Development and risk assessment of transgenic environmentally-friendly insect pest control methods for fruit flies and mosquitoes

各种害虫每年对农业造成重大损害，或将致命疾病传播给动物和人类。控制害虫种群的一个成功战略是基于不育昆虫技术，该技术利用释放大量饲养的、辐射绝育的雄性昆虫来引起不育交配，从而降低害虫种群水平。然而，辐射并不适用于每一种昆虫。因此，基于害虫遗传修饰的新策略已经开发或目前正在调查中，所提出的研究的目标是改善为增强生物控制程序而创建的基因工程（GE）昆虫的发展和生态安全性，包括SIT和基于条件致死的新策略。转基因昆虫释放计划的一个主要问题是转基因稳定性和维持其一致表达。转基因丢失或基因组内移动可能导致菌株属性的丧失，并可能最终导致种间移动，从而导致生态风险。为了解决潜在的转基因不稳定性，将在地中海、墨西哥和东方果蝇实蝇害虫物种中测试允许整合后固定的新转座子载体。此外，该系统还将在登革热和疟疾的携带者--伊蚊和按蚊这两种蚊子物种中建立。由于基因组位置效应抑制转基因表达，以及插入突变对宿主适应性和生存力产生负面影响，随机基因组插入对于GE菌株的开发也是有问题的。转基因表达减少可能导致条件致死个体的意外存活，或无法识别它们。为了将转基因载体靶向到对基因表达和宿主适应性具有最小负面影响的限定基因组插入位点，将开发重组酶介导的盒交换（RMCE）策略，其RMCE还将使靶点稳定，将在实蝇和蚊子物种中进行测试，并将有助于开发稳定的靶点菌株，用于有条件的致死生物防治。这将包括基于RNAi的致死性方法的分子和生物学评价。在所提出的昆虫中基于RNAi机制的致死性将增加物种特异性，并且相对于现有系统中的一个靶标具有多个致死性靶标。通过寻求提高转基因表达率和基于转座子的载体系统的稳定性，该提案通过减少田间释放后的非预期传播以及通过限制转基因渐渗的可能性来具体解决与新的转基因昆虫相关的问题。

Various species of pest insects cause substantial damage to agriculture every year, or transmit deadly diseases to animals and humans. A successful strategy to control pest insect populations is based on the Sterile Insect Technique (SIT), which uses the release of mass-reared, radiation sterilized male insects to cause infertile matings and thus reduce the pest population level. However, irradiation is not applicable to every insect species. Thus, new strategies based on genetic modifications of pest insects have been developed or are currently under investigation.The goal of the proposed research is to improve the development and ecological safety of genetically engineered (GE) insects created for enhanced biological control programs, including the SIT and new strategies based on conditional lethality. A major concern for GE insect release programs is transgene stability, and maintenance of their consistent expression. Transgene loss or intra-genomic movement could result in loss of strain attributes, and may ultimately lead to interspecies movement resulting in ecological risks. To address potential transgene instability, a new transposon vector that allows post-integration immobilization will be tested in the Mediterranean, Mexican and Oriental fruit fly tephritid pest species. In addition, the system will be established in the mosquito species Aedes and Anopheles – carriers of dengue and malaria.Random genomic insertion is also problematic for GE strain development due to genomic position effects that suppress transgene expression, and insertional mutations that negatively affect host fitness and viability. Diminished transgene expression could result in the unintended survival of conditional lethal individuals, or the inability to identify them. To target transgene vectors to defined genomic insertion sites having minimal negative effects on gene expression and host fitness, a recombinase-mediated cassette exchange (RMCE) strategy will be developed that. RMCE will also allow for stabilization of the target site, will be tested in tephritid and mosquito species, and will aid to the development of stabilized target-site strains for conditional lethal biocontrol. This will include a molecular and organismal evaluation of an RNAi-based lethality approach. Lethality based on an RNAi mechanism in the proposed insects would increase the species specificity and having multiple targets for lethality versus one target in existing systems. By seeking to improve transgene expressivity and stabilization of transposon-based vector systems, this proposal specifically addresses issues related to new GE insects by reducing their unintended spread after field release, and by limiting the possibilities for transgene introgression.