A. Preparación de la cultura de gametocitos: <ol><li> Llevan la sangre humana fresca y suero a 37 º C en un baño de agua. </li><li> En una campana de extracción, bajo condiciones estériles, pipetas de 1 mL de sangre caliente en un tubo eppendorf. Haga girar la sangre en una centrifugadora durante 5 minutos a 2200 rpm. Eritrocitos se sedimentan, retirar y desechar el suero. Lavado de eritrocitos con un volumen igual de suero de nuevo 3 veces para obtener la sangre de los gametocitos. </li><li> Recuperar la cultura de gametocitos de la incubadora de CO2 o un tarro de velas. Esta cultura se debe mantener en medio RPMI y se alimenta a los mosquitos en el día 16 de la cultura. Para determinar el% gametocytemia (el día 16 de la cultura) que una capa delgada sobre un portaobjetos con una gota de sangre y realizar una Geimsa mancha. Determinar el número de gametos en un campo de cultivo de parásitos. Por lo general, en el rango de 2 a 4%. Calcular el volumen de sangre total que se necesita para lograr el deseado gametocytemia% final (0,3 -1%). </li><li> Eliminar la mayor parte de los medios de comunicación de su cultura gametocitos utilizando una pipeta, teniendo cuidado de no perturbar o absorben los parásitos (parásitos aparecerá como negro, mientras que los medios de comunicación será la luz de color rosa / rojo). Pipeta el resto de los medios de comunicación y los parásitos en un tubo de 15 ml halcón. Girar en una centrífuga durante 5 minutos a 2200 rpm. Parásitos y eritrocitos de pellets, quite y deseche los medios de comunicación sobrenadante sin perturbar los parásitos. </li><li> Combine los parásitos con el volumen de sangre total preparado (de paso A2) para darle la gametocytemia final deseado de acuerdo a los cálculos anteriores. Pipeta para mezclar bien. (~ 1 minuto) </li></ol> Esta es ahora la muestra que se alimentan a su mosquitos. Mantener a 37 º C en un baño de agua. B. mosquitos Alimentación: <ol><li> Ponga su mosquitos Anopheles (~ 30-50) en vasos de cartón forrado de cera cubierta con red de malla asegurada por una banda de goma o una tapa de cartón suministrado con la taza. Nosotros usamos un aspirador a pilas para este paso. </li><li> Prepare la circulación del agua de baño de la inserción de las boquillas en ambos lados de los alimentadores de vidrio para tubo de goma. Conecte como alimentadores que necesite de una serie, un extremo de la serie debe estar conectado mediante un tubo al termostato del baño de agua (la parte que empuja el agua a través de los comederos), el otro extremo de la serie debe consistir en un tubo de goma sumergido en el baño para que el agua distribuida a vaciar de nuevo en el baño. A su vez en el baño hasta que el agua es de unos 37 º C y el agua circule a través de los alimentadores y de vuelta al baño. Esto es esencial para mantener caliente la sangre y mantener los parásitos vivos. </li><li> Prepare la membrana (nosotros usamos pequeños cuadrados de estirado parafina) se extiende por todo el alimentador de membrana de vidrio, utilizando la presión para sellar herméticamente la apertura del alimentador. Esta membrana se imitan la piel. </li><li> Coloque un alimentador de la membrana hacia abajo en cada taza de mosquitos y alimentadores seguro utilizando grapas o cinta adhesiva. La membrana debe sentarse al ras de la red para que los mosquitos pueden acceder a él desde el interior de la taza. </li><li> Cuidadosamente pipeta parásitos harina de sangre (de paso A5) en el cuello de la alimentación, lo que hace que la sangre recorre todo el camino a través del cuello en el depósito, en contacto con la membrana que los mosquitos pueden tener acceso a la sangre. Usamos unos 200 l de sangre por cada alimentador, pero esto puede variar. </li><li> Permita que los mosquitos se alimentan. Permitimos que la alimentación de ≈ 30 minutos (variable, dependiendo de theexperiment, para la mayoría de los experimentos, de vez en cuando el seguimiento de cómo muchos ya han tomado una comida mirando el contenido de sangre en el abdomen.) </li><li> Después de la alimentación, retire los alimentadores de las copas de los mosquitos. Apague el baño de agua y desconecte los alimentadores de la tubería. </li><li> Quitar la membrana y disfrutar alimentadores en 10% de lejía para desinfectar y enjuagar con agua. Coloque el material contaminado (membranas, toallas de papel, guantes, etc) en una pequeña bolsa de riesgo biológico y depositar en un recipiente grande de riesgo biológico. </li><li> Anestesiar a los mosquitos mediante la colocación de vasos en el refrigerador o cuarto frío. Una vez que los mosquitos son totalmente anestesiado, su transferencia en una placa de Petri incrustados en un cubo de hielo. Ordenar los mosquitos, mirando cuidadosamente el abdomen para detectar cualquier signo de una harina de sangre, y desechan las que no se alimentan. Algunos mosquitos serán completamente llena de sangre, mientras que otros muestran una estrecha banda de sangre ingerida, y todos son aceptables como &quot;alimentado&quot;. Ponga los mosquitos pasan a engrosar la vasos de cartón. </li><li> Proporcionar una comida de azúcar (un trozo de algodón empapado en 10% de sacarosa) a los mosquitos, y se incuba en condiciones normales de insectario durante 7 días. Todos los días, los mosquitos deben ser monitorizados por la tasa de mortalidad. Además, el algodón debe ser mojado con la sacarosa, por lo que no se les prive de los alimentos. Es mejor, sin embargo, al añadir una toalla de papel empapada en agua destilada, para mantener a los mosquitos húmedo. Estos mosquitos son P. falcipraum infectados, por lo que todos se debe tener cuidado para que no los mosquitos pueden escape de la copa. Lo mejor es poner los vasos en una protección cagefor doble pequeña. </li></ol> C. Disección midguts mosquito y la estimación de las cargas de ooquistes: <ol style="margin-top: 0in"><li style="tab-stops: list .5in"> Aspirar los mosquitos (desde el paso B) de vasos de cartón con un aspirador a pilas. Anestesiar a los mosquitos, que son en el interior del depósito de aspiración extraíble, al mudarse a una habitación fría o enterrar el depósito de hielo durante al menos 5 minutos o hasta que los mosquitos dejen de moverse. </li><li style="tab-stops: list .5in"> La transferencia de los mosquitos anestesiado desde el depósito hasta un plato de Petri de vidrio incrustados en el hielo. Propagación de los mosquitos de una sola capa y cubrir con una tapa de plato de plástico Petri. Los mosquitos que no se pueden acomodar en el plato de permanecer en el depósito, que se devuelve a la habitación fría o hielo. </li><li style="tab-stops: list .5in"> Utilizando unas pinzas de punta fina, la transferencia de un solo mosquito de la antena a 100 ul PBS contenida en un portaobjetos de vidrio montada en un microscopio de disección. Deja a los demás incluidos en el frío plato de petri. </li><li style="tab-stops: list .5in"> Utilizando unas pinzas de punta fina, mantener los mosquitos en el tórax y el abdomen posterior. Tire del cuerpo del otro hasta el intestino medio está expuesto. El uso de fórceps para extraer el exceso de tejido del intestino gut.The se parece a un blanco, en forma de saco del cuerpo. Retire el exceso de tejido de la gut.Mount el intestino en un pocillo de una placa de vidrio de 12 bien y sumergirse en l 50-10 del 0,2% mercurocromo. Aplastar el cuerpo y desechar el towel.Continue de papel húmeda para otros mosquitos hasta que la diapositiva de 12 y está lleno. Cubren las agallas montado con un cubreobjetos. </li><li style="tab-stops: list .5in"> Examinar las entrañas con un microscopio de luz, en busca de ooquistes ronda que se tiñen de color rosa brillante contra el tejido intestinal claro / rosa tenue. Contar con los ooquistes, registrar y seguir a la siguiente muestra. Seguir para todos los mosquitos de todos los tratamientos experimentales. </li><li style="tab-stops: list .5in"> Cuando haya terminado, pase de diapositivas limpiar con una toalla de papel. Coloque la toalla de papel con las tripas, y una toalla de papel con cadáveres, en la bolsa de riesgo biológico pequeñas y desechar en la basura. Se usan guantes durante todo el procedimiento. Hay que tener cuidado de no dejar que nadie se escape de mosquitos infectados. </li></ol>

<ol>
	<li>Avila, S. L., Ferreira, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+diagnosis:+a+review.">Malaria diagnosis: a review.</a> Braz J Med Biol Res. 29 (4), 431-443 (1996).</li><li>Bell, A. S., Ranford-Cartwright, 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=A+real-time+PCR+assay+for+quantifying+Plasmodium+falciparum+infections+in+the+mosquito+vector.">A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector.</a> Int J Parasitol. 34 (7), 795-802 (2004).</li><li>Collins, K. M., Hochberg, L. P., Ryan, P. R., Collins, W. E., Wirtz, R. A., Ryan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Plasmodium+malariae+infection+in+mosquito+vectors.">Quantification of Plasmodium malariae infection in mosquito vectors.</a> Ann Trop Med Parasitol. 98 (5), 469-472 (2004).</li><li>Czeher, C., Labbo, R., Djibrilla, A., Here, L., Arzika, I., Duchemin, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA+study+of+oocyst-sporozoite+transition+in+malaria+vectors.">ELISA study of oocyst-sporozoite transition in malaria vectors.</a> Parasite. 13 (3), 257-261 (2006).</li><li>Haghdoost, A. A., Mazhari, S., Bahadini, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+results+of+light+microscopy+with+the+results+of+PCR+method+in+the+diagnosis+of+Plasmodium+vivax.">Comparing the results of light microscopy with the results of PCR method in the diagnosis of Plasmodium vivax.</a> J Vector Borne Dis. 43 (2), 53-57 (2006).</li><li>Patsoula, E., Spanakos, G., Sofianatou, D., Parara, M., Vakalis, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-step,+PCR-based+method+for+the+detection+and+differentiation+of+Plasmodium+vivax.">A single-step, PCR-based method for the detection and differentiation of Plasmodium vivax.</a> Ann Trop Med Parasitol. 97 (1), 15-21 (2003).</li><li>Osta, M. A., Christophides, G. K., Kafatos, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mosquito+genes+on+Plasmodium+development.">Effects of mosquito genes on Plasmodium development.</a> Science. 303 (5666), 2030-2032 (2004).</li><li>Srinivasan, P., Abraham, E. G., Ghosh, A. K., Valenzuela, J., Ribeiro, J. M., Dimopoulos, G., Kafatos, F. C., Adams, J. H., Fujioka, H., Jacobs-Lorena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Plasmodium+and+Anopheles+transcriptomes+during+oocyst+differentiation.">Analysis of the Plasmodium and Anopheles transcriptomes during oocyst differentiation.</a> J Biol Chem. 279 (7), 5581-5587 (2004).</li></ol>

No puede haber algunas variaciones en la infección por Plasmodium y determinación fenotipo infección y los diferentes laboratorios pueden seguir diferentes técnicas.

protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination

Una vez que un gen es identificado como potencialmente resistentes a la malaria, que debe ser evaluado por su papel en la prevención de las infecciones por Plasmodium en el mosquito. Este protocolo se ilustra cómo la extensión de la infección por Plasmodium de mosquitos se puede probar. Las técnicas para la preparación del cultivo de gametocitos, la alimentación de los mosquitos membrana de sangre humana, y analizando los títulos virales en el intestino del mosquito se ha demostrado.

Watch this Scientific Journal Video about Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination at JoVE.com

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Protocolo de infecciones por Plasmodium falciparum en la determinación de fenotipo mosquitos y la infección

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

protocol-for-plasmodium-falciparum-infections-mosquitoes-infection

Research

JoVE Journal

Biology

17.8K vistas.  Johns Hopkins University. Una vez que un gen es identificado como potencialmente resistentes a la malaria, que debe ser evaluado por su papel en la prevención de las infecciones por Plasmodium en el mosquito. Este protocolo se ilustra cómo la extensión de la infección por Plasmodium de mosquitos se puede probar. Las técnicas para la preparación del cultivo de gametocitos, la alimentación de los mosquitos membrana de sangre humana, y analizando los títulos virales en el intestino del mosqui...

Video: Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

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.

Protocolo para las infecciones de dengue en mosquitos (Aedes aegypti) y determinación de fenotipo infección

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

Investigación

Biología

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

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 Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

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 Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocolo para ensayos de RNAi en mosquitos adultos (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Análisis de la elaboración de un fenotipo morfológico en función de la concentración de proteínas en la levadura en ciernes

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Un Protocolo de vídeo de la infección retroviral en Primaria Intestinal organoide Cultura

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

Un ensayo multi-detección para los mosquitos transmisores de la malaria

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

Protocolo de análisis de reloj para análisis de imagen: Plugins de ImageJ

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

Un protocolo de laboratorio vivienda de turquesa killis (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Oral infección bacteriana y vertimiento en Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protocolos para las pruebas de la toxicidad de la novela química insecticida para mosquitos

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

Forzado de la salivación como un método para analizar la competencia de vectores de mosquitos

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...