Browsing by Author "Compton, Austin"

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    The Beginning of the End: A Chromosomal Assembly of the New World Malaria Mosquito Ends with a Novel Telomere
    Compton, Austin; Liang, Jiangtao; Chen, Chujia; Lukyanchikova, Varvara; Qi, Yumin; Potters, Mark B.; Settlage, Robert; Miller, Dustin; Deschamps, Stephane; Mao, Chunhong; Llaca, Victor; Sharakhov, Igor V.; Tu, Zhijian Jake (Genetics Society of America, 2020-10-01)
    Chromosome level assemblies are accumulating in various taxonomic groups including mosquitoes. However, even in the few reference-quality mosquito assemblies, a significant portion of the heterochromatic regions including telomeres remain unresolved. Here we produce a de novo assembly of the New World malaria mosquito, Anopheles albimanus by integrating Oxford Nanopore sequencing, Illumina, Hi-C and optical mapping. This 172.6 Mbps female assembly, which we call AalbS3, is obtained by scaffolding polished large contigs (contig N50 = 13.7 Mbps) into three chromosomes. All chromosome arms end with telomeric repeats, which is the first in mosquito assemblies and represents a significant step toward the completion of a genome assembly. These telomeres consist of tandem repeats of a novel 30-32 bp Telomeric Repeat Unit (TRU) and are confirmed by analyzing the termini of long reads and through both chromosomal in situ hybridization and a Bal31 sensitivity assay. The AalbS3 assembly included previously uncharacterized centromeric and rDNA clusters and more than doubled the content of transposable elements and other repetitive sequences. This telomere-to-telomere assembly, although still containing gaps, represents a significant step toward resolving biologically important but previously hidden genomic components. The comparison of different scaffolding methods will also inform future efforts to obtain reference-quality genomes for other mosquito species.
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    Evaluation of ebony as a potential selectable marker for genetic sexing in Aedes aegypti
    Nikolouli, Katerina; Compton, Austin; Tu, Zhijian J.; Bourtzis, Kostas (2025-02-25)
    Background: Aedes aegypti is expected to invade previously unoccupied areas, mainly due to the climate change, the increase in travel and trade activities and the continuous transformation of the rural environment into urban areas. The sterile insect technique (SIT), which relies on the mass production and release of sterile males, is an environmentally friendly approach that can be applied for population control of Ae. aegypti. SIT programs can be greatly benefited by a genetic sexing strain (GSS) and a reliable sex sorting system to minimize any accidental female release. Visually detectable or conditionally lethal selectable markers can be used for the development of new GSSs. In this study, we evaluated the suitability and competence of a mutant Ae. aegypti ebony strain for the development of a new GSS. The ebony gene is known to be involved in the pigmentation pathway of several dipteran insects, including Ae. aegypti. Methods: An ebony gene knockout was developed though CRISPR/Cas9 mutagenesis. G0 individuals with the desired phenotype were crossed, and progeny were screened in every generation. PCR and sequencing were performed using gDNA from a pulled leg to determine the mutant genotype. Quality control tests, including pupae and adult recovery rates, male sex ratio and fecundity, were applied to the ebony mutant line to determine whether the mutation confers any fitness cost. Results: An Ae. aegypti ebony knockout mutant carrying a 5-bp deletion was obtained, which presented darker head and siphon phenotypes at the larval stage. However, genetic analysis revealed that this ebony mutation results in incomplete penetrance and variable expressivity. The establishment of a pure ebony mutant line was not possible because of the fitness costs conferred by the mutation. Conclusions: In this study, the adequacy and suitability of the ebony gene as a selectable marker for the development of a GSS in Ae. aegypti were assessed. Despite its clear phenotype early in larval development, the homozygous mutant line presented phenotypic inconsistency and loss of fertility. These drawbacks clearly indicate that this particular mutation is not suitable for the development of a new GSS. Nonetheless, it cannot be excluded that a different mutation will lead to a different expression and penetrance profile and a viable homozygous mutant line.
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    Expanding Genetic and Genomic Resources for Sex Separation and Mosquito Control Strategies
    Compton, Austin (Virginia Tech, 2021-10-26)
    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.
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    Expression of anti-chikungunya single-domain antibodies in transgenic Aedes aegypti reduces vector competence for chikungunya virus and Mayaro virus
    Webb, Emily M.; Compton, Austin; Rai, Pallavi; Chuong, Christina; Paulson, Sally L.; Tu, Zhijian; Weger-Lucarelli, James (Frontiers, 2023-06-12)
    Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related alphaviruses that cause acute febrile illness accompanied by an incapacitating polyarthralgia that can persist for years following initial infection. In conjunction with sporadic outbreaks throughout the sub-tropical regions of the Americas, increased global travel to CHIKV- and MAYV-endemic areas has resulted in imported cases of MAYV, as well as imported cases and autochthonous transmission of CHIKV, within the United States and Europe. With increasing prevalence of CHIKV worldwide and MAYV throughout the Americas within the last decade, a heavy focus has been placed on control and prevention programs. To date, the most effective means of controlling the spread of these viruses is through mosquito control programs. However, current programs have limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. We have previously identified and characterized an anti-CHIKV single-domain antibody (sdAb) that potently neutralizes several alphaviruses including Ross River virus and Mayaro virus. Given the close antigenic relationship between MAYV and CHIKV, we formulated a single defense strategy to combat both emerging arboviruses: we generated transgenic Aedes aegypti mosquitoes that express two camelid-derived anti-CHIKV sdAbs. Following an infectious bloodmeal, we observed significant reduction in CHIKV and MAYV replication and transmission potential in sdAb-expressing transgenic compared to wild-type mosquitoes; thus, this strategy provides a novel approach to controlling and preventing outbreaks of these pathogens that reduce quality of life throughout the tropical regions of the world.
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    Natural and Engineered Sex Ratio Distortion in Insects
    Compton, Austin; Tu, Zhijian (Frontiers, 2022-06-15)
    Insects have evolved highly diverse genetic sex-determination mechanisms and a relatively balanced male to female sex ratio is generally expected. However, selection may shift the optimal sex ratio while meiotic drive and endosymbiont manipulation can result in sex ratio distortion (SRD). Recent advances in sex chromosome genomics and CRISPR/Cas9-mediated genome editing brought significant insights into the molecular regulators of sex determination in an increasing number of insects and provided new ways to engineer SRD. We review these advances and discuss both naturally occurring and engineered SRD in the context of the Anthropocene. We emphasize SRD-mediated biological control of insects to help improve One Health, sustain agriculture, and conserve endangered species.
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    A Reexamination of Thioredoxin Reductase from Thermoplasma acidophilum, a Thermoacidophilic Euryarchaeon, Identifies It as an NADH-Dependent Enzyme
    Susanti, Dwi; Loganathan, Usha R.; Compton, Austin; Mukhopadhyay, Biswarup (ACS Publications, 2017-08-03)
    Flavin-containing Trx reductase (TrxR) of Thermoplasma acidophilum (Ta), a thermoacidophilic facultative anaerobic archaeon, lacks the structural features for the binding of 2′-phosphate of nicotinamide adenine dinucleotide phosphate (NADPH), and this feature has justified the observed lack of activity with NADPH; NADH has also been reported to be ineffective. Our recent phylogenetic analysis identified Ta-TrxR as closely related to the NADHdependent enzymes of Thermotoga maritima and Desulfovibrio vulgaris, both being anaerobic bacteria. This observation instigated a reexamination of the activity of the enzyme, which showed that Ta-TrxR is NADH dependent; the apparent Km for NADH was 3.1 μM, a physiologically relevant value. This finding is consistent with the observation that NADH:TrxR has thus far been found primarily in anaerobic bacteria and archaea.