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

点击翻译按钮获取中文摘要

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.