A. Herstellung von gametocyte Kultur: <ol><li> Bringen Sie frischen menschlichen Blut und Serum auf 37 o C in einem Wasserbad. </li><li> In einem Abzug, unter sterilen Bedingungen, pipet 1 ml warmes Blut in ein Eppendorf-Röhrchen. Drehen Sie das Blut in eine Zentrifuge für 5 Minuten bei 2200 Umdrehungen pro Minute. Erythrozyten sollten pelletiert; entfernen und entsorgen das klare Serum. Wash Erythozyten mit dem gleichen Volumen der neuen Serum 3 mal das ganze Blut für die gametocytes erhalten. </li><li> Rufen Sie die gametocyte Kultur aus dem CO 2-Inkubator oder Windlicht. Diese Kultur sollte auf RPMI Medien und gefüttert, um die Mücken am Tag 16 der Kultur gepflegt werden. Zur Bestimmung der gametocytemia% (am Tag 16 der Kultur) eine dünne Schicht auf eine Glasplatte mit einem Tropfen Blut und führen Sie eine Geimsa Fleck. Bestimmen Sie die Anzahl der Gameten in einem Feld des Parasiten Kultur. Es ist in der Regel im Bereich von 2 bis 4%. Berechnen Sie das Volumen von Vollblut, die Sie benötigen, um die gewünschte endgültige% gametocytemia (0,3 -1%) zu erreichen. </li><li> Entfernen Sie die meisten Medien von Ihrem gametocyte Kultur mit einer Pipette, dabei nicht zu stören oder zu saugen die Parasiten (Schmarotzer erscheint als schwarz, während die Medien werden rosa / rot). Pipettieren der Rest der Medien und Parasiten in ein 15 ml Falcon-Röhrchen. Spin in einer Zentrifuge für 5 Minuten bei 2200 Umdrehungen pro Minute. Parasiten und Erythrozyten Pellet, entfernen und entsorgen Medien Überstand ohne störende Parasiten. </li><li> Kombinieren Parasiten mit dem Volumen der vorbereiteten Vollblut (aus Schritt A2), um Ihnen die gewünschte endgültige gametocytemia nach den bisherigen Berechnungen. Pipettieren gründlich mischen. (~ 1 Minute) </li></ol> Dies ist nun das Beispiel, das Sie zu Ihrem Mücken ernähren. Halten bei 37 o C in einem Wasserbad. B. Fütterung Moskitos: <ol><li> Legen Sie Ihre Anopheles-Mücken (~ 30-50) in Wachs ausgekleideten Karton Tassen mit Netzgewebe durch ein Gummiband oder Pappdeckel mit Becher geliefert gesichert abgedeckt. Wir verwenden eine batteriebetriebene Sauger für diesen Schritt. </li><li> Bereiten Sie die Durchblutung Wasserbad durch Einfügen Düsen auf beiden Seiten des Glases Zubringer zu Gummischlauch. Schließen Sie so viele Anleger, wie Sie in einer Reihe müssen, ein Ende der Serie sollte durch Schlauch mit dem Thermostat des Wasserbades (der Teil, Wasser drückt durch den Zubringer) angeschlossen werden, sollten Sie das andere Ende der Reihe von Gummischlauch bestehen untergetaucht in der Wanne, damit der Kreislauf Wasser leer zurück ins Bad. Schalten Sie das Bad, bis das Wasser etwa 37 o C und das Wasser wird durch den Anleger im Umlauf und zurück in die Badewanne. Dies ist notwendig, um das Blut warm zu halten und halten die Parasiten am Leben. </li><li> Bereiten Sie die Membran (wir kleine Quadrate gestreckt Parafilm) durch Strecken rund um die Glasmembran Anleger, mit Druck dicht zu verschließen, um die Zuführung Öffnung. Diese Membran wird imitieren die Haut. </li><li> Legen Sie einen Feeder-Membran-Seite nach unten auf die einzelnen Moskito-Cup und sichere Anleger mit Klammern oder Klebeband. Die Membran sitzen sollte mit dem Netz bündig so Mücken kann man aus dem Inneren der Tasse zugreifen. </li><li> Sorgfältig Pipette Parasiten Blutmahlzeit (aus Schritt A5) in den Hals des Anlegers, so dass das Blut geht den ganzen Weg durch den Hals in das Reservoir, Kontaktierung der Membran, so dass die Mücken das Blut zugreifen können. Wir verwenden etwa 200 ul Blut pro System, aber das kann variieren. </li><li> Lassen Mücken zu füttern. Wir können sie für ≈ 30 Minuten Feed (variabel, je nach theexperiment; für die meisten Experimente, gelegentlich kontrollieren, wie viele haben bereits eine Mahlzeit suchen Sie in das Blut-Gehalt in den Bauch getroffen.) </li><li> Nach der Fütterung, Abrüsten von der Mücke Tassen. Schalten Sie das Wasserbad und ziehen Anleger von Schläuchen. </li><li> Entfernen Sie die Membran und genießen Anleger in 10% Bleichmittel zu dekontaminieren und mit Wasser abspülen. Kontaminierte Materialien (Membranen, Papiertücher, Handschuhe, etc.) in einen kleinen Beutel für biologisches Risikomaterial und Einzahlung in einen großen Behälter biohazard. </li><li> Anesthetize Mücken, indem Tassen im Kühlschrank oder Kühlraum. Sobald Mücken sind voll narkotisiert, übertragen Sie sie auf eine Petrischale in einem Eiskübel eingebettet. Sortieren Moskitos, suchen Sie genau auf den Bauch auf Anzeichen einer Blutmahlzeit, und entsorgen Sie diejenigen, die nicht eingezogen hat. Einige Moskitos voll engorged werden, während andere ein schmales verzehrte Blut zeigen wird, alle sind akzeptabel, &quot;gefüttert&quot;. Legen Sie die gefüttert Moskitos wieder in den Pappbecher. </li><li> Geben Sie einen Zucker essen (ein Stück Baumwolle in 10% Saccharose eingeweicht), um Mücken, und inkubieren unter normalen Bedingungen Insektarium für 7 Tage. Jeden Tag sollten die Mücken für die Sterblichkeitsrate überwacht werden. Auch sollte die Baumwolle nass mit der Saccharose werden, so dass sie nicht die Nahrung entzogen. Es ist besser, noch ein Papiertuch in destilliertem Wasser getränkt hinzuzufügen, zu halten die Mücken feucht. Diese Mücken sind P. falcipraum infiziert, so dass alle darauf geachtet werden muss, damit keine Mücken können escape aus dem Becher. Am besten ist es die Tassen in einem kleinen cagefor doppelten Schutz zu stellen. </li></ol> C. Dissecting Mücke Mitteldärmen und Schätzung oocyst Lasten: <ol style="margin-top: 0in"><li style="tab-stops: list .5in"> Absaugen Mücken (aus Schritt B) aus Pappbecher mit einer Batterie betrieben Aspirator. Anesthetize Mücken, die in dem herausnehmbaren Aspirator Reservoir sind, indem sie zu einem kalten Raum oder Vergraben des Reservoirs in Eis für mindestens 5 Minuten oder bis Mücken nicht mehr bewegt. </li><li style="tab-stops: list .5in"> Transfer betäubt Mücken aus dem Reservoir zu einer Glas-Petrischale in Eis eingebettet. Verbreiten Sie die Mücken zu einer einzigen Schicht und Abdeckung mit einer Kunststoff-Petrischale Deckel. Mücken, die nicht auf den Teller untergebracht werden können bleibt im Behälter, die kalten Raum oder Eis zurückgekehrt ist. </li><li style="tab-stops: list .5in"> Mit spitzen Pinzette, Übertragung einer Mücke aus der Schüssel zu 100 ul PBS auf einem Objektträger unter einem Dissektionsmikroskop montiert enthalten. Lassen Sie die anderen in der gekühlten Petrischale abgedeckt. </li><li style="tab-stops: list .5in"> Mit spitzen Pinzette, halten Mücken an der Brust und der hintere Bauch. Ziehen Sie den Körper auseinander, bis die Mitteldarm ausgesetzt ist. Verwenden einer Pinzette, um überschüssiges Gewebe aus gut.The gut entfernen sieht aus wie eine weiße, sackartige Körper. Entfernen Sie überschüssiges Gewebe aus der gut.Mount den Darm in eine Vertiefung einer 12-well Objektträger aus Glas und tauchen in 5-10 ul von 0,2% Mercurochrom. Squash die Karkasse und entsorgen auf feuchtem Papier towel.Continue für andere Mücken, bis der 12-Well-Rutsche ist voll. Bedecken Sie den Mut montiert mit einem Deckglas. </li><li style="tab-stops: list .5in"> Untersuchen Sie Mut unter einem Lichtmikroskop auf der Suche nach runden Oozysten, die leuchtend rosa gegen den klaren / zartrosa Darmgewebe Fleck. Graf Oozysten, aufnehmen und weiter auf die nächste Probe. Weiter für alle die Moskitos aus allen experimentellen Behandlungen. </li><li style="tab-stops: list .5in"> Wenn Sie fertig sind, wischen Sie gleiten sauber mit einem Papiertuch. Legen Sie das Papiertuch mit Mut und Papiertuch mit Kadavern, in kleine Beutel für biologisches Risikomaterial und entsorgen Sie in den Papierkorb. Handschuhe sind im gesamten Verfahren getragen. Es wird darauf geachtet, keine infizierten Mücken entweichen zu lassen. </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>

Es kann einige Variationen in der Plasmodium-Infektion und Infektion Phänotyp Bestimmung und verschiedenen Labors unterschiedliche Techniken zu folgen.

protocol for plasmodium falciparum infections in mosquitoes and infection phenotype determination

Sobald ein Gen als potentiell feuerfesten für Malaria identifiziert wird, muss er für seine Rolle bei der Verhinderung Plasmodium-Infektionen innerhalb der Mücke ausgewertet werden. Dieses Protokoll zeigt, wie das Ausmaß der Plasmodium-Infektionen von Mücken getestet werden kann. Die Techniken für die Vorbereitung der gametocyte Kultur-, Membran-Fütterung Moskitos menschlichem Blut und Testen von viralen Titer in der Mücke Mitteldarm demonstriert.

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

Protokoll für Plasmodium falciparum-Infektionen in Mücken und Infektionsbiologie Phänotyp Bestimmung

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

17.7K Aufrufe.  Johns Hopkins University. Sobald ein Gen als potentiell feuerfesten für Malaria identifiziert wird, muss er für seine Rolle bei der Verhinderung Plasmodium-Infektionen innerhalb der Mücke ausgewertet werden. Dieses Protokoll zeigt, wie das Ausmaß der Plasmodium-Infektionen von Mücken getestet werden kann. Die Techniken für die Vorbereitung der gametocyte Kultur-, Membran-Fütterung Moskitos menschlichem Blut und Testen von viralen Titer in der Mücke Mitteldarm demonstriert.

Serie: 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.

Protokoll für Dengue-Infektionen in Mücken (Aedes aegypti) und Infektionsbiologie Phänotyp Bestimmung

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 ...

Forschung

Biologie

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.

Protokoll für die RNAi-Assays in Adult Moskitos (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.

Ein Video-Protokoll von retrovirale Infektion in der Grunddarm Organoide Kultur

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.

Ein Multi-Nachweistest für Malaria tragenden Mücken

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.

Clock Scan Protocol für Bildanalyse: 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 ...

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.

Gezwungen Speichelfluss als Methode zur Vektorkompetenz von Stechmücken Analyse

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 Gametozytenkultur und Mückeninfektion durch künstliche Membranfütterung

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.

Nachweis von Plasmodium Sporozoiten in Anopheles-Mücken mit einem 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 ...

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.

Bestimmung der Temperaturpräferenz von Moskitos und anderen Ektothermen

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.

Quantifizierung der Fitnesskosten bei transgenen Aedes aegypti-Mücken

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