4 transactivators and UAS:reporter constructs by transfection of A. gambiae cell lines. Functional transactivators with differential activity will then be assayed in transgenic A. gambiae using a gut-specific promoter and the most sensitive UAS:reporter. The most efficient Gal4 transactivator in transgenic mosquitoes will then be used to develop fat body specific and hemocyte specific driver lines. Using responder lines carrying TEP1 and dsRNA cactus constructs, we will then examine the effect on parasite development when TEP1 is differentially expressed or cactus is specifically silenced in fat body, hemocytes and midgut. The principles developed will be applicable to other hematophagous insects. In addition, the lines developed can be used to study other parasites carried by mosquitoes and further aspects of insect biology related to tissue specific metabolism, including behaviour, insecticide resistance, aging and fertility.