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Title: Genomics of sex determination in the mosquito Aedes aegypti
Author: Turner, Joe
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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The mosquito Aedes aegypti is the principal vector of dengue, chikungunya and Zika arboviruses, all of which are growing public health concerns globally. Emerging technologies aim to control mosquito populations and limit disease transmission by releasing genetically modified mosquitoes, which could be improved by reliably manipulating sex determination. This relies on a strong understanding of the genetic basis of sex determination; however, little is known about this process in Ae. aegypti except that the male determining factor is located within a non-recombining, Y chromosome-like region on one chromosome called the M locus. In this thesis, I present experiments aimed at unravelling the nature of the Ae. aegypti M locus and analysing their impact on the evolution of mosquito sex chromosomes, with the intention of using the M locus as a target for sex-specific genetic modification. Initially, I attempt integration of a fluorescence gene in male mosquitoes by targeting male-biased genomic sequences thought to be within or linked to the M locus with the RNA-guided endonuclease system CRISPR/Cas9. This involved the establishment of a laboratory mosquito line expressing the Cas9 endonuclease in the germline to facilitate effective transgenesis. During this time, an M locus gene that acts as the sex determination switch, Nix , was discovered by other researchers. I designed experiments to further understand the nature of this gene by sequencing the region of the M locus in which it is situated. This resolved the gene structure of Nix , concluding that its intron is approximately 99 kb, which makes it one of the largest genes in the Ae. aegypti genome. The intron is enriched for repetitive DNA, suggesting that the inability of the M locus to recombine has led to the accumulation of transposable elements, in accordance with canonical models of sex chromosome evolution. These findings were later expanded on, and I describe my role in analysing an improved de novo genome assembly for Ae. aegypti , determining nearly the full sequence of a 1.5 Mb M locus region, and mapping it physically to the chromosome. In addition, I undertook further research to examine the differential sex-biased coverage, structural variation, and repeat content of the M locus and the rest of the genome. I found that male-biased sequence and transposable elements have accumulated on the wider M chromosome, which may be indicative of it transitioning to a fully male-limited Y chromosome. This analysis illustrated the importance of high quality genomics data for studying mosquito sex determination, and could help to improve sex-specific targeting of genetic vector control strategies in the future.
Supervisor: Darby, Alistair ; Betancourt, Andrea Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral