A. Propagate das Virus in den C6/36 Zelllinie. <ol><li> Zellen wachsen zu 80% Konfluenz in 75 cm 2-Kolben; </li><li> Entfernen Sie die Medien mit 3-4 ml im Kolben verbliebene; </li><li> Nehmen Sie 0,5 ml Lager Virus und fügen in die obige Zelle mit der Multiplizität der Infektion von 1,5 Viruspartikeln pro Zelle. Schütteln der Flasche für 15 Minuten langsam auf Raumtemperatur. </li><li> Inkubieren für 45 Minuten bei 5% CO 2 und 37 ° C </li><li> 30 ml Medien und Inkubation für 5 Tage. </li></ol> B. Vorbereiten der Mischung von Virus und Blut <ol><li> Lösen Sie die Zelle mit dem Schaber und übertragen Sie die Zelle und Medien, um die 50 ml konische Röhrchen; </li><li> Zentrifugat bei 800g für 10 Minuten; den Überstand, aber lassen Sie 1 ml Überstand mit dem Zellpellet; </li><li> Frieren Sie den oben Zellpellet in trockenen CO 2 und dann Tauwetter in den 37 ° C Wasserbad, wiederholen zwei vor drei Mal; </li><li> Zentrifugat bei 800g für 10 Minuten, nehmen Sie den Überstand und verbinden sich mit dem Überstand aus Schritt 3 gesammelt; </li><li> Sammeln menschlichem Vollblut mit dem gleichen Verfahren (Schritt 3) für die Herstellung von gametocyte Kultur Anopheles-Mücke mit Plasmodium falciparum infiziert; </li><li> Kombinieren Sie die gleiche Menge der oben menschlichem Vollblut mit dem Virus Überstand aus Schritt 4, und fügen menschlichem Serum (10% des gesamten Volumens). </li><li> Inkubieren Sie die obige Mischung von menschlichem Blut und Dengue-Virus für 30 Minuten bei 37 ° C Wasserbad </li></ol> C. Fütterung Moskitos Dieses Verfahren ist nahezu identisch zu dem, was in dem Abschnitt für Plasmodium falciparum-Infektionen in Mücken beschrieben worden. Wir Assay Mitteldarm Infektion an Tag 7, und der Speichel-Infektion an Tag 14 nach Blut-Fütterung. Alle Materialien, die in Kontakt mit dem Virus kam zunächst mit 75% Ethanol und dann mit 10% Bleichmittel behandelt. D. Assay der Virustiter in Mücke Gewebe <ol><li> Zwei oder drei Tage vor dem Sezieren von Moskito Gewebe wachsen C6/36 Zellen in einer 24-Well-Platte, so dass die Zelle 80% Konfluenz am Tag, wenn der Test durchgeführt wird erreicht; </li><li> Moskitos sind bei 4 ° C betäubt, getötet und tauchte sterile durch Einweichen in 75% Ethanol für 1 Minute. Dann Mücken wurden mit sterilem Wasser zweimal, bevor Dissektion der Mitteldarm oder Speicheldrüse unter dem Binokular gewaschen. Von diesem Schritt muss alle Schritte aus, um in einer sterilen Umgebung wie eine biologische Sicherheitswerkbank durchgeführt werden. Diese Gewebe werden in ein Röhrchen mit 150 ul Medium übertragen und homogenisiert mit einem Kontes Pellet Pistill Motor für 90 Sekunden; </li><li> Machen Sie eine 1:100 Verdünnung durch Zugabe von 10 ul des Homogenat in 990 ul Medien; </li><li> Machen Sie weiter 1: 10, 1:100 und 1:1000 Verdünnung mit dem Medium in der 96-Well-Platte; </li><li> Entfernen Sie die Medien in den 24-Well-Platte und 100 ul von jedem der vier oben genannten Verdünnungen auf die jeweils gut; </li><li> Schütteln Sie die Platte langsam für 15 Minuten bei Raumtemperatur, dann 45 Minuten bei 5% CO 2 inkubiert und 37 ° C; </li><li> 1ml von methycellulose Overlay (1%) in jede Vertiefung; </li><li> Die Platten bei 37 ° C mit 5% CO 2 für 5 Tage; </li><li> Die Schritte von hier aus benötigen keine sterile Umgebung, sondern wie bei jedem anderen Erreger Pflege muss jederzeit berücksichtigt werden. Discard methycellulose Overlay von jeder Platte und Blot, um überschüssige Medien entfernen </li><li> Add 1 ml Methanol / Aceton Fixativ (1:1) in jede Vertiefung. Halten Sie bei 4 ° C für 1 Stunde; </li><li> Abgießen Fixativ, und waschen Sie einmal mit 1X PBS; </li><li> Machen 1:1000 Verdünnung an den primären Antikörper gegen das Virus mit 5% blotto; </li><li> Add 200 ul verdünnt hyperimmune Flüssigkeit in jede Vertiefung, bei 37 ° C inkubieren für 1 Stunde; </li><li> Entfernen hyperimmune Flüssigkeit und waschen Teller mit 1 X PBS </li><li> Bereiten Sie eine 1:1000 Verdünnung von Ziegen-anti-Maus-Konjugat (KPL Cat # 074-1806) in 5% blotto (5g Magermilchpulver in 100 ml 1 X PBS), und fügen Sie dann 200 ul in jede Vertiefung und inkubieren 37 ° C für 1 Stunde; </li><li> Bereiten Substrat wie folgt: Fügen Sie 1 Tablette 3&#39;-Diaminobenzidin terrahydrochloride (Sigma Cat # D5905) in 20 ml 1 X PBS nach aufgelöst, und fügen Sie dann 8 ul von 30% Wasserstoffperoxidase </li><li> Geben Sie 200 ul der oben genannten Substrat in jede Vertiefung. Stehen lassen bei Raumtemperatur für 10 Minuten </li><li> Entfernen Substrat und Stop-Reaktion durch Zugabe von 1 ml destilliertem Wasser in jede Vertiefung </li><li> Abgießen und Blot-Platten zum Trocknen </li><li> Zählen Sie die Viruspartikel </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with 5% CO2, 37oC</td>
</tr>
<tr>
<td>50 mL conical tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>plastic</td>
</tr>
<tr>
<td>human serum and blood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>O+</td>
</tr>
<tr>
<td>pipette tip</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for tissue culture </td>
</tr>
<tr>
<td>pipettman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipetts</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass, sterile</td>
</tr>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>37C</td>
</tr>
<tr>
<td>centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>parafilm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>circulating water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>glass membrane feeders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with rubber tubing to fit feeders</td>
</tr>
<tr>
<td>mosquito cups</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75% ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10% bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue-culture plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>6-well, 24-well and 96-well plates</td>
</tr>
<tr>
<td>Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass</td>
</tr>
<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>fine-tip forceps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1X, sterile</td>
</tr>
<tr>
<td>dissecting microscope</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>C6/36 cells</td>
<td>cells</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For virus propagation</td>
</tr>
<tr>
<td>C6/36 medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>MEM with 10% heat inactivated FBS, 1% L-glutamine and non-essential amino acid and 1% (v/v) Penicillin-Streptomycin.</td>
</tr>
<tr>
<td>3&#x2019;-Diaminobenzidine terrahydrochloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5905</td>
<td>1 tablet in 20 ml of 1 X PBS, dissolve and then add 8 &#181;l of 30% hydrogen peroxidase</td>
</tr>
<tr>
<td>30% hydrogen peroxidase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A. aegypti</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>mosquito</td>
</tr>		</tbody>
		</table>
		</div>

protocol for dengue infections in mosquitoes (a. aegypti) and infection phenotype determination

Der Zweck dieses Verfahrens ist es, die Aedes Mücke mit Dengue-Virus zu infizieren in einem Labor untersuchen Zustand und die Infektion Niveau und Dynamik des Virus in der Mücke Gewebe. Dieses Protokoll wird routinemäßig zur Untersuchung von Moskito-Virus-Interaktionen, insbesondere für die Identifizierung neuer Host Faktoren, die in der Lage, Vektor-Kompetenz bestimmen werden. Der gesamte Versuch ist nach einem BSL2 Labor durchgeführt werden. Ähnlich wie bei Plasmodium falciparum-Infektionen, müssen angemessene Kleidung einschließlich der Handschuhe und Kittel jederzeit getragen werden. Nach dem Experiment, müssen alle Materialien, die in Kontakt mit dem Virus kam mit 75% Ethanol und gebleicht, bevor Sie mit normalem Waschen behandelt werden. Alle anderen Materialien müssen vor der Entsorgung zu autoklavieren.

Watch this Scientific Journal Video about Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination at JoVE.com

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

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

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 ...

protocol-for-dengue-infections-mosquitoes-aegypti-infection-phenotype

Research

JoVE Journal

Biology

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

17.5K Aufrufe.  Johns Hopkins University. Sobald ein Gen als potentiell feuerfesten für die Dengue-Virus identifiziert ist, muss er für seine Rolle bei der Verhinderung virale Infektionen innerhalb der Mücke ausgewertet werden. Dieses Protokoll zeigt, wie das Ausmaß der Dengue-Infektionen von Mücken getestet werden kann. Die Techniken für den Anbau bis das Virus in Kultur-, Membran-Fütterung Moskitos menschlichem Blut und Testen von viralen Titer in der Mücke Mitteldarm demonstriert.

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

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

Mikroinjektion von A. aegypti Embryonen, um transgene Moskitos erhalten

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Forschung

Biologie

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

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 ...

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

Dissection der Dünndarm-und Speicheldrüsen von Ae. aegypti Moskitos

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

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 ...

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

Verhinderung der Ausbreitung von Malaria und Dengue-Fieber mit Hilfe gentechnisch veränderter Moskitos

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

Ein besseres Mosquito: Identifizierung der Gene aktivieren Malaria und Dengue-Fieber Widerstand in A. gambiae und A. aegypti Moskitos

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi-vermittelte Gen-Knockdown und<em&gt; In Vivo</em&gt; Diurese Assay in Adult Female<em&gt; Aedes aegypti</em&gt; Moskitos

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

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, ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Pflege, Aedes Aegypti Moskitos mit Wolbachia infizierte

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

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

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

Protokolle für die Prüfung der Toxizität von neuartigen Insektizide Chemikalien, Mücken

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