Mosquitoes belonging to the genera Anopheles transmit malaria parasites, attributing the highest mortality of any vector-borne disease worldwide. Mosquitoes belonging to the genera Aedes transmit arboviruses including dengue, which has become the most important vector-borne virus due to a drastic surge in disease incidence. The scope of the studies in this dissertation is broad, with investigations bringing together elements of classical genetics, recent advances in sequencing and genome-editing technologies, and the use of modern forward genetics approaches. Chapter 2 of this dissertation explores the use of the Oxford Nanopore Sequencing Technology for the first time in mosquitoes. This new technology provides long reads that were used to piece together the AabS3 chromosomal assembly for Anopheles albimanus. The utility of this genomic resource is demonstrated by the discovery of novel telomeric repeats at the ends of the chromosomes that could have important implications in mosquito biology and control. Chapter 3 describes a forward genetics strategy called 'Marker-Assisted Mapping' (MAM) that enables high-resolution mapping of the causal gene locus of a mutant phenotype. The principle and effectiveness of MAM is first demonstrated by mapping a known transgene insertion. MAM is then used to identify cardinal as a candidate causal gene for the spontaneous red-eye (re) mutation. Genetic crosses between the re mutant and cardinal knocking out individuals generated using CRISPR/Cas9 confirmed that cardinal indeed is the causal gene for re mutation. Chapter 4 explores three innovative strategies for mosquito sex separation by exploiting several sex-linked marker lines. We show that by linking a transgenic marker to the male-determining locus (M locus), or by linking the male-determining Nix gene to a marker, males can be precisely separated from females. We also produce a two-marker transgenic line that allows for both non-transgenic male separation and for efficient line maintenance. Finally, we discuss further applications of the resources generated and future directions stemming from these findings. Altogether, the studies described in this dissertation contribute to the overall goal of understanding mosquito biology and of controlling mosquito-borne infectious diseases. / Doctor of Philosophy / Female mosquitoes bite and transmit deadly pathogens including the malaria parasite, and viruses such as dengue, Zika, and West Nile. Control programs that attempt to limit the spread of these deadly diseases rely on the control of mosquitoes themselves. These mosquito control methods have relied heavily on indoor and outdoor insecticidal spraying. However, the efficacy of these methods has been jeopardized by the increasing prevalence of insecticide resistance. Thus, it is necessary to implement other methods for effective mosquito control. Genetic control strategies such as the Sterile Insect Technique (SIT) and Wolbachia-based Incompatible Insect Technique (IIT) are excellent solutions to overcome the limitations of current control strategies. As female mosquitoes bite and transmit disease-causing pathogens, only males are released, which necessitate the separation of the non-biting males from females before release.
The aim of this work was to use recent technological advancements to better understand the genome and basic genetics of vector mosquito species, and to identify possible approaches to improve current sex separation practices. To develop a deep understanding of mosquito biology and genetics, it is crucial that a high-quality and accurate genome assembly is available. However, many mosquito genome assemblies remain fragmented. To address this limitation, we used recent advances in sequencing technologies to produce a high-quality genome assembly for the New World malaria mosquito, Anopheles albimanus. These sequencing and assembly efforts led to the discovery of novel telomere sequences at the ends of chromosomes, which could have implications for mosquito control.
Forward genetics, which identifies the gene(s) responsible for a given phenotype, has been hindered by the low recombination rate in the yellow and dengue fever mosquito, Aedes aegypti. We develop a Marker-Assisted Mapping (MAM) strategy to address this problem. We first demonstrate this method by mapping the known insertion of a transgene. MAM is then used to identify cardinal as a candidate causal gene for the spontaneous red-eye (re) mutation. MAM identification of the Cardinal gene was then verified by knocking out Cardinal, which represents the first successful gene mapping in Aedes aegypti using forward genetics. The MAM strategy has broad implications as it could enable the discovery of genes involved in important traits such as insecticide resistance.
To improve sex separation methods, we took advantage of several sex-linked transgenic lines to develop three novel strategies. First, we demonstrate that screening for a genetic marker that is tightly linked to the male-determining locus (M locus) is an effective approach to reduce female contamination. Second, we demonstrate that instead of linking a marker to the M locus, we can link the male-determining factor, Nix, to a genetic marker. When a Nix transgene is located adjacent to the red-eye locus with extremely tight linkage, the red-eye phenotype becomes a faithful marker for separation of males and females. Finally, we developed a two-marker genetic sexing strain that produces non-transgenic males that could be used for release, and transgenic marked males and females for efficient line maintenance.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/114585 |
| Date | 26 October 2021 |
| Creators | Compton, Austin |
| Contributors | Biochemistry, Tu, Zhijian, Slade, Daniel Joseph, Gillaspy, Glenda E., Zhu, Jinsong |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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