A. Preparação da cultura gametócitos: <ol><li> Trazem o sangue humano fresco e soro a 37 o C em banho-maria. </li><li> Em uma capela, sob condições estéreis, pipeta de 1 mL de sangue quente em um tubo eppendorf. Girar o sangue em uma centrífuga por 5 minutos a 2200 rpm. Eritrócitos deve ser peletizada, remover e descartar o soro claro. Lavar eritrócitos com um volume igual de novo soro 3 vezes para obter o sangue total para a gametócitos. </li><li> Recuperar a cultura gametócitos do CO 2 incubadora ou palmatória. Essa cultura deve ser mantida em RPMI mídia e alimentado para os mosquitos no dia 16 da cultura. Para determinar o gametocytemia% (no dia 16 de cultura) fazer um esfregaço fino sobre uma lâmina de vidro com uma gota de sangue e realizar uma Geimsa mancha. Determinar o número de gametas em um campo da cultura parasita. Geralmente é na faixa de 2-4%. Calcular o volume de sangue total que você vai precisar para alcançar o final desejado gametocytemia% (0,3 -1%). </li><li> Remover a maioria dos meios de comunicação de sua cultura gametócitos usando uma pipeta, tomando cuidado para não perturbar ou sugar os parasitas (parasitas aparecerá como preto, enquanto a mídia vai ser rosa claro / vermelho). Pipeta o resto da mídia e parasitas em um tubo falcon 15 ml. Girar em uma centrífuga por 5 minutos a 2200 rpm. Parasitas e eritrócitos vai pellet, remover e descartar media sobrenadante sem perturbar parasitas. </li><li> Combine parasitas com o volume de sangue total preparadas (de A2 etapa) dar-lhe a gametocytemia final desejada de acordo com os cálculos anteriores. Pipeta para misturar bem. (~ 1 minuto) </li></ol> Este é agora o exemplo que você vai alimentar a sua mosquitos. Manter a 37 º C em banho-maria. Alimentação mosquitos B.: <ol><li> Ponha seu mosquitos Anopheles (~ 30-50) em copos de cera revestido de papelão coberto com malha de redes garantidos por uma faixa de borracha ou tampa de papelão fornecido com copo. Nós usamos um aspirador a pilhas para esta etapa. </li><li> Prepare o banho-maria a circulação através da inserção de bicos em cada lado de alimentadores de vidro para tubos de borracha. Conectar como alimentadores quantas forem necessárias em uma série, um fim da série deve ser conectado por tubulação para o circulador do banho de água (a parte que empurra a água através do feeders), a outra extremidade da série deve ser composto de tubos de borracha submerso no banho para que a água distribuída aos vazios de volta para banho. Ligue o banho até que a água é de cerca de 37 º C ea água está a circular através do alimentadores e de volta para o banho. Isto é essencial para manter o aquecimento de sangue e manter os parasitas vivos. </li><li> Prepare a membrana (usamos pequenos quadrados de parafilme esticado) esticando todo o alimentador de membrana de vidro, usando a pressão para selar firmemente a abertura do alimentador. Esta membrana vai imitar a pele. </li><li> Coloque um alimentador da membrana voltada para baixo em cada copo mosquito e alimentadores seguro usando grampos ou fita adesiva. A membrana deve sentar-se alinhada com a rede para os mosquitos podem acessá-lo de dentro do copo. </li><li> Cuidadosamente pipeta refeição parasita sangue (de A5 passo) para o pescoço do alimentador, certificando-se o sangue vai todo o caminho através do pescoço para dentro do reservatório, entrar em contato com a membrana de modo que o mosquito pode acessar o sangue. Usamos cerca de 200 mL de sangue por alimentador, mas isso pode variar. </li><li> Permitir que mosquitos para se alimentar. Nós lhes permitem alimentar para ≈ 30 minutos (variável, dependendo theexperiment; para a maioria dos experimentos, ocasionalmente monitoramento quantos já tomaram uma refeição por olhar para o conteúdo sangüíneo no abdômen.) </li><li> Após a alimentação, retire copos alimentadores do mosquito. Desligue o banho de água e desconecte alimentadores de tubos. </li><li> Remover a membrana e mergulhe alimentadores em lixívia a 10% para descontaminar e enxágüe com água. Lugar material contaminado (membranas, toalhas de papel, luvas, etc) em um pequeno saco de risco biológico e depósito em um recipiente grande risco biológico. </li><li> Anestesiar os mosquitos colocando copos na geladeira ou câmara fria. Uma vez que os mosquitos estão totalmente anestesiados, transferi-los em uma placa de Petri incorporado em um balde de gelo. Ordenar mosquitos, olhando atentamente para o abdômen para qualquer sinal de uma refeição de sangue, e descartar aqueles que não alimentam. Alguns mosquitos será totalmente ingurgitadas, enquanto outros mostram uma estreita faixa de sangue ingerido, todos são aceitáveis ​​como &quot;alimentados&quot;. Coloque os mosquitos alimentado de volta para os copos de papelão. </li><li> Fornecer uma refeição de açúcar (um pedaço de algodão embebido em sacarose 10%) para os mosquitos, e incubar em condições normais de insetário durante 7 dias. Todos os dias, os mosquitos devem ser monitorados para a taxa de mortalidade. Além disso, o algodão deve ser molhado com a sacarose, para que eles não são privados de alimentos. É melhor, mas, para adicionar uma toalha de papel embebido em água destilada, para manter os mosquitos úmido. Esses mosquitos são P. falcipraum infectados, para que todos os cuidados devem ser tomados para que não os mosquitos podem ESCAPe do copo. É melhor colocar os copos em uma proteção cagefor pequena dupla. </li></ol> C. Dissecting intestino médio do mosquito e estimar as cargas de oocistos: <ol style="margin-top: 0in"><li style="tab-stops: list .5in"> Aspirar mosquitos (da etapa B) de copos de papelão usando um aspirador a pilhas. Anestesiar os mosquitos, que estão dentro do reservatório aspirador removível, mudando-se para uma sala fria ou enterrar o reservatório no gelo por pelo menos 5 minutos ou até que os mosquitos parar de se mover. </li><li style="tab-stops: list .5in"> Transferência de mosquitos anestesiados do reservatório para uma placa de Petri de vidro embebidas em gelo. Espalhe os mosquitos em uma única camada e cubra com uma tampa de plástico placa de Petri. Mosquitos que não podem ser acomodados no prato permanecer no reservatório, que é retornado ao quarto frio ou gelo. </li><li style="tab-stops: list .5in"> Usando uma pinça de ponta fina, a transferência de um único mosquito do prato para 100 ul PBS contidos em uma lâmina de vidro montada em um microscópio de dissecação. Deixe os outros coberta na placa de Petri refrigeradas. </li><li style="tab-stops: list .5in"> Usando uma pinça de ponta fina, manter os mosquitos no tórax e do abdome posterior. Puxar o corpo para além até o intestino médio está exposto. Use uma pinça para remover o excesso de tecido do intestino gut.The se parece com um branco, saco-como corpo. Retire o excesso de tecido do intestino gut.Mount em um poço de uma lâmina de vidro de 12 bem e mergulhe em mL 10/05 de 0,2% mercurocromo. Squash a carcaça e descartar em towel.Continue papel úmido para outros mosquitos até o slide 12 bem-estar cheio. Cobrir a coragem montado com uma lamela de vidro. </li><li style="tab-stops: list .5in"> Examine coragem em microscópio de luz, à procura de oocistos redonda que mancha rosa brilhante contra o tecido do intestino clara / rosada. Contagem de oocistos, e continuar a gravar a próxima amostra. Continue para todos os mosquitos de todos os tratamentos experimentais. </li><li style="tab-stops: list .5in"> Quando terminar, limpe lâmina limpa com uma toalha de papel. Coloque a toalha de papel com coragem, e toalha de papel com carcaças, em saco de biohazard pequeno e descartar no lixo. As luvas são usadas durante todo o processo. Cuidado é tomado para não deixar escapar 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>

Pode haver algumas variações na infecção de Plasmodium e determinação fenótipo infecção e laboratórios diferentes podem seguir diferentes técnicas.

protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination

Uma vez que um gene é identificada como potencialmente refratário para a malária, deve ser avaliado pelo seu papel na prevenção de infecções Plasmodium dentro do mosquito. Este protocolo ilustra como a extensão de infecções plasmodium de mosquitos podem ser ensaiadas. As técnicas de preparação da cultura gametócitos, membrana alimentarem de sangue humano os mosquitos, e assaying títulos viral no intestino médio do mosquito são demonstradas.

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 para Infecções Plasmodium falciparum em mosquitos e Infecção Determinação Fenótipo

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

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JoVE Journal

Biology

17.7K visualizações.  Johns Hopkins University. Uma vez que um gene é identificada como potencialmente refratário para a malária, deve ser avaliado pelo seu papel na prevenção de infecções Plasmodium dentro do mosquito. Este protocolo ilustra como a extensão de infecções plasmodium de mosquitos podem ser ensaiadas. As técnicas de preparação da cultura gametócitos, membrana alimentarem de sangue humano os mosquitos, e assaying títulos viral no intestino médio do mosquito são demonstradas.

Vídeo: 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 infecções por dengue em mosquitos (A. aegypti) e Determinação Fenótipo Infecção

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 ...

Biologia

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 Ensaios RNAi em mosquitos adultos (A. gambiae)

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

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.

Um Protocolo de vídeo de infecção retroviral na Primária Intestinal organóide 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.

Um ensaio para detecção multi-malária mosquitos transmissores

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 varredura do relógio para análise de imagem: plugins 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 ...

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.

Forçado a salivação como um método para analisar a competência vetorial 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 ...

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

Plasmodium falciparum Cultura gametocito e infecção por mosquitos através da alimentação artificial

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.

Detecção de esporozoítos de Plasmodium em mosquitos Anopheles usando um ensaio imunoenzimático

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female ...

Determining Temperature Preference of Mosquitoes and Other Ectotherms

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on their fitness, as well as other characteristics. Consequently, many such ectotherms seek out their optimal temperature range. Although temperature preferences of mosquitoes and other insects have been well studied, the traditional experimental setup is performed using a temperature gradient on an aluminum surface in a highly enclosed space. In some cases, this equipment restricts many natural behaviors, such as flying, which may be important in preference selection.
The objective of this study is to observe insect preference for air temperature by using a two-chamber apparatus with sufficient room for flight. The two chambers consist of independent temperature-controlled incubators, each with a large aperture. The incubators are connected by these apertures using a short acrylic bridge. Inside the incubators are two netted cages, linked via the apertures and bridge, allowing the insects to freely fly between the different conditions. The acrylic bridge also acts as a temperature gradient between the two incubators.
Due to the spacious area in the cage and easy construction, this method can be used to study any small ectotherm and/or any manipulation which may alter temperature preference including sensory organ manipulation, diet, gut flora, and endosymbiont presence at biosafety levels 1 or 2 (BSL 1 or 2). Additionally, the apparatus can be used for the study of pathogen infection using further containment (e.g., inside of a biosafety cabinet) at BSL 3.

Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors.

Determinando a preferência de temperatura de mosquitos e outros ectotérmicos

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on ...

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life parameter data from genetically modified mosquitoes and comparing them to mosquitoes lacking transgenes from the same genetic background. This manuscript illustrates how to measure common life history traits in the mosquito Aedes aegypti, including fecundity, wing size and shape, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. The representative results quantify fitness costs associated with either a gene knock-out or a single insertion of a gene drive element. Standardizing how life parameter data are collected is important because such data may be used to compare the health of transgenic mosquitoes generated across studies or to model the transgene fixation rate in a simulated wild-type mosquito population. Although this protocol is specific for transgenic Aedes aegypti, the protocol may also be used for other mosquito species or other experimental treatment conditions, with the caveat that certain biological contexts may require special adaptations.

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

Quantificando os custos de aptidão em mosquitos Aedes aegypti transgênicos

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life ...