scholarly journals Disrupting female flight in the vector Aedes aegypti

2019 ◽  
Author(s):  
Sarah O’Leary ◽  
Zach N. Adelman
Keyword(s):  
Aedes Aegypti ◽  
Flight Muscle ◽  
Population Control ◽  
Gene Knockout ◽  
Control Strategies ◽  
Specific Gene ◽  
Source Reduction ◽  
Guide Rnas ◽  
Control Flight ◽  
Female Specific

AbstractAedes aegypti is a vector of dengue, chikungunya, and Zika viruses. Current vector control strategies such as community engagement, source reduction, and insecticides have not been sufficient to prevent viral outbreaks. Thus, interest in novel strategies involving genetic engineering is growing. Female mosquitoes rely on flight to mate with males and obtain a bloodmeal from a host. We hypothesized that knockout of genes specifically expressed in female mosquitoes associated with the indirect flight muscles would result in a flightless female mosquito. With the CRISPR-Cas9 system, we performed embryonic microinjections of Cas9 protein and guide RNAs specific to genes hypothesized to control flight in mosquitoes, and have obtained genetic knockouts in several genes specifically expressed in the flight-muscle, including those specific to female flight muscle. Analysis of the phenotype of these female-specific gene knockout mutants resulted in flightless females and flying males. While further assessment is required, this work lays the groundwork for a mechanism of population control that is female-specific for the Ae. aegypti vector.

scholarly journals A functional requirement for sex-determination M/m locus region lncRNA genes in Aedes aegypti female larvae

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Keshava Mysore ◽  
Limb K. Hapairai ◽  
Ping Li ◽  
Joseph B. Roethele ◽  
Longhua Sun ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Sex Determination ◽  
Population Control ◽  
Control Strategies ◽  
Adult Males ◽  
Non Coding Rna ◽  
Female Specific ◽  
Interfering Rna ◽  
Long Non Coding Rna ◽  
Male Mosquitoes

AbstractAlthough many putative long non-coding RNA (lncRNA) genes have been identified in insect genomes, few of these genes have been functionally validated. A screen for female-specific larvicides that facilitate Aedes aegypti male sex separation uncovered multiple interfering RNAs with target sites in lncRNA genes located in the M/m locus region, including loci within or tightly linked to the sex determination locus. Larval consumption of a Saccharomyces cerevisiae (yeast) strain engineered to express interfering RNA corresponding to lncRNA transcripts resulted in significant female death, yet had no impact on male survival or fitness. Incorporation of the yeast larvicides into mass culturing protocols facilitated scaled production and separation of fit adult males, indicating that yeast larvicides could benefit mosquito population control strategies that rely on mass releases of male mosquitoes. These studies functionally verified a female-specific developmental requirement for M/m locus region lncRNA genes, suggesting that sexually antagonistic lncRNA genes found within this highly repetitive pericentromeric DNA sequence may be contributing to the evolution of A. aegypti sex chromosomes.

scholarly journals CRISPR/Cas9 knockout of female-biased genes AeAct-4 or myo-fem in Ae. aegypti results in a flightless phenotype in female, but not male mosquitoes

2020 ◽  
Vol 14 (12) ◽  
pp. e0008971
Author(s):  
Sarah O’Leary ◽  
Zach N. Adelman
Keyword(s):  
Flight Muscle ◽  
Control Strategies ◽  
Loss Of Function ◽  
Wild Type ◽  
Vector Species ◽  
Source Reduction ◽  
Flight Muscles ◽  
Control Flight ◽  
Reduced Rate ◽  
Male Mosquitoes

Aedes aegypti is a vector of dengue, chikungunya, and Zika viruses. Current vector control strategies such as community engagement, source reduction, and insecticides have not been sufficient to prevent viral outbreaks. Thus, interest in novel strategies involving genetic engineering is growing. Female mosquitoes rely on flight to mate with males and obtain a bloodmeal from a host. We hypothesized that knockout of genes specifically expressed in female mosquitoes associated with the indirect flight muscles would result in a flightless female mosquito. Using CRISPR-Cas9 we generated loss-of-function mutations in several genes hypothesized to control flight in mosquitoes, including actin (AeAct-4) and myosin (myo-fem) genes expressed specifically in the female flight muscle. Genetic knockout of these genes resulted in 100% flightless females, with homozygous males able to fly, mate, and produce offspring, albeit at a reduced rate when compared to wild type males. Interestingly, we found that while AeAct-4 was haplosufficient, with most heterozygous individuals capable of flight, this was not the case for myo-fem, where about half of individuals carrying only one intact copy could not fly. These findings lay the groundwork for developing novel mechanisms of controlling Ae. aegypti populations, and our results suggest that this mechanism could be applicable to other vector species of mosquito.

scholarly journals Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

2015 ◽  
Vol 112 (13) ◽  
pp. 4038-4043 ◽  
Author(s):  
Sanjay Basu ◽  
Azadeh Aryan ◽  
Justin M. Overcash ◽  
Glady Hazitha Samuel ◽  
Michelle A. E. Anderson ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Specific Gene ◽  
Gene Drive ◽  
Transcription Activator ◽  
End Joining ◽  
Site Specific ◽  
Gene Insertion ◽  
Guide Rnas ◽  
Essential Components ◽  
Drive Systems

Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24–90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2–3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

scholarly journals Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice

Gene Therapy ◽  
2021 ◽  
Author(s):  
Sam Hana ◽  
Michael Peterson ◽  
Helen McLaughlin ◽  
Eric Marshall ◽  
Attila J. Fabian ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Low Cost ◽  
High Specificity ◽  
Specific Gene ◽  
Preclinical Research ◽  
Guide Rnas ◽  
Neuronal Gene ◽  
Sgrna Design

AbstractCRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research.

scholarly journals Germline Cas9 Expression Yields Highly Efficient Genome Engineering in a Major Worldwide Disease Vector, Aedes aegypti

2017 ◽  
Author(s):  
Ming Li ◽  
Michelle Bui ◽  
Ting Yang ◽  
Bradley J. White ◽  
Omar S. Akbari
Keyword(s):  
Aedes Aegypti ◽  
High Throughput ◽  
Disease Transmission ◽  
Genome Engineering ◽  
Population Control ◽  
Control Strategies ◽  
Morphological Development ◽  
Disease Vector ◽  
Genome Modifications ◽  
Gene Drives

AbstractThe development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti. Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we built, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a first step towards the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.Significance StatementAedes aegypti is the principal vector of multiple arboviruses that significantly affect human health including dengue, chikungunya, and zika. Development of tools for efficient genome engineering in this mosquito will not only lay the foundation for the application of novel genetic control strategies that do not rely on insecticides, but will also accelerate basic research on key biological processes involved in disease transmission. Here, we report the development of a transgenic CRISPR approach for rapid gene disruption in this organism. Given their high editing efficiencies, the Cas9 strains we developed can be used to quickly generate novel genome modifications allowing for high-throughput gene targeting, and can possibly facilitate the development of gene drives, thereby accelerating comprehensive functional annotation and development of innovative population control strategies for Ae. aegypti.

scholarly journals A conserved female-specific larval requirement for MtnB function facilitates sex separation in multiple species of disease vector mosquitoes

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Keshava Mysore ◽  
Longhua Sun ◽  
Joseph B. Roethele ◽  
Ping Li ◽  
Jessica Igiede ◽  
...  
Keyword(s):  
Chromosome Evolution ◽  
Sex Chromosome ◽  
Population Control ◽  
Control Strategies ◽  
Sex Chromosome Evolution ◽  
Disease Vector ◽  
Sex Separation ◽  
Multiple Species ◽  
Female Specific ◽  
Male Mosquitoes

Abstract Background Clusters of sex-specific loci are predicted to shape the boundaries of the M/m sex-determination locus of the dengue vector mosquito Aedes aegypti, but the identities of these genes are not known. Identification and characterization of these loci could promote a better understanding of mosquito sex chromosome evolution and lead to the elucidation of new strategies for male mosquito sex separation, a requirement for several emerging mosquito population control strategies that are dependent on the mass rearing and release of male mosquitoes. This investigation revealed that the methylthioribulose-1-phosphate dehydratase (MtnB) gene, which resides adjacent to the M/m locus and encodes an evolutionarily conserved component of the methionine salvage pathway, is required for survival of female larvae. Results Larval consumption of Saccharomyces cerevisiae (yeast) strains engineered to express interfering RNA corresponding to MtnB resulted in target gene silencing and significant female death, yet had no impact on A. aegypti male survival or fitness. Integration of the yeast larvicides into mass culturing protocols permitted scaled production of fit adult male mosquitoes. Moreover, silencing MtnB orthologs in Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus revealed a conserved female-specific larval requirement for MtnB among different species of mosquitoes. Conclusions The results of this investigation, which may have important implications for the study of mosquito sex chromosome evolution, indicate that silencing MtnB can facilitate sex separation in multiple species of disease vector insects. Graphical Abstract

scholarly journals Control Strategy for Aedes aegypti (Linnaeus, 1762) Population

2021 ◽  
Author(s):  
Taiana Gabriela Barbosa de Souza ◽  
Eduardo José de Arruda ◽  
Raphael Antônio Borges Gomes ◽  
Alex Martins Machado ◽  
Antônio Pancrácio de Souza
Keyword(s):  
Aedes Aegypti ◽  
Control Strategy ◽  
Population Control ◽  
Control Strategies ◽  
Breeding Sites ◽  
The World ◽  
Adaptive Ability ◽  
High Degree ◽  
New Strategies ◽  
Vectorial Competence

The mosquito Aedes aegypti (Diptera: Culicidae), is adapted to different environments, mainly urban ones. They have a high degree of vectorial competence for viral diseases, especially Dengue, the arbovirus with the highest number of cases in the world. The adaptive ability of this insect and the abundance of breeding sites have undermined attempts at population’s control, resulting in a high degree of infestation in many regions of the world, resulting in a Dengue endemic. It is important to understand the different nuances of the insect in order to understand the adaptive capacity of this vector, through the knowledge of his behavior, to propose new strategies and engagement of population in proactive actions that allow the population control of this vector, especially in periods of greater proliferation. This chapter discusses population control strategies, in different scenarios and carried out by different researchers, mainly in Brazil.

scholarly journals Predicting Aedes aegypti infestation using landscape and thermal features

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Camila Lorenz ◽  
Marcia C. Castro ◽  
Patricia M. P. Trindade ◽  
Maurício L. Nogueira ◽  
Mariana de Oliveira Lage ◽  
...  
Keyword(s):  
Remote Sensing ◽  
Aedes Aegypti ◽  
Vector Control ◽  
Land Surface ◽  
Urban Areas ◽  
Negative Binomial ◽  
Control Strategies ◽  
Winter Season ◽  
Landsat 8 ◽  
Thermal Features

AbstractIdentifying Aedes aegypti breeding hotspots in urban areas is crucial for the design of effective vector control strategies. Remote sensing techniques offer valuable tools for mapping habitat suitability. In this study, we evaluated the association between urban landscape, thermal features, and mosquito infestations. Entomological surveys were conducted between 2016 and 2019 in Vila Toninho, a neighborhood of São José do Rio Preto, São Paulo, Brazil, in which the numbers of adult female Ae. aegypti were recorded monthly and grouped by season for three years. We used data from 2016 to 2018 to build the model and data from summer of 2019 to validate it. WorldView-3 satellite images were used to extract land cover classes, and land surface temperature data were obtained using the Landsat-8 Thermal Infrared Sensor (TIRS). A multilevel negative binomial model was fitted to the data, which showed that the winter season has the greatest influence on decreases in mosquito abundance. Green areas and pavements were negatively associated, and a higher cover of asbestos roofs and exposed soil was positively associated with the presence of adult females. These features are related to socio-economic factors but also provide favorable breeding conditions for mosquitos. The application of remote sensing technologies has significant potential for optimizing vector control strategies, future mosquito suppression, and outbreak prediction.

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