Name: inoculating anopheles gambiae mosquitoes with beads to induce and measure the melanization immune response
Uploaded: 2017-01-12T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response at JoVE.com

This research was possible through funding from the University of Neuch&#226;tel. We would like to thank all the students that helped in improving this technique, namely our colleague Kevin Thievent. We would also like to thank the members of the Thomas Lab for making their laboratory available. We would like to thank Janet Teeple for her help with mosquito rearing. We would also like to thanks Loyal Hall in the laboratory of Pr. Tom Baker for his help in the preparation of the micro capillary glass tubes.

1. Solución salina para inyección y disección <ol><li> Preparar la solución salina mediante la adición de NaCl, KCl, CaCl 2 y a agua destilada para obtener NaCl 1,3 mM, KCl 0,5 mM, y CaCl 0,2 mM 2 a pH = 6,8. </li><li> Añadir 1 ml de solución de verde 0,1% de metilo a 99 mililitros de la solución salina para colorear las perlas transparentes. Esta es la &quot;solución de inyección&quot; de 0,001% verde de metilo. </li><li> A continuación, añadir 5 ml de solución de verde 0,1% de metilo a 45 ml de la solución salina. Esta &quot;solución disección&quot; 0,01% verde de metilo es de 10 veces más concentrado en verde de metilo para facilitar la coloración de las perlas y su observación durante la disección. NOTA: Filtro de esterilizar el tampón si es necesario. </li></ol> 2. Preparación Capilar <ol><li> Tubos de vidrio capilar se Heat-pull para obtener una punta muy fina ligeramente más grandes que los granos más pequeños (φ = 40 micras) 42. </li><li> Abierta y unaJUSTE cada capilar rompiendo la punta con unas pinzas o por aserrado la punta fuera. </li></ol> 3. Mosquito Crianza <ol><li> Trasera A. gambiae a 26 ± 1 ° C, 70 ± 5% de humedad relativa, y una luz 12:12 h: oscuridad ciclo. Mantenga las hembras adultas y darles acceso a una solución de azúcar al 10%. </li></ol> 4. Selección del grano con el capilar <ol><li> Verter 0.009 g de perlas cargadas negativamente (40 a 120 micras de diámetro) en un plato de Petri de 5 cm y añadir 5 ml de solución de inyección 30 min con antelación. NOTA: El objetivo es permitir que el tinte de color Las cuentas para hacer la selección del grano para la inyección más fácil. Autoclave las cuentas si no vienen en un frasco estéril. </li><li> El uso de un capilar montado en un bulbo de pipeta capilar, seleccionar visualmente el grano más pequeño en 0,1 l de la solución salina con un microscopio binocular. NOTA: Cuando la inoculación de varias cuentas al mismo tiempo, seleccione tres cuentas de un volumen total de 0,1 l.</li></ol> 5. Manejo del mosquito y la inoculación <ol><li> El uso de un aspirador de insectos, coloque cada mosquito en un tubo de 50 ml. </li><li> Chill cada mosquito brevemente colocando el tubo en hielo triturado (2-5 min). </li><li> Coloque un mosquito en su lado derecho bajo el microscopio binocular. </li><li> Bajo un microscopio estereoscópico, inocular el mosquito con el capilar por firmemente perforación a través de la parte izquierda de la cutícula cavidad torácica y luego inyectar el líquido y el talón. NOTA: Tenga cuidado de no dañar los músculos de vuelo mediante la celebración de los capilares como perpendicularmente al mosquito como sea posible. </li><li> Retire el capilar. </li><li> El uso de pinzas entomología de peso pluma, colocar cada mujer individualmente en un vaso de plástico de 180 ml cubierto con un mosquitero; darles acceso a una solución de azúcar al 10%. NOTA: Se debe tener precaución con la solución de azúcar, como el mosquito puede quedar atrapado en gotas de la solución azucarada. </li></ol> 6. MosquitoLa disección y Fotografía del grano <ol><li> Compruebe si los mosquitos están vivos 24 horas después de la inoculación. Si es así, comprobar su estado y mantener sólo los mosquitos que son capaces de volar. </li><li> Congelación de estos mosquitos a -20 ° C. NOTA: Se pueden conservar varios días a meses antes de la disección, si es necesario. </li><li> Retire las alas de los mosquitos utilizando pinzas bajo un microscopio binocular en la ampliación de 32X. Fijar las alas en portaobjetos de vidrio con cinta adhesiva transparente. Anote el código utilizado para cada individuo de mosquitos. </li><li> Ponga los portaobjetos de vidrio en un escáner y escanearlas a 1.200 dpi. NOTA: Compruebe que el código es legible para cada individuo de mosquitos. </li><li> En un portaobjetos de microscopio de vidrio y en la solución de la disección, el uso de fórceps para separar el tórax de la cabeza y el abdomen. </li><li> Abrir el tórax con una pinza para recuperar las perlas. NOTA: los granos que no se mueven en el abdomen, no están en contacto entre sí y la mayoría de ellos flotan libremente en tque hemolinfa. Tenga cuidado de no romper el cordón con las pinzas, como una perla no melanized puede ser difícil de encontrar. Espere 1 a 5 min para los granos para obtener color. </li><li> La transferencia de los granos en una gota de la solución de la disección en un portaobjetos de vidrio, antes de tomar la fotografía. NOTA: No es necesario lavar las perlas. </li><li> El uso de un microscopio equipado con una cámara, tomar una imagen digital de cada perla con un ajuste de la iluminación estándar y una ampliación de 400X. NOTA: No cambie la iluminación entre las diferentes imágenes para permitir su comparación. </li></ol> 7. Análisis de la foto: Bead melanización <ol><li> Abra el software de análisis de imágenes. Haga clic en &quot;archivo&quot; y luego en &quot;abrir&quot; para encontrar una imagen de perlas. </li><li> Haga clic en &quot;Analizar&quot;. Haga clic en &quot;mediciones establecidas.&quot; Marque la casilla &quot;valor medio gris&quot; en la ventana de &quot;medidas de ajuste&quot;. </li><li> Haga clic en el icono de la herramienta de selección círculo. Seleccione el perímetro del talón; permanecer enlado del talón y evitar el halo luminoso alrededor de ella. </li><li> Haga clic en el menú &quot;Analizar&quot;. Haga clic en &quot;medida&quot;. Obtener el valor de gris medio y el tamaño del área de sección transversal de la perla. <ol><li> Para obtener el valor melanización de cada cuenta, realice la siguiente operación usando una calculadora o calculadora de software: &quot;256 - valor medio gris.&quot; La medida melanización es un valor entre 0 y 256, donde 0 representa una imagen totalmente blanco. </li></ol></li></ol>

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J., George, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Manipulation&amp;#34;+without+the+parasite:+altered+feeding+behaviour+of+mosquitoes+is+not+dependent+on+infection+with+malaria+parasites.">34;Manipulation&amp;#34; without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites.</a> P Roy Soc B-Biol Sci. 280 (1763), 20130711 (2013).</li><li>Voordouw, M. J., Lambrechts, L., Koella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+maternal+effects+after+stimulation+of+the+melanization+response+in+the+yellow+fever+mosquito+Aedes+aegypti.">No maternal effects after stimulation of the melanization response in the yellow fever mosquito Aedes aegypti.</a> Oikos. 117 (8), 1269-1279 (2008).</li><li>Paskewitz, S., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Plasmodium+refractory+and+susceptible+strains+of+Anopheles+gambiae+to+inoculated+Sephadex+beads.">Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to inoculated Sephadex beads.</a> Dev comp immunol. 18 (5), 369-375 (1994).</li></ol>

The authors declare that they have no competing financial interests.

Esta técnica de inyección es útil para estimular y estudiar la melanization respuesta en los mosquitos. Por ejemplo, aquí se estudió el efecto de la carga de estímulos inmune. El paso crítico en este procedimiento es para inocular apropiadamente el mosquito. Cualquier daño excesivo a los músculos de vuelo o al propio mosquito puede prevenir el mosquito de la alimentación o puede acabar con él antes de la disección. Un segundo paso clave es mantener a los mosquitos en el hielo el ...

Los insectos se basan en las respuestas inmunitarias para protegerse contra los parásitos y patógenos 1 - 3 que violan a través de su cutícula o el epitelio del intestino medio 4. En los mosquitos, estas respuestas son eficaces contra las bacterias, virus 5 6 7 filaria, nematodos, parásitos de la malaria y 1,8,9. En los mosquitos, una respuesta inmune clave es la encapsulación de partículas extrañas con melanina 10 - 12. Esta encapsulación puede ocurrir en el intestino medio o en la hemolinfa sistema de circulación de 10 a 12. Este melanization respuesta es el resultado de la pro-fenoloxidasa cascada 10 - 12, y puede conducir a la muerte de los parásitos o para su fagocitosis. En los mosquitos adultos, donde el número de células de hemocitos es limitado, melanización es una respuesta humoral, al igual que contra los parásitos Plasmodium o nematodos filarias 7.En algunos otros insectos, que está directamente las células de hemocitos que se reúnen alrededor del parásito a melanize ellas 7. Además, la melanina es también esencial para varios otros procesos fisiológicos como la producción de huevos y heridas de cicatrización de la cutícula 7. La estimulación de la respuesta inmune se utiliza como una herramienta para estudiar la inmunidad de insectos en varios sistemas modelo de salud pública agrícola y 13 - 18. Se utiliza en los mosquitos Anopheles gambiae, el principal vector de la malaria en África, para estudiar las interacciones huésped-parásito 14 - 16,19. Estas técnicas se basan en la capacidad de los insectos para detectar parásitos con sus receptores de reconocimiento de patrones (PRR) 2. Los mosquitos también pueden detectar otras moléculas que interfieren con su biología, tales como patrones moleculares asociados a patógenos (PAMP), o detectar sus propias células dañadas debido a la liberación de colágeno y ácidos nucleicos. La célula inmune mosquitos tales como los hemocitos se utilizan para la detección de 20 - 23. Las principales vías de señalización son inmunes IMD, peaje, JAK / STAT 24, y la interferencia de ácido ribonucleico (RNAi) 25,26. Tanto peaje y vías IMD influyen en la melanization respuesta e interactúan con el pro-fenoloxidasa en cascada 10 - 12. La herramienta estándar que se utiliza para estimular la respuesta melanización es la inoculación de un mosquito con una pequeña gota en la hemolinfa de la cavidad torácica. El grado de encapsulación de melanina a continuación, se puede medir 19 después de recuperar el cordón a través de la disección del mosquito. En la mayoría de estudios, sólo un talón se inyectó por mosquito 15,16,27, pero la inyección de más cuentas es posible con el fin de estudiar los límites de la respuesta melanización 19. Estas perlas se inyectan usando una solución de inyección (suero fisiológico) para limitar la perturbación de la fisiología y mosquitola desecación del mosquito 15,16,27. Un colorante se añade a esta solución para facilitar la selección de perlas. Es lo mismo para la solución de disección se utiliza para recuperar el 15,16,27 talón. La ventaja de la inoculación de los insectos con los estímulos no patógena es la capacidad de concentrarse en el efecto directo sobre la respuesta inmune. No hay efectos que complican debido a la patogenicidad del parásito 28, inmunosupresión 29 - 31, o el 31 de evasión inmune - de 34. Además, las consecuencias de las estimulaciones en otros rasgos de historia de vida, tales como la longevidad o la fecundidad, pueden también ser estudiados. Por lo tanto, los investigadores que estudian la ecología evolutiva pueden requerir este tipo de herramientas 2,35,36. Por ejemplo, los abejorros inmuno-desafió tienen una vida más corta en virtud de la inanición. efectos negativos similares de estímulos inmunes y las implementaciones se han observado en diferentes modelos de invertebrados, a menudo resulta en un cortoer vida útil o el éxito reproductivo menos 13,27,37. Tales estudios pueden llevarse a cabo en diversos entornos 2,4,38. Estimular la inmunidad es también de interés para los que se centran directamente en la inmunopatología 39,40. Este protocolo se basa en la inoculación de los granos con los mosquitos para estimular la respuesta melanización y directamente medir la cantidad de melanina. Esto permite el estudio cuantitativo y cualitativo de la melanization respuesta en diferentes parámetros experimentales. Tal herramienta se puede extender a la estimulación de otras respuestas inmunes, tales como la respuesta a las bacterias antibacteriano 41 muertos por calor. También puede llevarse a cabo en muchos entornos ecológicos.

<table><tbody><tr><td>Microcapillary glass tubes GB120TF-10</td><td>science-products.com</td><td>GB120TF-10</td><td>http://www.science-products.com/Products/CatalogG/Glass/Glass.html</td></tr><tr><td>Microcaps Capillary pipette bulb</td><td>Drumond</td><td>1-000-9000</td><td></td></tr><tr><td>negatively charged Sephadex CM C-25 beads</td><td>Sigma-Aldrich, Steinheim, Germany</td><td>C25120 SIGMA</td><td>need few to start</td></tr><tr><td>Methyl green</td><td>Sigma-Aldrich</td><td>323829 ALDRICH</td><td>need few to start</td></tr><tr><td>Software ImageJ</td><td>opensource</td><td></td><td>Version 1.47f7 or later</td></tr></tbody></table>

inoculating anopheles gambiae mosquitoes with beads to induce and measure the melanization immune response

La estimulación de la respuesta inmune es una herramienta común en estudios de invertebrados para examinar la eficacia y los mecanismos de la inmunidad. Esta estimulación se basa en la inyección de partículas no patógenos en los insectos, como las partículas serán detectados por el sistema inmune y inducirán la producción de efectores inmunes. Nos centramos aquí en la estimulación de la respuesta melanización en el mosquito Anopheles gambiae. Los resultados melanization respuesta en la encapsulación de partículas extrañas y parásitos con una capa oscura de melanina. Para estimular esta respuesta, los mosquitos se inoculan con los granos en la cavidad torácica utilizando tubos de vidrio microcapilar. Entonces, después de 24 horas, los mosquitos se disecan para recuperar las perlas. El grado de melanización de la perla se mide usando software de análisis de imágenes. Perlas no tienen los efectos patógenos de los parásitos, o su capacidad para evadir o suprimen la respuesta inmune. Estas inyecciones son una manera de Measure eficacia inmunológica y el impacto de los estímulos inmunes en otros rasgos de historia de vida, tales como la fecundidad o la longevidad. No es exactamente lo mismo que estudiar directamente las interacciones huésped-parásito, pero es una interesante herramienta para estudiar la inmunidad y su ecología evolutiva.

<html> Los mosquitos no todos hicieron melanize las perlas de la misma manera, ya que algunas cuentas fueron cubiertas con menos melanina que otros (Figura 1). De hecho, algunas perlas permanecieron azul debido a la falta de melanización, mientras que otros eran completamente oscuro (Figura 1). El valor melanización fue estandarizado por interpolación lineal a un valor entre 0 (lo que correspondía a una perla azul y unmelanized) y 100 (que corresponde...

Watch this Scientific Journal Video about Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response at JoVE.com

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

Inoculador<em&gt; Anopheles gambiae</em&gt; Los mosquitos con los granos para inducir y medir la respuesta inmune melanización

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

7.9K vistas.  Université de Neuchâtel. Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

Video: Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

La inyección de una. Los embriones stephensi para generar la malaria resistente a los mosquitos

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

Investigación

Biología

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

Integración mediada por φC31 dirigida por sitios e intercambio de casetes en anopheles vectores de malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

Genética

Microinjection Method for Anopheles gambiae Embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

Método de microinyección para embriones de Anopheles gambiae

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Un<em&gt; In Vitro</em&gt; Modelo para medir la respuesta inmune a la malaria en el contexto del VIH Co-infección

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Inmunología e Infección

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

El aislamiento de las células mieloides de ratón de la piel y el drenaje de ganglios linfáticos Siguiendo intradérmica La inmunización con viva atenuada<em&gt; Plasmodium</emLos esporozoitos&gt;

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Seguimiento In Vivo del Desarrollo del Edema y Patología Microvascular en un Modelo de Malaria Cerebral Experimental usando Imágenes de Resonancia Magnética

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Medicina

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Ensayo estándar de alimentación por membrana para la detección de la infección por Plasmodium falciparum en mosquitos vectores Anopheles

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Generación de esporozoítos de Plasmodium berghei modificados genéticamente

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi gatillo en la entrega<em&gt; Anopheles gambiae</em&gt; Las pupas

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

Inoculador

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