Developing transgene-free CRISPR-based genetic sexing systems for insect pest control

地中海果蝇、头角杆菌和亚洲虎蚊、白纹伊蚊是全球最具侵入性和侵略性的害虫，对欧洲和中东地区构成重大威胁。杀虫剂作为主要的控制策略，由于耐药性的发展正在迅速失去效力，这就是为什么迫切需要新的和可持续的控制策略。最有希望的方法之一是遗传控制（GC），这是一种物种特异性、自我分散和环境友好的生物控制形式。一个著名的例子是昆虫不育技术（sterile insect technique， SIT），将绝育的雄虫释放到目标种群中，造成不育交配，减少种群规模。几十年来，SIT已经成功地用于对抗像C. capitata这样的害虫，并激发了几个类似的研究项目来控制流行病学上重要的蚊子物种，包括伊蚊。蚊。所有GC策略都希望只释放雄性，因为释放雌性可能会增加疾病传播或作物损害。然而，对于大多数物种来说，实现大规模快速、高效和可靠的性别分离的技术还不具备，这阻碍了从成功的试点项目向全面的大规模控制措施的过渡。在所有可能的性别分离解决方案中，遗传性别菌株（GSS）可能是最可扩展和最可靠的方法。GSS在常染色体上携带一个可选择的隐性基因突变，并在男性特异性染色体上携带该基因的功能拷贝。因此，雄性都是野生型，而雌性都是突变型。迄今为止最成功的GSS是地中海果蝇的VIENNA 8株，它携带两个偶联的可选择标记。雌性对温度敏感致死（tsl），可以通过提高饲养温度来有效和经济地消灭。此外，雌性蛹为白色（wp），而不是野生型的棕色。这种结构代表了一个完美的性别系统，但对tsl突变的遗传或机制理解目前尚不清楚。本项目的目标是确定medfly tsl表型的分子基础（目标1），并建立编辑重要害虫物种C. capitata（目标2）和Ae. Y染色体上的性别特异性区域和M位点的工具和基础知识。白纹伊蚊（目标3）。研究结果将为今后开发可持续防治农业害虫和蚊子的方法奠定基础。

The Mediterranean fruit fly, Ceratitis capitata, and the Asian tiger mosquito, Aedes albopictus, are among the most invasive and aggressive insect pest species globally and are significant threats to Europe and the Middle East. Insecticides, as the primary control strategy, are rapidly losing their effectiveness due to the development of resistance, which is why new and sustainable control strategies are urgently needed. One of the most promising methods is genetic control (GC), a form of biological control that is species-specific, self-dispersing, and environmentally friendly. A well-known example of GC is the sterile insect technique (SIT), in which sterilized males are released into the target population to cause infertile mating and a reduction of the population size. The SIT has been successfully used for decades to combat insect pests like the C. capitata and inspired several similar research projects to control epidemiologically important mosquito species, including Ae. albopictus. All GC strategies want to release only males, as released females could increase disease transmission or crop damage. However, a technology that enables fast, efficient, and reliable sex separation on a large scale is not yet available for most species and prevents the transition from successful pilot programs to comprehensive large-scale control measures. Among all possible solutions for sex separation, genetic sexing strains (GSS) are probably the most scalable and reliable approach. GSS carry a mutation in a selectable recessive gene on an autosome and a functional copy of the gene on a male-specific chromosome location. As a result, males are all wild type, while females are mutant. The most successful GSS to date is the VIENNA 8 strain of the Mediterranean fruit fly, which carries two coupled, selectable markers. The females are temperature-sensitive lethal (tsl) and can be eliminated efficiently and cost-effectively by increasing the rearing temperature. In addition, female pupae are white (wp) instead of wild-type brown. This architecture represents a perfect sexing system, but a genetic or mechanistic understanding of the tsl mutation is currently unknown. The goals of the proposed project are to identify the molecular basis for the medfly tsl phenotype (objective 1) and to establish tools and basic knowledge for editing sex-specific regions on the Y chromosome and the M locus in the important insect pest species C. capitata (objective 2) and Ae. albopictus (objective 3), respectively. The results will enable the development of sustainable control against invasive agricultural pest insects and mosquitoes in the future.