A. Preparazione della cultura gametocyte: <ol><li> Portare fresco sangue umano e nel siero a 37 ° C in un bagno d&#39;acqua. </li><li> In una cappa aspirante, in condizioni sterili, pipettare 1 ml di sangue caldo in una provetta Eppendorf. Spin il sangue in una centrifuga per 5 minuti a 2200 giri al minuto. Eritrociti dovrebbero essere trasformati in pellet; rimuovere ed eliminare il siero chiaro. Lavare erythocytes con un uguale volume di nuovo siero 3 volte per ottenere il sangue intero per la gametociti. </li><li> Recuperare la cultura gametocyte dal CO 2 incubatore o portacandele. Questa cultura deve essere mantenuta in RPMI media e alimentato le zanzare nel giorno 16 della cultura. Per determinare la gametocytemia% (il giorno 16 della cultura), preparare uno striscio sottile su un vetrino con una goccia di sangue e di eseguire una Geimsa macchia. Determinare il numero di gameti in un campo della cultura parassita. Di solito è nel range di 2 - 4%. Calcolare il volume di sangue intero che sarà necessario per ottenere il desiderato gametocytemia% finale (0,3 -1%). </li><li> Rimuovere la maggior parte dei media dalla vostra cultura gametocyte utilizzando una pipetta, facendo attenzione a non disturbare o aspirare i parassiti (parassiti apparirà come nero, mentre il supporto verrà rosa chiaro / rosso). Pipettare il resto dei media e parassiti in una provetta da 15 ml falco. Spin in una centrifuga per 5 minuti a 2200 giri al minuto. Parassiti e eritrociti si pellet, rimuovere e scartare i media sopranatante senza disturbare i parassiti. </li><li> Combina i parassiti con il volume di preparato sangue intero (da A2 passo) per darvi la gametocytemia finale desiderato in base ai calcoli precedenti. Pipetta per mescolare completamente. (~ 1 minuto) </li></ol> Questo è ora il campione si feed al tuo zanzare. Mantenere a 37 ° C in un bagnomaria. B. Alimentazione zanzare: <ol><li> Metti il tuo zanzare Anopheles (~ 30-50) in cera alberato bicchieri di carta coperto con rete rete protetta da un elastico o coperchio di cartone forniti con tazza. Usiamo un a batteria Aspiratore per questo passo. </li><li> Preparare la circolazione bagnomaria con l&#39;inserimento di ugelli su entrambi i lati di alimentatori di vetro per tubi in gomma. Collegare come alimentatori di cui hai bisogno in una serie, una fine della serie devono essere collegati da tubi al circolatore del bagnomaria (la parte che spinge l&#39;acqua attraverso gli alimentatori), l&#39;altra estremità della serie dovrebbe essere costituito da tubi di gomma immersi nella vasca da bagno per permettere all&#39;acqua circolare per svuotare di nuovo in bagno. Accendere il bagno fino a quando l&#39;acqua è di circa 37 ° C e l&#39;acqua circola attraverso gli alimentatori e di nuovo al bagno. Ciò è essenziale per mantenere il sangue caldo e mantenere i parassiti vivi. </li><li> Preparare la membrana (usiamo piccoli quadrati di distese parafilm) con l&#39;alimentatore che si estende intorno membrana di vetro, utilizzando la pressione per sigillare ermeticamente l&#39;apertura di alimentazione. Questa membrana sarà imitare la pelle. </li><li> Posizionare un alimentatore membrana rivolto verso il basso su ogni tazza di zanzara e alimentatori sicuro utilizzando fascette o nastro. La membrana deve sedersi a filo con la rete in modo zanzare può accedere dall&#39;interno della coppa. </li><li> Con attenzione pipetta parassita-farina di sangue (da A5 passo) nel collo dell&#39;alimentatore, assicurandosi che il sangue va tutto il senso attraverso il collo nel serbatoio, contattando la membrana in modo le zanzare possono accedere al sangue. Utilizziamo circa 200 ml di sangue per alimentatore, ma questo può variare. </li><li> Permettono di nutrire le zanzare. Permettiamo loro di mangimi per ≈ 30 minuti (variabile, a seconda theexperiment; per la maggior parte degli esperimenti, a volte monitoraggio quanti hanno già preso un pasto guardando il contenuto di sangue in addome). </li><li> Dopo l&#39;alimentazione, togliere alimentatori dalle coppe zanzara. Spegnere il bagno d&#39;acqua e staccare alimentatori da tubo. </li><li> Rimuovere la membrana alimentatori e immergersi nel 10% di candeggina per decontaminare e sciacquare con acqua. Luogo materiale contaminato (membrane, asciugamani di carta, guanti, ecc) in una piccola borsa rischio biologico e depositare in un recipiente di grandi dimensioni a rischio biologico. </li><li> Anestetizzare le zanzare mettendo coppe in frigo o in camera fredda. Una volta che le zanzare sono completamente anestetizzati, trasferirli su una piastra di Petri incorporato in un secchiello del ghiaccio. Ordina zanzare, guardando attentamente l&#39;addome per qualsiasi segno di un pasto di sangue, e scartare quelli che non sono stati caricati. Alcune zanzare sarà completamente gonfio mentre altri mostrano una stretta banda di sangue ingerito, tutti sono accettabili come &quot;nutriti&quot;. Mettere le zanzare reimmessa nelle tazze di cartone. </li><li> Fornire un pasto zucchero (un pezzo di cotone imbevuto di 10% saccarosio) alle zanzare, e incubare in normali condizioni insectary per 7 giorni. Ogni giorno, le zanzare devono essere monitorati per il tasso di mortalità. Inoltre, il cotone dovrebbe essere bagnato con il saccarosio, in modo che non siano privati ​​di cibo. E &#39;meglio, ancora, per aggiungere un tovagliolo di carta imbevuto di acqua distillata, per mantenere le zanzare umido. Queste zanzare sono P. falcipraum infetti, in modo che tutti la cura deve essere presa in modo che le zanzare non possono ESCAPe dalla tazza. E &#39;meglio mettere le tazze in una protezione cagefor piccolo doppio. </li></ol> C. Dissecting midguts zanzara e la stima dei carichi oocisti: <ol style="margin-top: 0in"><li style="tab-stops: list .5in"> Aspirare zanzare (dal passaggio B) da bicchieri di carta con un aspiratore a batteria. Anestetizzare le zanzare, che sono all&#39;interno del serbatoio estraibile aspiratore, passando a una stanza fredda o seppellire il serbatoio in ghiaccio per almeno 5 minuti o fino a quando le zanzare si fermano. </li><li style="tab-stops: list .5in"> Trasferimento zanzare anestetizzati dal serbatoio di un piatto di vetro Petri incorporati nel ghiaccio. Diffondere le zanzare per un singolo strato e coprire con un coperchio di plastica capsula di Petri. Zanzare che non possono essere ospitati sul piatto rimangono nel serbatoio, che viene restituita alla camera fredda o ghiaccio. </li><li style="tab-stops: list .5in"> Utilizzando pinze a punta fine, il trasferimento di una zanzara dal piatto a 100 ul di PBS contenute su un vetrino montato sotto un microscopio dissezione. Lasciare gli altri coperti nel piatto freddo di Petri. </li><li style="tab-stops: list .5in"> Utilizzando pinze a punta fine, tenere le zanzare al torace e l&#39;addome posteriore. Tirare il corpo senza fino a quando il midgut è esposto. Usare pinze per rimuovere il tessuto in eccesso dal budello gut.The appare come un bianco, sac-come corpo. Rimuovere il tessuto in eccesso dal gut.Mount l&#39;intestino in un pozzetto di un 12-ben vetrino e immergere in 5-10 microlitri di 0.2% mercurocromo. Squash e gettare la carcassa in towel.Continue carta umida per altre zanzare fino al 12-scivolo bene è pieno. Coprire il coraggio montato con un coprioggetto di vetro. </li><li style="tab-stops: list .5in"> Esaminare fegato al microscopio ottico, alla ricerca di oocisti rotonda che macchia rosa brillante contro il chiaro / tenue tessuto intestinale rosa. Conte oocisti, registrare e continuare con il campione successivo. Proseguire per tutte le zanzare da tutti i trattamenti sperimentali. </li><li style="tab-stops: list .5in"> Al termine, asciugare vetrino pulito con un tovagliolo di carta. Posizionare il tovagliolo di carta con le budella, e tovagliolo di carta con le carcasse, in piccolo sacchetto e gettarlo nella spazzatura. I guanti sono indossati in tutto procedura. Si fa attenzione a non lasciare alcuna fuga le zanzare infette. </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>

Ci possono essere delle variazioni in Plasmodium l&#39;infezione e il fenotipo determinazione infezione e laboratori diversi possono seguire diverse tecniche.

protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination

Una volta che un gene è identificato come potenzialmente refrattaria per la malaria, deve essere valutato per il suo ruolo nella prevenzione delle infezioni da Plasmodium all&#39;interno della zanzara. Questo protocollo illustra come l&#39;entità delle infezioni plasmodio di zanzare possono essere analizzati. Le tecniche per la preparazione della coltura gametocyte, nutrendosi di sangue umano membrana zanzare, e saggio titoli virale nel midgut zanzara sono dimostrati.

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

Protocollo per Plasmodium falciparum infezioni in Fenotipo Determinazione Zanzare e infezione

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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17.7K Views.  Johns Hopkins University. Una volta che un gene è identificato come potenzialmente refrattaria per la malaria, deve essere valutato per il suo ruolo nella prevenzione delle infezioni da Plasmodium all'interno della zanzara. Questo protocollo illustra come l'entità delle infezioni plasmodio di zanzare possono essere analizzati. Le tecniche per la preparazione della coltura gametocyte, nutrendosi di sangue umano membrana zanzare, e saggio titoli virale nel midgut zanzara sono dimostrati.

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.

Protocollo per la dengue in Zanzare (A. aegypti) e Fenotipo Determinazione infezione

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 ...

Ricerca

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.

Protocollo per saggi RNAi in zanzare adulte (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.

Un protocollo video di infezione retrovirale in Primaria intestinale 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 test multi-rilevamento per la malaria Trasmissione Zanzare

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.

Protocollo di controllo dell&#39;orologio per l&#39;analisi delle immagini: plugin 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.

Costretto la salivazione come un metodo per analizzare la competenza vettoriale delle zanzare

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 Coltura di gametociti e infezione da zanzare attraverso l'alimentazione artificiale della membrana

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.

Rilevamento di Plasmodium Sporozoites nelle zanzare Anopheles utilizzando un test di immunoassorbimento enzimatico

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.

Determinazione della preferenza di temperatura di zanzare e altri ectotermi

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.

Quantificazione dei costi di fitness nelle zanzare transgeniche Aedes aegypti

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