Profiling gene expression in spermatogenesis in model and pest insects

现代农业面临的一个主要挑战是控制害虫种类，同时将对环境的不利影响降至最低。虫害会造成重大的经济损失，要么会吞噬作物，要么会降低作物的质量，使作物无法销售。昆虫也是疾病的媒介，传播人类和动物的病原体以及植物疾病。昆虫不育技术(SIT)是控制害虫种群的一种有效、物种特异性和环境友好的方法。SIT涉及在大范围内释放数百万不育昆虫，以与现有的本地昆虫交配。与不育的雄性交配的本地雌性会产生不能存活的后代，导致目标害虫种群的下降。在传统的SIT中，昆虫是通过辐射灭菌的，这降低了释放的昆虫的竞争力，而且破坏性太大，不适用于许多昆虫物种。该研究项目的工业合作伙伴Oxitec已经证明，通过使用转基因昆虫可以实现重大改进。转基因昆虫带有一个容易识别的标记(因此它们可以与野生动物区分开来)，很容易被区分为雄性和雌性，除非(在工厂里)喂食膳食补充剂，否则会产生不能存活的后代。理想情况下，被释放的果蝇应该是天生不育的。此外，理想情况下，工厂化饲养的放生雄性的精子将被标记，这样就可以很容易地辨别出是否有特定的野生雌性与野生雄性或放生的雄性交配。果蝇是一种无害的昆虫，上个世纪以来一直是遗传学研究的支柱。果蝇作为模式生物的研究为人类疾病生物学的研究以及对害虫生物学的理解提供了信息。在果蝇中，我们可以在几乎任何细胞中表达我们喜欢的任何基因，除了某些精子发生阶段。果蝇精子发生的基因功能分析一直受到阻碍，因为我们在这个过程中缺乏随意表达基因的能力。许多基因在精子产生过程中表达，虽然在确定它们的功能方面已经取得了重大进展，但更复杂的遗传操作技术将极大地促进进一步的功能分析。在这个项目中，我们将研究模式昆虫黑腹果蝇和两种害虫--头状蜡虫(地中海果蝇)和埃及伊蚊(黄热病蚊子)--精子形成不同阶段的基因表达模式。我们将解剖睾丸，并确定这三个物种在精子产生过程的不同阶段的基因表达谱。来自果蝇的数据将扩展我们目前的理解，提供更多阶段特定的信息。对于害虫物种，我们的项目将是首次对精子发生过程中的基因表达进行基因组规模分析。在项目结束时，我们将生成这三个物种在精子发生过程中的基因表达水平的概况。我们将定义许多睾丸表达基因的精确转录结构，因此我们的数据将为所有三个物种的基因组注释提供信息。我们将选择一些睾丸表达的基因进行进一步的验证，并将产生一系列昆虫，使我们能够在睾丸细胞中特定地表达我们选择的任何基因。这将促进学术实验室(以及世界各地的其他果蝇实验室)的基础研究，以及工业伙伴在开发不育雄性昆虫以控制种群策略方面的应用研究。

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.