Transposon-based forward and reverse genetics in Anopheles mosquitoes. David A. O'Brochta^1,2, Kristina L. Pilitt¹, Robert A. Harrell, II¹, Channa Aluvihare¹, Robert T. Alford¹. 1) Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, MD; 2) Department of Entomology, University of Maryland, College Park, MD.

   The growing abundance of insect genome sequence data is creating a large demand for functional genomics tools, methods and technologies that can be used in these non-model systems. Transposon-based technologies such as gene- and enhancer-traps are particularly powerful functional genomics technologies and the purpose of this study was to develop these technologies for Anopheles stephensi mosquitoes, which are important vectors of human malaria. The mobility properties of integrated piggyBac elements in An. stephensi were tested by crossing piggyBac-containing lines with piggyBac transposase-expressing jumpstarter lines. piggyBac was found to be efficiently remobilized and conducive to being used to create gene- and enhancer-trap systems. A Gal4-based enhancer-trap system was created consisting of six transgenic lines of Anopheles stephensi, each with a single piggyBac-Gal4 element in a unique genomic location, six lines with a single piggyBac-UAStdTomato element and two lines, each with a single Minos transposable element containing the piggyBac-transposase gene under the regulatory control the hsp70 promoter from Drosophila melanogaster. From five genetic screens for larval- and adult-specific enhancers 314 progeny were recovered from 24,250 total progeny (1.3%) with unique patterns of tdTomato expression arising from the influence of an enhancer. The frequency of piggyBac remobilization and enhancer detection was 2.5-3 fold higher in female germ-lines compared to male germ-lines. A small collection of enhancer-trap lines are described in which Gal4 expression occurred in adult female salivary glands, midgut and fat body, either singly or in combination. These three tissues play critical roles during the infection of Anopheles stephensi by malaria-causing Plasmodium parasites. This system and the lines generated using it will be valuable resources to ongoing mosquito functional genomics efforts.