This research was possible through funding from the University of Neuch&#226;tel. We would like to thank all the students that helped in improving this technique, namely our colleague Kevin Thievent. We would also like to thank the members of the Thomas Lab for making their laboratory available. We would like to thank Janet Teeple for her help with mosquito rearing. We would also like to thanks Loyal Hall in the laboratory of Pr. Tom Baker for his help in the preparation of the micro capillary glass tubes.

1. soluzione salina per l&#39;iniezione e la dissezione <ol><li> Preparare la soluzione salina aggiungendo NaCl, KCl, e CaCl 2 per acqua distillata per ottenere 1,3 mM NaCl, 0,5 mM KCl, e 0,2 mM CaCl 2 a pH = 6,8. </li><li> Aggiungere 1 ml di 0,1% di metil soluzione verde a 99 ml della soluzione salina per colorare le perle trasparenti. Questo è il metile verde &quot;soluzione per l&#39;iniezione&quot; 0,001%. </li><li> Quindi, aggiungere 5 ml di 0,1% di metil soluzione verde a 45 ml di soluzione salina. Questa metil verde &quot;soluzione dissezione&quot; 0,01% è 10 volte più concentrato in verde metile per facilitare la colorazione delle perle e la loro osservazione durante la dissezione. NOTA: Filtro sterilizzare il buffer se necessario. </li></ol> 2. capillare Preparazione <ol><li> Tubi di vetro microcapillare Calore-pull per ottenere una finissima punta leggermente più grande delle piccole perle (φ = 40 micron) 42. </li><li> Aprire e unEGOLARE ogni capillare rompendo la punta con una pinzetta o segando la punta. </li></ol> 3. Mosquito allevamento <ol><li> Posteriore A. gambiae a 26 ± 1 ° C, 70 ± 5% di umidità relativa, e una luce 12:12 hr: ciclo scuro. Mantenere femmine adulte e dare loro l&#39;accesso a una soluzione zuccherina al 10%. </li></ol> 4. Le selezioni Bead con la capillare <ol><li> Versare 0,009 g di perle di carica negativa (40-120 micron di diametro) in una capsula di 5 centimetri di Petri e aggiungere 5 ml di soluzione iniettabile di 30 minuti in anticipo. NOTA: L&#39;obiettivo è quello di lasciare il colorante colore le perline per effettuare la selezione tallone per l&#39;iniezione più facile. Autoclave le perle se non sono disponibili in un barattolo sterile. </li><li> Utilizzando un capillare montato in un bulbo capillare pipetta, selezionare visivamente il più piccolo tallone in 0,1 ml di soluzione salina al microscopio binoculare. NOTA: quando inoculando varie perline contemporaneamente, selezionare tre perle da un volume totale di 0,1 ml.</li></ol> 5. Manipolazione Mosquito e inoculazione <ol><li> Utilizzando un aspiratore insetto, inserire ogni zanzara in un tubo da 50 ml. </li><li> Raffreddare ogni zanzara brevemente ponendo la provetta in ghiaccio tritato (2-5 min). </li><li> Inserire una zanzara sul lato destro al microscopio binoculare. </li><li> Sotto uno stereomicroscopio, inoculare la zanzara con il capillare saldamente forando il lato sinistro della cuticola cavità toracica e poi iniettato il liquido e il tallone. NOTA: Fare attenzione a non danneggiare i muscoli del volo tenendo il capillare come perpendicolarmente alla zanzara possibile. </li><li> Rimuovere il capillare. </li><li> Utilizzando pinze entomologia piuma, posizionare ogni femmina singolarmente in un bicchiere di plastica 180 ml coperta con zanzariere; dare loro accesso a una soluzione di zucchero 10%. NOTA: E &#39;necessaria cautela con la soluzione di zucchero, come la zanzara può rimanere bloccati sulle gocce di soluzione di zucchero. </li></ol> 6. MosquitoDissezione e Fotografia Bead <ol><li> Verificare che le zanzare sono vivi 24 ore dopo l&#39;inoculazione. Se è così, controllare la loro condizione e mantenere solo le zanzare che sono in grado di volare. </li><li> Congelare queste zanzare a -20 ° C. NOTA: Possono essere conservate per diversi giorni o mesi prima della dissezione, se necessario. </li><li> Rimuovere le ali delle zanzare utilizzando pinzette sotto un microscopio binoculare a 32X di ingrandimento. Fissare le ali su vetrini con nastro adesivo trasparente. Annotare il codice utilizzato per ogni singolo zanzara. </li><li> Mettere i vetrini in uno scanner e la scansione a 1.200 dpi. NOTA: Verificare che il codice è leggibile per ogni singolo zanzara. </li><li> Su un vetrino per microscopio e in soluzione dissezione, utilizzare pinze per separare il torace dalla testa e l&#39;addome. </li><li> Aprire il torace con una pinza per recuperare le perline. NOTA: Beads che non si muovono all&#39;addome, non sono in contatto tra di loro e la maggior parte di essi galleggiare liberamente in tegli Emolinfa. Fare attenzione a non rompere il tallone con le pinze, come una perla non melanized può essere difficile da trovare. Attendere da 1 a 5 minuti per perline per ottenere il colore. </li><li> Trasferire le perle in una goccia di soluzione di dissezione su un vetrino da microscopio in vetro, prima di scattare la foto. NOTA: Non c&#39;è bisogno di lavare le perline. </li><li> Utilizzando un microscopio dotato di una fotocamera, scattare una immagine digitale di ciascuna sfera in un ambiente illuminazione standard e l&#39;ingrandimento 400X. NOTA: Non modificare l&#39;illuminazione tra le diverse immagini per consentire loro confronto. </li></ol> 7. Analisi Immagine: Bead melanizzazione <ol><li> Aprire il software di analisi delle immagini. Fare clic su &quot;File&quot; e poi su &quot;Apri&quot; per trovare un&#39;immagine tallone. </li><li> Clicca su &quot;analizzare&quot;. Clicca su &quot;misure fissate.&quot; Barrare la casella &quot;valore medio grigia&quot; nella finestra &quot;misure fissate&quot;. </li><li> Fare clic sull&#39;icona dello strumento selezione cerchio. Selezionare il perimetro tallone; rimanere inlato del tallone e di evitare la alone luminoso intorno. </li><li> Fare clic sul menu &quot;analizzare&quot;. Clicca su &quot;misura&quot;. Ottenere il valore di grigio medio e le dimensioni della sezione trasversale del cordone. <ol><li> Per ottenere il valore melanizzazione di ciascuna sfera, eseguire la seguente operazione utilizzando una calcolatrice o un calcolatore software: &quot;256 - valore medio grigio.&quot; La misura melanizzazione è un valore compreso tra 0 e 256, con 0 che rappresenta un&#39;immagine completamente bianca. </li></ol></li></ol>

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J., George, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Manipulation&amp;#34;+without+the+parasite:+altered+feeding+behaviour+of+mosquitoes+is+not+dependent+on+infection+with+malaria+parasites.">34;Manipulation&amp;#34; without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites.</a> P Roy Soc B-Biol Sci. 280 (1763), 20130711 (2013).</li><li>Voordouw, M. J., Lambrechts, L., Koella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+maternal+effects+after+stimulation+of+the+melanization+response+in+the+yellow+fever+mosquito+Aedes+aegypti.">No maternal effects after stimulation of the melanization response in the yellow fever mosquito Aedes aegypti.</a> Oikos. 117 (8), 1269-1279 (2008).</li><li>Paskewitz, S., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Plasmodium+refractory+and+susceptible+strains+of+Anopheles+gambiae+to+inoculated+Sephadex+beads.">Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to inoculated Sephadex beads.</a> Dev comp immunol. 18 (5), 369-375 (1994).</li></ol>

The authors declare that they have no competing financial interests.

Questa tecnica di iniezione è utile per stimolare e studiare la risposta melanizzazione in zanzare. Ad esempio, qui abbiamo studiato l&#39;effetto del carico stimoli immunitario. Il passo fondamentale in questa procedura è per inoculare correttamente la zanzara. Qualsiasi danni eccessivi ai muscoli del volo o per la zanzara si può impedire la zanzara da alimentazione o può uccidere prima della dissezione. Un secondo passo fondamentale è quello di mantenere le zanzare sul ghiaccio abbast...

Gli insetti si basano su risposte immunitarie per proteggersi contro i parassiti e patogeni 1 - 3 che violano attraverso la loro cuticola o il loro midgut dell&#39;epitelio 4. In zanzare, queste risposte sono efficaci contro i batteri, virus 5 6, nematodi filarial 7, e parassiti della malaria 1,8,9. In zanzare, una risposta immunitaria chiave è l&#39;incapsulamento di corpi estranei con la melanina 10 - 12. Questo incapsulamento può accadere nel midgut o in emolinfa sistema di circolazione 10 - 12. Questa risposta melanizzazione è il risultato del pro-phenoloxidase cascata 10 - 12, e può portare alla morte dei parassiti o alla loro fagocitosi. In zanzare adulte, in cui viene limitato il numero di cellule ematociti concentrati, melanizzazione è una risposta umorale, come contro i parassiti Plasmodium o nematodi filarial 7.In alcuni altri insetti, è direttamente le cellule hemocytes che si riuniscono intorno al parassita di melanize loro 7. Inoltre, la melanina è essenziale anche per molti altri processi fisiologici come la produzione di uova e ferite cuticola guarigione 7. La stimolazione della risposta immunitaria è utilizzato come strumento per studiare l&#39;immunità insetto in diversi sistemi modello sanità pubblica agricola e 13 - 18. E &#39;utilizzato in Anopheles gambiae zanzare, il principale vettore della malaria in Africa, per studiare le interazioni ospite-parassita 14 - 16,19. Queste tecniche si basano sulla capacità di insetti per individuare parassiti con i loro recettori pattern recognition (PRR) 2. Le zanzare possono anche rilevare altre molecole che interferiscono con la loro biologia come modelli molecolari associati ai patogeni (PAMPs), o rilevare le proprie cellule danneggiate a causa del rilascio di collagene ed acidi nucleici. La cellula immunitaria della zanzaras quali gli emociti sono utilizzati per la rilevazione 20 - 23. Le principali vie di segnalazione immunitario sono Imd, Toll, JAK / STAT 24, e l&#39;interferenza acido ribonucleico (RNAi) 25,26. Sia Toll e percorsi IMD influenzano la risposta melanizzazione e interagiscono con il pro-phenoloxidase cascata 10 - 12. Lo strumento standard utilizzato per stimolare la risposta melanizzazione è l&#39;inoculazione di una zanzara con un piccolo tallone nel emolinfa della cavità toracica. Il grado di melanina incapsulamento può quindi essere misurato 19 dopo aver ottenuto il tallone attraverso la dissezione della zanzara. Nella maggior parte degli studi, solo una perlina stato iniettato per zanzare 15,16,27, ma iniettando più perline è possibile al fine di studiare i limiti della risposta melanizzazione 19. Queste perle sono iniettati con una soluzione iniettabile (fisiologica) per ridurre i disturbi della fisiologia e zanzarel&#39;essiccazione della zanzara 15,16,27. Un colorante viene aggiunto a questa soluzione per facilitare la selezione tallone. È lo stesso per la soluzione dissezione utilizzato per recuperare il 15,16,27 tallone. Il vantaggio di inoculare insetti con stimoli non patogeno è la capacità di mettere a fuoco l&#39;effetto diretto sulla risposta immunitaria. Non ci sono effetti di complicazione a causa di parassiti patogenicità 28, immunosoppressione 29 - 31, o l&#39;evasione immune 31 - 34. Inoltre, le conseguenze delle stimolazioni su altri tratti della storia di vita, come la longevità o la fecondità, possono anche essere studiati. Così, i ricercatori che studiano l&#39;ecologia evolutiva possono richiedere tali strumenti 2,35,36. Ad esempio, i bombi immuno-sfidato hanno una durata di vita ridotta sotto fame. effetti negativi simili di stimolazione del sistema immunitario e le implementazioni sono state osservate in diversi modelli di invertebrati, spesso con conseguente breveer la durata della vita o il successo riproduttivo meno 13,27,37. Tali studi possono essere condotti in diversi ambienti 2,4,38. Stimolare l&#39;immunità è anche di interesse per coloro concentrarsi direttamente su immunopatologia 39,40. Questo protocollo si basa sulla inoculazione di perline con zanzare per stimolare la risposta melanizzazione e direttamente misurare la quantità di melanina. In questo modo lo studio quantitativo e qualitativo della risposta melanizzazione in contesti diversi sperimentali. Tale strumento può essere esteso alla stimolazione di altre risposte immunitarie, come la risposta antibatterico per batteri 41 termicamente uccisi. Può anche essere condotta in molti ambienti ecologici.

<table><tbody><tr><td>Microcapillary glass tubes GB120TF-10</td><td>science-products.com</td><td>GB120TF-10</td><td>http://www.science-products.com/Products/CatalogG/Glass/Glass.html</td></tr><tr><td>Microcaps Capillary pipette bulb</td><td>Drumond</td><td>1-000-9000</td><td></td></tr><tr><td>negatively charged Sephadex CM C-25 beads</td><td>Sigma-Aldrich, Steinheim, Germany</td><td>C25120 SIGMA</td><td>need few to start</td></tr><tr><td>Methyl green</td><td>Sigma-Aldrich</td><td>323829 ALDRICH</td><td>need few to start</td></tr><tr><td>Software ImageJ</td><td>opensource</td><td></td><td>Version 1.47f7 or later</td></tr></tbody></table>

inoculating anopheles gambiae mosquitoes with beads to induce and measure the melanization immune response

La stimolazione della risposta immunitaria è un attrezzo comune negli studi invertebrati per esaminare l&#39;efficacia ei meccanismi di immunità. Questa stimolazione è basato sull&#39;iniezione di particelle non patogeni in insetti, come le particelle vengono rilevati dal sistema immunitario e indurrà la produzione di effettori immunitari. Ci concentriamo qui sulla stimolazione della risposta melanizzazione nella zanzara Anopheles gambiae. I risultati di risposta melanizzazione nel incapsulamento di particelle estranee e parassiti con uno strato scuro di melanina. Per stimolare questa risposta, le zanzare vengono inoculati con perline all&#39;interno della cassa toracica utilizzando tubi di vetro microcapillari. Poi, dopo 24 ore, le zanzare sono sezionati per recuperare le perline. Il grado di melanizzazione del cordone viene misurata utilizzando il software di analisi delle immagini. Beads non hanno gli effetti patogeni di parassiti, o la loro capacità di eludere o sopprimere la risposta immunitaria. Queste iniezioni sono un modo per misure efficacia immunitario e l&#39;impatto della stimolazione del sistema immunitario su altri tratti della storia di vita, come ad esempio la fecondità e la longevità. Non è esattamente lo stesso di studiare direttamente le interazioni ospite-parassita, ma è uno strumento interessante per studiare l&#39;immunità e la sua ecologia evolutiva.

<html> Zanzare non tutti hanno melanize le sfere nello stesso modo, come alcuni perle sono state coperte con meno melanina di altri (Figura 1). Infatti, alcune perle rimaste blu a causa di una mancanza di melanizzazione, mentre altri sono stati completamente scuro (Figura 1). Il valore melanizzazione stato standardizzato mediante interpolazione lineare per un valore compreso tra 0 (che corrisponde ad un cordone blu e unmelanized) e 100 (corrispondente ad ...

Watch this Scientific Journal Video about Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response at JoVE.com

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

inoculando<em&gt; Anopheles gambiae</em&gt; Le zanzare con perline di indurre e misurare la risposta immunitaria melanizzazione

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

inoculating-anopheles-gambiae-mosquitoes-with-beads-to-induce-measure

Research

JoVE Journal

Immunology and Infection

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

7.9K Views.  Université de Neuchâtel. Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

Video: Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

Iniezione di An. Gli embrioni stephensi per generare la malaria resistenti zanzare

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

Ricerca

Biologia

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

Integrazione mediata dal sito φC31 e scambio di cassette in Anopheles Vettori della malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

Genetica

Microinjection Method for Anopheles gambiae Embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

Metodo di microiniezione per embrioni di Anopheles gambiae

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Un<em&gt; In Vitro</em&gt; Modello per la misurazione Risposte immunitarie Malaria nel contesto della coinfezione da HIV

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Immunologia e infezioni

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Mieloide isolamento delle cellule da topo pelle e drenante linfonodi seguito intradermica immunizzazione con vivo attenuato<em&gt; Plasmodium</em&gt; sporozoiti

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Tracciamento in vivo di sviluppo dell&#39;edema e patologia microvascolare in un modello di malaria cerebrale sperimentale mediante l&#39;utilizzo di immagini a risonanza magnetica

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Medicina

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Test standard di alimentazione a membrana per la rilevazione dell'infezione da Plasmodium falciparum in vettori di zanzare Anopheles

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Generazione di sporozoiti geneticamente modificati di Plasmodium berghei

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAi trigger consegna in<em&gt; Anopheles gambiae</em&gt; Pupe

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

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Capitoli in questo video

Iniezione di An. Gli embrioni stephensi per generare la malaria resistenti zanzare

Integrazione mediata dal sito φC31 e scambio di cassette in Anopheles Vettori della malaria

Metodo di microiniezione per embrioni di Anopheles gambiae

Un

Mieloide isolamento delle cellule da topo pelle e drenante linfonodi seguito intradermica immunizzazione con vivo attenuato

Tracciamento in vivo di sviluppo dell'edema e patologia microvascolare in un modello di malaria cerebrale sperimentale mediante l'utilizzo di immagini a risonanza magnetica

Test standard di alimentazione a membrana per la rilevazione dell'infezione da Plasmodium falciparum in vettori di zanzare Anopheles

Generazione di sporozoiti geneticamente modificati di Plasmodium berghei

RNAi trigger consegna in

Somministrazione efficace di interferenza orale dell'RNA (RNAi) alle zanzare Anopheles gambiae adulte