Developing a Gene Silencing Technology for Insect Vectors of Disease

The study of insect gene function provides a crucial step towards understanding physiology, behaviour, immunology and disease transmission processes in this very diverse and successful group of animals. Armed with this knowledge we can develop models to fight disease and strategies to control pest insect populations. Obtaining this knowledge, however, is not as simple as it could be. In this proposal we will develop a new technology to make the study of insect genes easier. The publication of the first complete genome sequence was a defining moment in history. However, simply knowing the sequence of a gene is not enough to explain its function. Luckily a powerful technique was found to interrogate gene function on an individual gene basis. This tool exploits an ancient cellular antiviral defence response: RNA interference (RNAi). When a cell encounters RNA in a double-stranded (ds) form (as in viral infections), it processes the RNA and uses it to 'switch off' or silence a specific gene in the host cell whose sequence complements that of the dsRNA. By artificially synthesising dsRNA with a known sequence and introducing it to target cells, it is possible to understand the role of a specific gene by observing the consequences of its loss of activity. RNAi and other so-called reverse genetics techniques are thus revolutionizing biological sciences. Many of the organisms selected for genome sequencing represent species that either inflict suffering (e.g. the mosquito Anopheles gambiae) or that provide spectacular models for human physiology and disease (e.g. the fruitfly Drosophila melanogaster). Indeed the dsRNA technique was rapidly adapted for use in the Anopheles mosquito, and it is the application of RNAi technologies in insects that provides the focus for our project. The dsRNA delivery method for insects is a little complicated. Insects are most commonly injected with, or occasionally fed, dsRNA. While the great majority of insects so far addressed are amenable to dsRNA-mediated RNAi, insects below a certain size suffer high mortality associated with injection injury and anaesthesia (and one must inject an awful lot of them), whereas large insects require expensive quantities of dsRNA to be synthesised. Other factors, such as the relatively short duration of the silencing effect (that may not suit long-lived insects) means that the technology in its current state is inappropriate for many insect species. If we are to make the most of emerging insect genetic information, RNAi methods must evolve to accommodate a wider variety of species. This project will develop a new RNAi technique that relies on the in vivo synthesis of dsRNA by transgenic symbiotic gut bacteria, and its ingestion by the insect host. The dsRNA will be directed against genes of the insect, leading to a knockdown effect that will reveal the role of the target gene. Our model insect will be Rhodnius prolixus; a large, long-lived blood-sucking bug that has evolved a symbiotic relationship with Rhodococcus rhodnii bacteria. Newly-hatched insects are free from symbiotic bacteria and must acquire them through ingestion of R. rhodnii - contaminated faeces from other insects. This means that dsRNA expressing bacteria have the potential to spread naturally through a colony of insects. The technique should reduce insect handling and associated mortality, and boost cost-efficiency. The hurdles we face are in ensuring adequate and stable transformation of the bacteria and the expression of dsRNA, the retention of their symbiotic characteristics and fitness, their ability to repopulate insects, and in maintaining the fitness of their insect hosts. We envisage that this new technique would not only improve reverse genetics studies in insects and widen the range of species that can be studied, but also that it may eventually form the basis of a novel and highly specific pest control strategy that will target genes essential to insect survival or reproduction.

Our aim is to develop a new gene-silencing (RNAi) technology that overcomes many of the practical problems currently faced by insect molecular biologists. We will take the laborious, expensive tasks of dsRNA synthesis and its delivery to the target insect out of the hands of human operators and delegate them to symbiotic gut bacteria. The remarkable ability of the RNAi effect to spread systemically in most insects means that it should be possible to genetically modify the symbiotic bacterium R. rhodniii to express dsRNA, reintroduce it by feeding into the gut of the insect R. prolixus, and obtain a body-wide knockdown of the target gene. Two well-described test genes with obvious phenotypes have been chosen: Vitellogenin 1 (a yolk protein transporter) and Nitrophorin-2 (which codes for a salivary anticoagulant). We will test bacterial conjugation as a method to introduce the dsRNA gene construct into R. rhodnii, and assess the stability of dsRNA expression and overall fitness of resulting transgenic bacteria. To avoid dsRNA degradation, we will generate mutant bacteria lacking RNase-III prior to expressing the dsRNA. By including a fluorescent marker gene (EGFP), we will follow the success of gut recolonization by these bacteria, principally via fluorescence microscopy, observing recolonization in both symbiont-free and normal insects. The stability, reproducibility and efficiency of the RNAi effect (gauged by real-time PCR, by in-situ visualization of gene transcription (FISH) and by phenotype analyses) will be compared with traditional dsRNA delivery. Finally, we will microscopically assess gut health and the fitness of the insects, and investigate whether it is possible to spread a stable knockdown effect though an insect population via coprophagic bacterial transmission. Our ultimate goal is to spin out this technology to any insect harbouring culturable symbionts, and further, to assess the applicability of the technique as a highly specific pest control agent.