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

各种害虫每年都会对农业造成重大破坏，或将致命疾病传播给动物和人类。一种成功的控制害虫种群的策略是基于不育昆虫技术(SIT)，该技术利用大量饲养的、辐射不育的雄性昆虫的释放来导致不育交配，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正在研究中。拟议研究的目标是改善为加强生物防治计划而创建的基因工程(GE)昆虫的发育和生态安全性，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的稳定性，并保持其一致的表达。转基因丢失或基因组内移动可能导致菌株属性的丧失，并最终可能导致物种间的移动，从而导致生态风险。为了解决潜在的转基因不稳定性，一种新的转座子载体将在地中海、墨西哥和东方果蝇Tephritid害虫物种中进行测试，该载体允许整合后固定化。此外，该系统将在登革热和疟疾携带者伊蚊和按蚊物种中建立。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合性和生存能力产生负面影响，随机插入基因组对GE菌株的发展也是有问题的。转基因表达减弱可能导致条件性致死个体意外存活，或者无法识别他们。为了将转基因载体定向到对基因表达和宿主适合度具有最小负面影响的特定基因组插入位置，将开发一种重组酶介导盒交换(RMCE)策略。RMCE还将允许稳定目标地点，将在粉虱和蚊子物种中进行测试，并将帮助开发稳定的目标地点菌株，用于有条件的致命生物防治。这将包括对基于RNAi的致命性方法的分子和组织评估。在拟议的昆虫中，基于RNAi机制的致命性将增加物种特异性，与现有系统中的一个目标相比，具有多个致命性目标。通过寻求提高基于转座子的载体系统的转基因表达能力和稳定性，该建议专门解决了与新的转基因昆虫相关的问题，减少了它们在田间释放后的意外传播，并限制了转基因导入的可能性。

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.