Los autores desean agradecer a John Frey y Ben Peterson para la cría de mosquitos. También queremos agradecer a Christine Davis y Gary Radice para ayudar con las micrografías hemocitos. Esta investigación fue financiada por la Universidad de Richmond Arte y Ciencia de becas de verano para AA Qayum y la facultad de conceder A. Telang.

1. Preparación antes de la recolección de hemocitos <ol><li> Prepare una solución diluyente hemolinfa que consta de Schneider media del 60%, 10% de suero fetal bovino (SFB), y el 30% de tampón citrato (anticoagulante; 98 mM NaOH, 186 mM NaCl, 1,7 mM EDTA, 41 mM de ácido cítrico, pH 4,5) 11. Hacer 1,0 ml alícuotas que se pueden almacenar a -20 ° C. Use cada alícuota de una sola vez (ver Materiales). </li><li> Preparar portaobjetos de vidrio (75 x 25 x 1 mm) al anotar un círculo de 1 cm de diámetro en un extremo con una herramienta de vidrio grabado. Por otra parte, portaobjetos con dos pre-grabado círculos se pueden comprar (por ejemplo, Fisher Scientific o fuentes EMS). Una placa de vidrio por los mosquitos serán necesarios. </li><li> Preparar un conjunto de agujas de vidrio (ver Materiales) tiró de acuerdo con los siguientes valores sugeridos (Sutter Instruments, modelo P-87): <table border="1"><tbody><tr><td> El calor de rampa 5 </td></tr><tr><td> Tire: 45 </td></tr><tr><td> Vel: 75 </td></tr><tr><td> Tiempo: 175 </td></tr><tr><td> Presión de 580 </td></tr></tbody></table> Utilizando los valores recomendados en este tirador, construimos las agujas que son alrededor de 500 mm de longitud total, con longitudes de caña de unos 3 mm. Pre-retiró las agujas también se pueden comprar (por ejemplo, Tritech de Investigación). </li></ol> 2. Preparación de microinyector antes de la recolección de hemocitos <ol><li> Insertar una aguja se detuvo en el porta-agujas microinyector de acuerdo a las instrucciones del fabricante. Rellene el microinyector (ver Materiales) tubo, jeringa y aguja de vidrio tirado con aceite mineral de acuerdo a las instrucciones del fabricante. </li></ol> 3. Preparación de los mosquitos hembra antes de la recolección de hemocitos <ol><li> Aspirar 15 hembras adultas en pequeños contenedores jaula sólida que puede estar en contacto con el hielo. Jaula de sumergirse con los mosquitos en el hielo lo suficientemente profundo para que toda la altura de la jaula y todos los lados están en contacto con el hielo. Esto asegurará que todos los mosquitos se exponen al frío. Fría los mosquitos anestesiar durante unos 8 minutos o hasta que ya no son móviles. </li><li> Coloque un mosquito en un portaobjetos de la depresión. Esta diapositiva será el sitio para las inyecciones. </li></ol> 4. Colección de hemocitos <ol><li> Establecer el microinyector tomar hasta 12 l de diluyente hemolinfa utilizando una aguja de vidrio tirado e inyectar la solución en el mosquito entre los segmentos abdominales VII y VIII. Alrededor de 3 mosquitos pueden ser inyectados con 3-3,5 l de un total de 12 l de volumen ocupado. El mosquito debe ser agitado en su lugar con una pinza mientras que la inyección se lleva a cabo. Un pequeño volumen de diluyente debe permanecer en la punta de la aguja con el fin de evitar que el aceite mineral de ser inyectada. Después de la inyección en el abdomen debería ser &quot;lleno&quot; o hinchado. </li><li> Lugar inyectado mosquitos en un recipiente limpio por separado en el hielo para recuperar durante 5 min. La incubación de la solución diluyente anticoagulante en el interior del mosquito ayudar a desalojar hemocitos adherirse al tejido interno. El promedio de 3 a 5 mosquitos se inyectan nuevos durante cada período de 5 minutos de recuperación. </li><li> Después de 5 minutos de recuperación, corte con tijeras de las piernas, las alas y la punta del abdomen (en el octavo segmento) de cada uno de los mosquitos de uno en uno con unas tijeras micro. Esto se debe hacer en la copa de recuperación se coloca en hielo. El corte de las patas y las alas reduce la probabilidad de que las escalas que se transfieren a las diapositivas de la colección. Nota: no corte las piernas y las alas muy cerca de los sitios de fijación torácica o corre el riesgo de la creación de otras aberturas de la hemolinfa de escapar. Hemolinfa sólo debe ser recogida a través de la abertura creada en el extremo del abdomen. </li><li> Lavar un portaobjetos de vidrio microscopio grabado con el 70% de etanol y se seca con un Kimwipe. Una posición de inyección y se recuperó de mosquitos en la diapositiva en el borde exterior del círculo grabado. Establecer microinyector para recoger 12 l de diluyente hemolinfa fresca e inyectar este volumen en el mosquito en la parte lateral de su mesotórax. Entregar más, pero no todo el volumen de diluyente (alrededor de 8 a 10 l) con el fin de evitar que el aceite mineral de ser inyectada. Un pequeño volumen de diluyente debe permanecer en la punta de la aguja. </li><li> La posición del mosquito por lo que la apertura abdominal corte en el borde del círculo de 1 cm. Después de la inyección de la hemolinfa diluida debe ser recogida dentro del área marcada. Una vez que se retira la aguja de la inyección mesothoracic, reanudar la celebración de los mosquitos entre las dos ramas de la pinza y apretar suavemente el abdomen con las pinzas para dirigir la hemolinfa en el área del círculo grabado. </li><li> Permitir hemolinfa diluida para secar en la diapositiva durante unos 10 minutos o hasta que esté seco de forma visible. </li></ol> 5. La fijación y la tinción de hemocitos <ol><li> Tinción de cada diapositiva por separado con HEMA3 7 (ver Materiales): Dip slide en fijador de 5 veces durante 1 segundo cada vez, Dipslide en la solución de I 3 veces durante 1 segundo cada uno y, por último, inmersión en solución de diapositivas II 3 veces durante 1 segundo cada uno. Enjuague la parte de atrás de la corredera con agua destilada. La parte delantera de la diapositiva no deben enjuagarse. Blot-seca la parte delantera de la diapositiva fuera del círculo que contiene grabados de tejidos teñidos. </li><li> Aplicar medio de montaje (por ejemplo, Shur / Monte o Polymount) alrededor del centro de grabado y colocar un cubreobjetos sobre ella. Presione suavemente hacia abajo en la hoja de la cubierta (25 x 25 mm) para que el montaje se extiende a medio y toda el área cubierta por la hoja de la cubierta (incluyendo círculo grabado que contiene tejido teñido). Permitir que se sequen por un mínimo de 3 horas o toda la noche. Hemocitos debe ser visto dentro de 2 semanas después de la recogida para una visualización óptima y de imagen. </li></ol> 6. Resultados representante Ejemplos de hemocitos teñidos fijos y HEMA3 recogidos de una hembra de Aedes aegypti adultos se muestran en la Figura 1-C. La figura 1A muestra una hemocitos que hemos identificado como una prohemocyte sobre la base de su pequeño tamaño, esférica y la forma esferoidal de alta nuclear de citoplasma (550x de aumento) 11. La figura 1B muestra una hemocitos clasificado como un oenocytoid en función de su forma esferoidal y el citoplasma alta proporción nuclear (550x aumentos). Por último, la Figura 1C muestra una hemocitos de granulocitos y tipo (550x aumentos). Los granulocitos son más granular en la naturaleza y tienden a ser más ameboides en la forma y el comportamiento, ya que se adhieren a las superficies de vidrio. Utilizando los criterios publicados previamente para la tipificación de hemocitos y la recuperación de 11, nuestro método de recolección de producido el mismo número de hemocitos totales y también encontró que los granulocitos constituyen el mayor porcentaje de hemocitos un total de 11 (Fig. 2). <img src="/files/ftp_upload/2772/2772fig1.jpg" alt="Figura 1" /> Figura 1. Imágenes de microscopio de luz de un representante HEMA3 prohemocyte fija, manchado (A), oenocytoid (B), y de granulocitos (C) de una Ae. aegypti hembra adulta. C = citoplasma, núcleo N = G = gránulos. <img src="/files/ftp_upload/2772/2772fig2.jpg" alt="Figura 2"/> Figura 2. Recuento total ± SE de todos los hemocitos y granulocitos obtenidos por los mosquitos hembra de nuestra dilución de la hemolinfa y el método de recolección.

<ol>
	<li>Enayati, A., Hemingway, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+management:+past,+present,+and+future.">Malaria management: past, present, and future.</a> Annu. Rev. Entomol. 55, 569-569 (2010).</li><li>Medlock, J., Luz, P. M., Struchiner, C. J., Galvani, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+transgenic+mosquitoes+on+dengue+virulence+to+humans+and+mosquitoes.">The impact of transgenic mosquitoes on dengue virulence to humans and mosquitoes.</a> The American Naturalist. 174, 565-565 (2009).</li><li>Baton, L. A., Garver, L., Xi, Z., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+studies+on+the+innate+immunity+of+disease+vectors.">Functional genomics studies on the innate immunity of disease vectors.</a> Insect Sci. 15, 15-15 (2008).</li><li>Thomas, R. E., Wu, W. K., Verleye, D., Rai, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midgut+basal+lamina+thickness+and+dengue-1+virus+dissemination+rates+in+laboratory+strains+of+Aedes+albopictus+(Diptera:+Culicidae).">Midgut basal lamina thickness and dengue-1 virus dissemination rates in laboratory strains of Aedes albopictus (Diptera: Culicidae).</a> J. Med. Entomol. 30, 326-326 (1993).</li><li>Bartholomay, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+infection+responses+in+the+haemocytes+of+the+mosquito,+Aedes+aegypti.">Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti.</a> Insect Mol. Biol. 16, 761-761 (2007).</li><li>Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+cellular+immune+response.">The insect cellular immune response.</a> Insect Sci. 15, 1-1 (2008).</li><li>Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+mortality+in+immune+challenged+mosquitoes+(Aedes+aegypti)+correlates+with+a+decrease+in+haemocyte+numbers.">Age-associated mortality in immune challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers.</a> Cell. Microbiol. 7, 39-39 (2005).</li><li>Chen, C. C., Laurence, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+on+humoral+encapsulation+of+microfilariae:+establishment+of+technique+and+description+of+reaction.">In vitro study on humoral encapsulation of microfilariae: establishment of technique and description of reaction.</a> International Journal for Parasitology. 17, 781-781 (1987).</li><li>Beerntsen, B. T., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dirofilaria+immitis:+effects+on+hemolymph+polypeptide+synthesis+in+Aedes+aegypti+during+melanotic+encapsulation+reactions+against+Microfilariae.">Dirofilaria immitis: effects on hemolymph polypeptide synthesis in Aedes aegypti during melanotic encapsulation reactions against Microfilariae.</a> Exp. Parasitol. 71, 406-406 (1990).</li><li>Hillyer, J. F., Schmidt, S. L., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+innate+immune+response+by+the+mosquito+Aedes+aegypti+is+mediated+by+hemocytes+and+independent+of+Gram+type+and+pathogenicity.">The antibacterial innate immune response by the mosquito Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity.</a> Microb. Infect. 6, 448-448 (2004).</li><li>Castillo, J. C., Robertson, A. E., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hemocytes+from+the+mosquitoes+Anopheles+gambiae+and+Aedes+aegypti.">Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.</a> Insect Biochem. Mol. Biol. 36, 891-891 (2006).</li></ol>

El método de recogida de hemocitos se describe aquí es una modificación de los métodos publicados anteriormente y permite aislar los preparativos limpia de hemocitos de los mosquitos Aedes, con menos pasos. Aunque nuestro interés particular se encuentra en la caracterización de las poblaciones de hemocitos en los mosquitos Aedes, creemos que esta técnica se puede aplicar a los grupos de mosquitos otros después de los ensayos iniciales se llevan a cabo para determinar los volú...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Medio de Schneider </td><td> Sigma-Aldrich </td><td> S0146 </td><td></td></tr><tr><td> De suero fetal bovino </td><td> Sigma-Aldrich </td><td> F0643 </td><td></td></tr><tr><td> Hema3 kit de tinción </td><td> Pescador </td><td> 123-869 </td><td></td></tr><tr><td> Agujas de vidrio (Borosilicato con filamento) </td><td> Sutter Instrumento </td><td> BF100-78-10 </td><td> 1.0mm OD y ID 0.78mm </td></tr><tr><td> Aguja extractor </td><td> Sutter Instrumentos </td><td> Modelo P-87 </td><td></td></tr><tr><td> Microinyector </td><td> Tritech Investigación </td><td> Minj-PD </td><td></td></tr><tr><td> Porta-agujas </td><td> Tritech Investigación </td><td> Minj-4 </td><td></td></tr></tbody></table>

a protocol for collecting and staining hemocytes from the yellow fever mosquito aedes aegypti

Los mosquitos son vectores de una serie de patógenos que causan enfermedades como el virus de la fiebre amarilla, parásitos de la malaria y filarias. Los laboratorios están investigando anti-patógeno componentes del sistema inmune innato en la especie vector de la enfermedad con la esperanza de la generación de mosquitos transgénicos que son refractarios a los agentes patógenos como 1, 2. El sistema inmune innato de los mosquitos se compone de varias líneas de defensa 3. Patógenos que logran escapar de la barrera impuesta por el mosquito del epitelio del intestino medio-alineado 4 entrar en la hemolinfa y el encuentro hemocitos circulantes, componentes celulares importantes que encapsulan y patógenos engullir 5, 6. Los investigadores no han encontrado pruebas de los tejidos hematopoyéticos en los mosquitos y la evidencia actual sugiere que el número de hemocitos se fija en la emergencia de adultos y los números en realidad puede disminuir a medida que el mosquito 7. La capacidad de recoger e identificar correctamente los hemocitos de los insectos de importancia médica es un paso esencial para los estudios en la inmunidad celular. Sin embargo, el pequeño tamaño de los mosquitos y el limitado volumen de hemolinfa plantean un desafío a la recogida de las células inmunitarias. Dos métodos establecidos para la recolección de hemocitos mosquitos incluyen la expulsión de la hemolinfa de un corte trompa 8, y el desplazamiento de volumen (perfusión), en el que la solución salina es inyectada en la región necklike membranosa entre la cabeza y el tórax (es decir, el cuello uterino) y la hemolinfa perfundidos recogidos se a partir de una apertura de partido en una región distal del abdomen 9, 10. Estas técnicas, sin embargo, están limitadas por la baja recuperación de los hemocitos y la posible contaminación por las células de grasa corporal, respectivamente 11. Más recientemente, un método conocido como de inyección de alta / recuperación de la recuperación mejorada de inmunocitos por el uso de tampones anticoagulante, mientras que la reducción de los niveles de contaminación de las escalas y los tejidos internos 11. Mientras que el método permite un mejor método de recopilación y mantenimiento de hemocitos para cultivo primario, que conlleva una serie de medidas de inyección y recolección que no son necesarios si el objetivo posterior es recoger, fijar y teñir hemocitos para el diagnóstico. Aquí, demostramos nuestro método de recolección de la hemolinfa de mosquitos que combina la simplicidad de la perfusión, usando tampones anticoagulante en lugar de solución salina, con la precisión de las técnicas de inyección de alta para aislar a los preparativos limpia de hemocitos en los mosquitos Aedes.

Watch this Scientific Journal Video about A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti at JoVE.com

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Un método simplificado pero preciso para recoger y mancha hemocitos mosquito se describe. Nuestro método combina la simplicidad de la perfusión con la precisión de las técnicas de inyección de alta para aislar a los preparativos limpia de hemocitos en los mosquitos Aedes. Este método facilita el conocimiento de los estudios que requieren los tipos de hemocitos y su abundancia.

Un protocolo para la recolección y la tinción de los hemocitos del mosquito de la fiebre amarilla Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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Research

JoVE Journal

Immunology and Infection

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

15.2K vistas.  University of Richmond. Un método simplificado pero preciso para recoger y mancha hemocitos mosquito se describe. Nuestro método combina la simplicidad de la perfusión con la precisión de las técnicas de inyección de alta para aislar a los preparativos limpia de hemocitos en los mosquitos Aedes. Este método facilita el conocimiento de los estudios que requieren los tipos de hemocitos y su abundancia.

Video: A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Protocolo para la producción de un cruce genético de los parásitos de la malaria de roedores

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Investigación

Inmunología e Infección

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

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.

Un ensayo de bioluminiscencia de calcio para el análisis funcional de los Mosquitos ( Aedes aegypti) Y Tick ( Rhipicephalus microplus) G receptores acoplados a proteínas

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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.

Un protocolo Chelex simple para la extracción de ADN a partir de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Un protocolo para el análisis de la hepatitis C de replicación del virus

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

Extracción de hemocitos de larvas de Drosophila melanogaster para infección microbiana y análisis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Co de tinción de los vasos sanguíneos y fibras nerviosas en el tejido adiposo

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Prueba IN Vitro ELISA para evaluar la potencia de la vacuna contra la rabia

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Análisis de competencia vectorial en mosquitos Aedes aegypti con el virus Zika

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Propagación del parásito microsporidiano Edhazardia aedis en mosquitos Aedes aegypti

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Alimentación y cuantificación de sangre de origen animal y comidas artificiales en mosquitos Aedes aegypti

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...