JoVE General
RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes
1Biology Department, New Mexico State University, 2Molecular Biology Department, New Mexico State University
In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.
JoVE Immunology and Infection
A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti
Department of Biology, University of Richmond
A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.
JoVE Immunology and Infection
Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors
Microbiology, Immunology, and Pathology, Colorado State University
Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.
JoVE Immunology and Infection
Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes
Department of Entomology, Virginia Tech
Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.
JoVE Immunology and Infection
Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes
1Department of Molecular Biology and Biochemistry, University of California, Irvine, 2Department of Microbiology and Molecular Genetics, University of California, Irvine
Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.
JoVE Immunology and Infection
A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors
1Department of Entomology, Texas A&M University (TAMU), 2Department of Molecular and Cellular Medicine, Texas A&M University (TAMU)
This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.
JoVE General
Protocol for Mosquito Rearing (A. gambiae)
Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University
This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.
JoVE General
Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)
In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.
JoVE General
Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)
The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.
JoVE Editorial
May 2011: This Month in JoVE
The main highlights for our May issue include methods for measuring cognition in zero gravity, isolating mosquito immune cells, engineering recombinant SARS vaccines, and detecting tumors with thermal imaging. In addition, procedures for isolating neural stem cells from human fetal brain and culturing antigen-presenting liver cells will also be released.
JoVE General
A Simple Protocol for Extracting Hemocytes from Wild Caterpillars
Department of Biological Sciences, The George Washington University
Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.
JoVE General
High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization
Department of Entomology, Virginia Tech
Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.
JoVE Immunology and Infection
A Simple Chelex Protocol for DNA Extraction from Anopheles spp.
1Malaria Institute at Macha, 2Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health
A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.
JoVE General
Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq
Department of Biology, New Mexico State University
A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.
JoVE Editorial
September 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
This September in JoVE, researchers from the School of Medicine at the Free University of Berlin demonstrate a novel method for studying how stroke patients compensate for visual field defects. To do this, our authors make use of a driving simulator complete with brakes, a steering wheel, and turn signals. Using driving simulation software and sophisticated eye tracking, researchers can compare the gaze behavior of stroke patients as they navigate through virtual driving courses with varying degrees of complexity. Though posterior cerebral artery infarction can lead to similar visual deficits in patients, some are able to navigate through the driving courses by developing compensatory eye movements, while others crash into dangerous obstacles, like wild boars. Through the analysis of compensatory gaze behavior employed by patients, our authors see great potential for using driving simulation as a tool to rehabilitate stroke patients trying to overcome the blind spots in their visual fields.
JoVE General
Injection of dsRNA into Female A. aegypti Mosquitos
1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI)
Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.
JoVE General
Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination
Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University
Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.
JoVE General
Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes
Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University
In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.
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