Genetic modification of the homomorphic sex chromosomes in the yellow fever mosquito Aedes aegypti provides novel insights and tools for biocontrol

Abstract

Aedes aegypti is a highly adaptable and invasive species largely responsible for the spread of Zika, yellow fever, chikungunya, and dengue viruses. Current methods of preventing the spread of arboviral diseases rely primarily on population control of the mosquito vector. These practices are insufficient as millions of arboviral infections still occur each year. Genetic biocontrol, which includes the mass release of sterile, incompatible, or modified males, is a promising approach. However, to prevent the release of females that can potentially transmit pathogens, efficient and accurate sex separation of the non-biting males from the biting females is essential. Genetic sexing strains often rely on marker genes linked to the sex chromosomes. Sex linkage is more challenging in Ae. aegypti than many other species due to the homomorphic nature of their sex chromosomes. Homomorphic sex chromosomes are largely undifferentiated from each other and contain only small non-recombining sex-specific regions. In Ae. aegypti, the sex specific regions include either the dominant male determining locus (M-locus) or its counterpart, the m-locus. Further understanding the sex loci and the basic biology of sex determination can facilitate improved sex separation strategies. Recent developments surrounding the sex determination pathway of Ae. aegypti and other mosquito species is reviewed in this thesis. To provide new insights, we knocked out the male determining factor in the RED strain of Ae. aegypti and we were able to obtain both males and females with two M chromosomes. We found that the m-locus is not required for either male or female survival, however it may impact the fitness and fertility in females. Additional studies to further characterize Ae. aegypti homomorphic sex chromosomes has also uncovered the presence of recessive lethal alleles (RLAs) near the M-locus. We have developed lines that leverage these recessive alleles as well as regions of suppressed recombination to introduce chromosomal incompatibility, termed the Differential Elimination of MARKed sex chromosomes (DeMARK). In some DeMARK lines, we link fluorescent reporters to naturally occurring or engineered RLAs to ensure males never inherit the transgene and females always inherit it. This concept can be used on its own as a means of sex separation, or it can be combined with conditional gene expression as a means of sex-specific selection. We review existing methods of conditional expression that can be used for this purpose, and present a novel method of conditional selection in Ae. aegypti. By knocking out an essential metabolic gene, sugarlethal (sgll), we have created an auxotrophic phenotype that can be rescued by dietary supplementation of activated vitamin B6. Further, we have established a male-specific rescue for this phenotype that can be used for conditional sex separation in various ways. Overall, we present multiple contributions to the genetic tools, strains, and basic knowledge to facilitate the population control of the yellow fever mosquito, Ae. aegypti.