Profiling gene expression in spermatogenesis in model and pest insects

A major challenge for modern agriculture is to control pest species while minimising the adverse consequences to the environment. Insect pests cause significant economic damage, either by eating the crop or reducing quality such that the crop is not marketable. Insects also act as disease vectors, transmitting human and animal pathogens as well as plant diseases. The Sterile Insect Technique (SIT) is an effective, species-specific and environmentally friendly method for controlling pest populations. SIT involves releasing millions of sterile insects over a wide area to mate with the native insects that are present. Native females that mate with the sterile males produce non viable offspring, leading to a decline in the target pest population. In classical SIT, insects are sterilised with irradiation which reduces the competitiveness of the released insects and is too damaging to be applied to many insect species. Oxitec, the industrial partner for this research project, has demonstrated that major improvements are possible through the use of genetically engineered insects. The genetically engineered insects carry an easily identifiable marker (so they can be distinguished from wild animals), can be readily sorted into males vs females and produce inviable offspring unless fed a dietary supplement (in the factory). Ideally the released flies would be inherently male sterile. Also ideally the sperm from factory-reared released males would be marked so it would be possible to easily discern whether any particular wild female had mated with a wild male or a released male. The fruit fly Drosophila melanogaster is a harmless insect that has been a mainstay of genetics research for the last century. Research on Drosophila as a model organism has informed research in human disease biology as well as in understanding the biology of pest insects. In Drosophila we can express any gene we like in almost any cell, with the notable exception of certain spermatogenesis stages. Analysis of gene function in Drosophila spermatogenesis has been hampered by our lack of ability to express genes at will during this process. Many genes are expressed during sperm production, and, while significant progress has been made in determining their functions, more sophisticated genetic manipulation techniques will greatly facilitate further functional analyses. In this project we will investigate gene expression patterns in various stages of sperm formation in the model insect Drosophila melanogaster and in two insect pests, Ceratitis capitata (Mediterranean fruit fly) and Aedes aegypti (Yellow fever mosquito). We will dissect the testes and determine gene expression profiles at various stages of the sperm production process for all three species. The data from Drosophila will extend our current understanding to give much more stage specific information. For the pest insect species our project will be first genome scale analysis of gene expression in spermatogenesis. At the end of the project we will have generated profiles of gene expression levels during spermatogenesis in the three species. We will have defined the precise transcript structures of many testis-expressed genes, thus our data will inform genome annotations for all three species. We will select some testis-expressed genes for further validation, and will generate lines of insects that allow us to express any gene of our choice specifically in the cells of the testes. This will facilitate both the basic research of the academic lab (and other Drosophila labs worldwide) and the applied research of the industrial partner in developing sterile male insects for population control strategies.

Drosophila melanogaster is well known as a model organism for research into basic cellular genetic processes; this has informed analysis of human diseases, including neural degeneration, cancer etc. Drosophila, as a harmless insect, also provides an excellent model and basic research platform for investigating economically damaging pest insects. Oxitec is using methods developed in Drosophila to generate genetically modified pest insects for use in 'Release of Insects carrying a Dominant Lethal' (RIDL), a modification of the Sterile Insect Technique. This project is directed towards identifying gene expression patterns in spermatogenesis for basic Drosophila research, and to improve RIDL. We will determine spermatogenic gene expression profiles in Drosophila and two pest insects (C. capitata and A. aegypti). RNA-seq of dissected testis samples will reveal which genes are expressed by male germline cells at early, mid and late spermatogenic stages, adding temporal resolution to help us understand gene function during spermatogenesis. Very little temporally-delineated data exists for spermatogenic stages in Drosophila, and no data at all yet exist for testis-specific gene expression in these pest insects. Combining data from three insects span the dipteran lineage will allow us to investigate evolutionary conservation of testis gene expression. We will identify transcriptional start sites, and splice junctions, in testes, of testis specific genes, and select a few genes for further validation. Eventually we will produce a set of transgenic insects to allow regulated ectopic expression at particular stages, specifically in the male germline. This will facilitate further Drosophila research by giving us more flexible tools for investigating gene function in the testis. It will facilitate RIDL product development for the industrial partner by allowing them to engineer repressible male sterility into medfly and mosquitoes, and hopefully other pest insects.

This LINK Project will generate profiles of gene expression levels during spermatogenesis in three species; the model insect Drosophila melanogaster and in two insect pests, Ceratitis capitata (Mediterranean fruit fly) and Aedes aegypti (Yellow fever mosquito). The data from Drosophila will extend our current understanding to give much more stage specific information and this will be the first genome scale analysis of gene expression in spermatogenesis in a pest insect. The data generated will inform genome annotations for all three species. Studying expression across a range of dipteran species will reveal interesting data on the conservation or divergence of gene expression patterns during evolution. The advances in understanding of gene expression in spermatogenesis will facilitate both the basic research of the academic lab, other Drosophila labs worldwide, and applied labs developing sterile male insects for population control strategies. The industry partner, Oxitec, will benefit from the data and experience of the Project to improve the range of transgenic insect product profiles that it can offer for the suppression of insect pests. Oxitec's suite of genetic technologies, RIDL, will make sterile insect technique (SIT) more cost-effective and applicable to a wider range of pests. RIDL is in development and could be commercialised in the next 2-3 years, bringing revenues and employment into the UK life science sector. This Project could add tools to the product offer, potentially strengthening its efficacy and range of application, which would be realised in 5-10 years. Oxitec is developing products for agriculture and public health. Potential beneficiaries of the Project are its collaborators including the US Department of Agriculture (USDA), the Ministry of Health of Malaysia and a multinational agribusiness. RIDL is being developed to control the Aedes mosquitoes that spread dengue and chikungunya, diseases which are not well controlled by insecticides and for which there are no vaccines or specific medications. Potential indirect beneficiaries are the populations at risk of dengue (2.5 billion people) as well as the national and local government agencies in disease-endemic countries such as Malaysia, India and Singapore responsible for protecting them. In the global agricultural economy, producers, traders and consumers will benefit from greater availability of the clean and sustainable control method SIT and the ability to export pest-free produce. Oxitec is working with the USDA to supply technology into fruit fly and cotton pest control, not only in the USA but also in Guatemala and Mexico where SIT facilities are located. Other SIT programmes exist to protect crops in countries as diverse as Brazil, Argentina, Spain, Israel, Japan and Australia which could all benefit. The environment will benefit directly from the development of new, clean, species-specific methods of controlling key pest species, which will allow the eventual reduction in chemical usage and a move to more sustainable agricultural systems. The USDA recently completed an Environmental Impact Statement on the use of equivalent technology in some agricultural pests and concluded (Record of Decision, May 2009) 'that the use of such technology was not merely acceptable, but in fact was the environmentally preferred alternative' (http://www.aphis.usda.gov/plant_health/ea/geneng.shtml). UK scientists will benefit directly and indirectly from the knowledge-generating and knowledge-sharing activities that are integral to the Collaboration. The Collaboration will be managed to ensure exchange of sophisticated technical skills and knowledge between the academic and private sectors by networking activities, meetings and short term exchanges. The SME will gain from the specialist science in the University sector while academic researchers will be aware of the market needs and drivers in the field of application of their technology.