Browsing by Author "Aryan, Azadeh"

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    Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology
    Martin-Martin, Ines; Leon, Paola Carolina Valenzuela; Amo, Laura; Shrivastava, Gaurav; Iniguez, Eva; Aryan, Azadeh; Brooks, Steven; Kojin, Bianca B.; Williams, Adeline E.; Bolland, Silvia; Ackerman, Hans; Adelman, Zach N.; Calvo, Eric (Cell Press, 2022-04-12)
    Saliva from mosquitoes contains vasodilators that antagonize vasoconstrictors produced at the bite site. Sialokinin is a vasodilator present in the saliva of Aedes aegypti. Here, we investigate its function and describe its mechanism of action during blood feeding. Sialokinin induces nitric oxide release similar to substance P. Sialokinin-KO mosquitoes produce lower blood perfusion than parental mosquitoes at the bite site during probing and have significantly longer probing times, which result in lower blood feeding success. In contrast, there is no difference in feeding between KO and parental mosquitoes when using artificial membrane feeders or mice that are treated with a substance P receptor antagonist, confirming that sialokinin interferes with host hemostasis via NK1R signaling. While sialokinin-KO saliva does not affect virus infection in vitro, it stimulates macrophages and inhibits leukocyte recruitment in vivo. This work highlights the biological functionality of salivary proteins in blood feeding.
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    Gene editing in Aedes aegypti
    Aryan, Azadeh (Virginia Tech, 2013-10-08)
    Aedes aegypti (Ae. aegypti) is one of the most important vectors of dengue, chikungunya and yellow fever viruses. The use of chemical control strategies such as insecticides is associated with problems including the development of insecticide resistance, side effects on animal and human health, and environmental concerns. Because current methods have not proven sufficient to control these diseases, developing novel, genetics-based, control strategies to limit the transmission of disease is urgently needed. Increased knowledge about mosquito-pathogen relationships and the molecular biology of mosquitoes now makes it possible to generate transgenic mosquito strains that are unable to transmit various parasites or viruses. Ae. aegypti genetic experiments are enabled, and limited by, the catalog of promoter elements available to drive transgene expression. To find a promoter able to drive robust expression of firefly (FF) luciferase in Ae. aegypti embryos, an experiment was designed to compare Ae. aegypti endogenous and exogenous promoters. The PUb promoter was found to be extremely robust in expression of FF luciferase in different stages of embryonic development from 2-72 hours after injection. In subsequent experiments, transformation frequency was calculated using four different promoters (IE1, UbL40, hsp82 and PUb) to express the Mos1 transposase open reading frame in Mos1-mediated transgenesis. Germline transformation efficiency and size of transgenic cluster were not significantly different when using endogenous Ae. aegypti PUb or the commonly used exogenous Drosophila hsp82 promoter to express Mos1 transposase. This study also describes the development of new tools for gene editing in the Ae. aegypti mosquito genome and the use of these tools to design an efficient gene drive system in this mosquito. Homing endonucleases (HEs) are selfish elements which catalyze double-stranded DNA (dsDNA) breaks in a sequence-specific manner. The activities of four HEs (Y2-I-AniI, I-CreI, I-PpoI, and I-SceI) were investigated for their ability to catalyze the excision of genomic segments from the Ae. aegypti genome. All four enzymes were found to be active in Ae. aegypti; however, the activity of Y2-I-AniI was higher compared to the other three enzymes. Single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways were identified as mechanisms to repair HE-induced dsDNA breaks. TALE nucleases (TALENs) are a group of artificial enzymes capable of generating site-specific DNA lesions. To examine the ability of TALENs for gene editing in Ae. aegypti, a pair of TALENs targeted to the kmo gene were expressed from a plasmid following embryonic injection. Twenty to forty percent of fertile G0 produced white-eyed progeny which resulted from disruption of the kmo gene. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. A small deletion of one to seven bp occurred at the TALEN recognition site. These results show that TALEN and HEs are highly active in the Ae. aegypti germline and can be used for gene editing and gene drive strategies in Ae. aegypti.
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    Germline excision of transgenes in Aedes aegypti by homing endonucleases
    Aryan, Azadeh; Anderson, Michelle A. E.; Myles, Kevin M.; Adelman, Zach N. (Nature Publishing Group, 2013-04)
    Aedes (Ae.) aegypti is the primary vector for dengue viruses (serotypes1-4) and chikungunya virus. Homing endonucleases (HEs) are ancient selfish elements that catalyze double-stranded DNA breaks (DSB) in a highly specific manner. In this report, we show that the HEs Y2-I-AniI, I-CreI and I-SceI are all capable of catalyzing the excision of genomic segments from the Ae. aegypti genome in a heritable manner. Y2-I-AniI demonstrated the highest efficiency at two independent genomic targets, with 20-40% of Y2-I-AniI-treated individuals producing offspring that had lost the target transgene. HE-induced DSBs were found to be repaired via the single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways in a manner dependent on the availability of direct repeat sequences in the transgene. These results support the development of HE-based gene editing and gene drive strategies in Ae. aegypti, and confirm the utility of HEs in the manipulation and modification of transgenes in this important vector.
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    Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight
    Aryan, Azadeh; Anderson, Michelle A. E.; Biedler, James K.; Qi, Yumin; Overcash, Justin M.; Naumenko, Anastasia N.; Sharakhova, Maria V.; Mao, Chunhong; Adelman, Zach N.; Tu, Zhijian Jake (NAS, 2020-06-12)
    A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegypti. Nix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sexconversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus,was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.
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    Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9
    Martin-Martin, Ines; Aryan, Azadeh; Meneses, Claudio; Adelman, Zach N.; Calvo, Eric (PLOS, 2018-09)
    Background Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis. Methodology/Principal findings Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this nonmodel organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed. Conclusion and significance The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions.
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    TALEN-Based Gene Disruption in the Dengue Vector Aedes aegypti
    Aryan, Azadeh; Anderson, Michelle A. E.; Myles, Kevin M.; Adelman, Zach N. (PLOS, 2013-03-21)
    In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been slow. TALE nucleases (TALENs) have been used with great success in a number of organisms to generate site-specific DNA lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20–40% of fertile survivors produced kmo alleles that failed to complement an existing khw mutation. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1–7 bp specifically at the selected target site. White-eyed individuals could also be recovered following a blind intercross of G1 progeny, yielding several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are performed in this important disease vector.