Gene expression in male germline of pest insects

A major challenge for modern agriculture is to control pest species while minimising the adverse consequences to the environment. An attractive option is the Sterile Insect Technique (SIT) in which sterile insects are released to mate with wild pest insects [1]. Successful SIT programs have been conducted against the New World screwworm across the Americas and against Medfly worldwide. We have demonstrated that major improvements to this technique are possible through the use of genetically engineered insects [2]. Oxitec is developing this 'RIDL' technology to provide improved methods for the control of insects of agricultural and medical importance. This is clearly dependent on the ability to construct suitable genetically modified (GM) insects. Oxitec's present strains are based on expression in somatic cells, but more sophisticated manipulation of fertility and reproduction will clearly require, or at least be facilitated by, germline expression of novel sequences. Presently our ability to do this in pest insects is extremely limited. The aim of this project is to investigate conservation and expression of germline-specific genes, particularly in males, and use this new basic knowledge to build advanced expression systems for both basic and applied purposes. For many purposes we need regulated, i.e. conditional, gene expression. The GAL4-UAS bipartite expression system is an extremely powerful and flexible method for investigating and analysing gene expression in Drosophila. We have used the tTA-tetO ('tet-off') system, which, while similar has the additional advantage of being repressed by tetracycline (provided in the diet). The only promoter known to work in the male germline for ectopic expression in multiple insect species is that of beta2-tubulin, a testis-specific beta-tubulin. Constructs using a beta2-tubulin promoter fragment driving expression of a fluorescent reporter have been shown by ourselves and others to give fluorescent sperm in Drosophila, Medfly and Aedes and Anopheles mosquitoes, however neither beta2tubulin-GAL4 and beta2tubulin-tTA work for expression of ectopic genes in testes (our unpublished data, in several species). In brief, the GAL4 or tTA protein is expressed too late - giving no time for activation of target genes before the entire transcription machinery shuts down at the onset of the meiotic divisions. Expression in the Drosophila male germline can be accomplished using bam-GAL4; bam expresses much earlier in spermatogenesis than beta2-tubulin. However, bam is a poorly conserved gene whose homologs are not easily recognised in other species. The solution is clear: we need a better knowledge of gene expression during spermatogenesis, particularly of early genes. Studying this across a range of species will also reveal interesting data on conservation, or otherwise, of both regulatory and coding sequence. As well as being an important area of basic science, this is of practical interest to Oxitec; we would very much like to understand cross-species function so that we can build single constructs that will work across a range of species. The student will investigate germline expression of homologs of early acting Drosophila genes, in several species. We will begin with Aedes aegypti, as this species (i) has been sequenced (ii) is of commercial interest to Oxitec and (iii) has particularly amenable testis structure, where the cysts of progressively older cells are stacked in order, so the timing of gene expression is particularly easy to visualise. Within the timescale of this studentship, genome sequence will become available for some other major agricultural pests, e.g. Medfly, and one or more Lepidopteran, and we will therefore extend our studies into these species as the project progresses. [1] Sterile Insect Technique Dyck, V.A. et al. Eds.; Springer, 2005. [2] Thomas, D.D.; Donnelly, C.A.; Wood, R.J.; Alphey, L.S. Science 2000, 287, 2474-2476