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

地中海实蝇（Ceratitis capitata）和亚洲虎蚊（Aedes albopictus）是全球最具侵略性和侵略性的害虫物种之一，对欧洲和中东构成重大威胁。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其有效性，这就是为什么迫切需要新的和可持续的控制策略。最有前途的方法之一是遗传控制（GC），这是一种生物控制形式，具有物种特异性，自分散性和环境友好性。GC的一个众所周知的例子是不育昆虫技术（SIT），其中不育的雄性被释放到目标种群中，导致不育交配和种群规模的减少。几十年来，SIT已经成功地用于防治像C。capitata，并启发了几个类似的研究项目，以控制流行病学上重要的蚊子物种，包括Ae。白纹伊蚊所有GC策略都希望只释放雄性，因为释放的雌性可能会增加疾病传播或作物损害。然而，一种能够大规模快速、有效和可靠地进行性别分离的技术还没有应用于大多数物种，这阻碍了从成功的试点计划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，遗传性别鉴定菌株（GSS）可能是最可扩展和可靠的方法。GSS携带常染色体上的选择性隐性基因中的突变和雄性特异性染色体位置上的基因的功能拷贝。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是地中海果蝇的维也纳8菌株，其携带两个偶联的选择标记。雌虫是温度敏感致死性的，提高饲养温度可以有效地消除雌虫。此外，雌性蛹是白色（wp）而不是野生型棕色。这种结构代表了一个完美的性别鉴定系统，但目前对tsl突变的遗传或机制的理解还不清楚。拟议项目的目标是确定地中海果蝇tsl表型的分子基础（目标1），并建立编辑重要昆虫害虫物种C的Y染色体和M基因座上的性别特异性区域的工具和基础知识。capitata（目标2）和Ae.白纹伊蚊（目标3）。研究结果将使未来对入侵农业害虫和蚊子的可持续控制得以发展。

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.