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. Kochsalzlösung für die Injektion und Dissection <ol><li> Bereiten Sie die Salzlösung durch Zugabe von NaCl, KCl und CaCl 2 zu destilliertem Wasser 1,3 mM NaCl zu erhalten, 0,5 mM KCl und 0,2 mM CaCl 2 bei pH = 6,8. </li><li> 1 ml 0,1% Methylgrün Lösung auf 99 ml der Kochsalzlösung die transparenten Kügelchen zu färben. Dies ist die 0,001% Methylgrün &quot;Injektionslösung&quot;. </li><li> Dann werden 5 ml 0,1% Methylgrün Lösung zu 45 ml der Salzlösung. Diese 0,01% Methylgrün &quot;dissection Lösung&quot; ist 10mal konzentrierter in Methylgrün die Färbung der Kügelchen und deren Beobachtung während der Dissektion zu erleichtern. HINWEIS: Filter sterilisieren den Puffer, falls erforderlich. </li></ol> 2. Kapillar-Vorbereitung <ol><li> Wärme-Pull - Rohre Mikrokapillare Glas mit einer sehr feinen Spitze etwas größer als die kleinste Kügelchen (φ = 40 &amp; mgr; m) 42 zu erhalten. </li><li> Offene und eindjust jede Kapillare, indem die Spitze mit einer Pinzette zu brechen oder durch die Spitze Absägen. </li></ol> 3. Mosquito aufrichten <ol><li> Hinten A. gambiae bei 26 ± 1 ° C, 70 ± 5% relative Luftfeuchtigkeit und eine 0.12 Stunden Licht: Dunkel - Zyklus. Halten erwachsenen Weibchen und geben ihnen den Zugang zu einer 10% igen Zuckerlösung. </li></ol> 4. Bead Auswahl mit dem Kapillar <ol><li> Gießen 0,009 g negativ geladenen Kügelchen (40-120 &amp; mgr; m im Durchmesser) in einer 5 cm Petrischale und 5 ml Injektionslösung 30 Minuten im Voraus. HINWEIS: Das Ziel ist der Farbstoff zu lassen, um die Perlen Farbe Perlen Auswahl für die Injektion zu erleichtern. Autoclave die Perlen, wenn sie eine sterile Glas nicht kommen. </li><li> Verwendung einer Kapillare in einer Glühbirne Kapillarpipette montiert, visuell wählen die kleinste bead in 0,1 &amp; mgr; l der Salzlösung unter einem binokularen Mikroskop. HINWEIS: Wenn Sie gleichzeitig mehrere Perlen Impfen, wählen drei Perlen aus einem Gesamtvolumen von 0,1 ul.</li></ol> 5. Mosquito Handhabung und Inokulation <ol><li> Unter Verwendung eines Insekten Absauger, platzieren Sie jede Mücke in einem 50-ml-Tube. </li><li> Kühlen Sie jede Mücke kurz durch das Rohr in zerstoßenem Eis legen (2-5 min). </li><li> Legen Sie eine Mücke auf der rechten Seite unter dem Binokular. </li><li> Unter einem Stereomikroskop, impfen die Mücke mit der Kapillare fest durch die linke Seite der Brusthöhle Kutikula Piercing und dann die Flüssigkeit und den Wulst zu injizieren. HINWEIS: Achten Sie darauf, nicht wie möglich an die Mücke, indem die Kapillare als senkrecht die Flugmuskeln zu beschädigen. </li><li> Entfernen Sie die Kapillare. </li><li> Mit Feder Entomologie Pinzette, legen jedes Weibchen einzeln in einem 180 ml Kunststoffbecher mit Moskitonetz bedeckt; geben ihnen den Zugang zu einer 10% igen Zuckerlösung. HINWEIS: Vorsicht ist mit der Zuckerlösung erforderlich, da die Mücke auf Zuckerlösung Tropfen stecken bleiben. </li></ol> 6. MosquitoDissection und Bead Fotografie <ol><li> Überprüfen Sie, ob die Mücken am Leben 24 Stunden nach der Impfung sind. Wenn ja, überprüfen Sie ihren Zustand und halten nur die Mücken, die fähig sind zu fliegen. </li><li> Frieren Sie diese Mücken bei -20 ° C. HINWEIS: Sie können mehrere Tage bis Monate vor Dissektion gehalten werden, wenn nötig. </li><li> Entfernen Sie die Moskitos Flügel Pinzette unter dem Binokular auf das 32-fache Vergrößerung verwendet wird. Befestigen Sie die Flügel auf Glasobjektträger mit durchsichtigem Klebeband. Schreiben Sie den Code unten für jede einzelne Mücke verwendet. </li><li> Setzen Sie die Glasobjektträger in einem Scanner und scannen sie bei 1.200 dpi. HINWEIS: Überprüfen Sie, dass der Code für jede einzelne Mücke lesbar ist. </li><li> Auf einem Glasobjektträger und in Sezieren Lösung, verwenden Pinzette den Thorax aus dem Kopf und Bauch zu trennen. </li><li> Öffnen Sie den Thorax mit einer Pinzette die Perlen zu holen. HINWEIS: Perlen, die auf den Bauch nicht bewegen, sind nicht in Kontakt miteinander und die meisten von ihnen schweben frei in ter Hämolymphe. Achten Sie darauf, nicht den Wulst mit der Zange zu brechen, als ein nicht-melanisierten Wulst hart sein kann zu finden. Warten Sie 1 bis 5 min für Perlen Farbe zu erhalten. </li><li> Übertragen Sie die Perlen in einem Tropfen Dissektion Lösung auf einem Glasobjektträger, bevor Sie das Bild. HINWEIS: Keine Notwendigkeit, die Perlen zu waschen. </li><li> Mit einem Mikroskop mit einer Kamera ausgestattet, nehmen Sie ein digitales Bild von jedem Wulst an einer Standardbeleuchtung Einstellung und 400-facher Vergrößerung. HINWEIS: Die Beleuchtung zwischen den verschiedenen Bilder nicht ändern ihren Vergleich zu ermöglichen. </li></ol> 7. Bildanalyse: Bead Melanisierung <ol><li> Öffnen Sie die Bildanalyse-Software. Klicken Sie auf &quot;Datei&quot; und dann auf &quot;Öffnen&quot; eine Perle Bild zu finden. </li><li> Klicken Sie auf &quot;zu analysieren.&quot; Klicken Sie auf &quot;Set-Messungen.&quot; Markieren Sie das &quot;bedeuten Grauwert&quot; in der Gruppe &quot;Set-Messungen&quot; Fenster. </li><li> Klicken Sie auf das Kreis-Auswahl-Werkzeug-Symbol. Wählen Sie den Wulst Umfang; bleiben inSeite der Wulst und vermeiden Sie den leuchtenden Halo umgeben. </li><li> Klicken Sie auf das &quot;analysieren&quot; -Menü. Klicken Sie auf &quot;Messen&quot;. Erhalten des mittleren Grauwert und die Größe der Querschnittsfläche der Sicke. <ol><li> Um den melanization Wert jeder Perle zu erhalten, führen Sie die folgende Operation einen Rechner oder Rechner-Software: &quot;256 - Grauwert bedeuten.&quot; Die melanization Maßnahme ist ein Wert zwischen 0 und 256, wobei 0 ein völlig weißes Bild darstellt. </li></ol></li></ol>

<ol>
	<li>Dong, Y., Aguilar, R., Xi, Z., Warr, E., Mongin, E., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anopheles+gambiae+immune+responses+to+human+and+rodent+Plasmodium+parasite+species.">Anopheles gambiae immune responses to human and rodent Plasmodium parasite species.</a> PLoS pathog. 2 (6), e52 (2006).</li><li>Sadd, B. M., Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PERSPECTIVE:+Principles+of+ecological+immunology.">PERSPECTIVE: Principles of ecological immunology.</a> Evolutionary Appl. 2 (1), 113-121 (2008).</li><li>Crompton, P. D., Moebius, 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=Malaria+Immunity+in+Man+and+Mosquito:+Insights+into+Unsolved+Mysteries+of+a+Deadly+Infectious+Disease*.">Malaria Immunity in Man and Mosquito: Insights into Unsolved Mysteries of a Deadly Infectious Disease*.</a> Annu Rev Immuno. 32 (1), 157-187 (2014).</li><li>Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EVOLUTIONARY+ECOLOGY+OF+INSECT+IMMUNE+DEFENSES.">EVOLUTIONARY ECOLOGY OF INSECT IMMUNE DEFENSES.</a> Annu Rev Entomol. 50 (1), 529-551 (2005).</li><li>Hillyer, J. F., Schmidt, S. L., Christensen, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+phagocytosis+and+melanization+of+bacteria+and+Plasmodium+sporozoites+by+hemocytes+of+the+mosquito+Aedes+aegypti.">Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti.</a> J parasito. 89 (1), 62-69 (2003).</li><li>Carissimo, G., Pondeville, E., 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=Antiviral+immunity+of+Anopheles+gambiae+is+highly+compartmentalized,+with+distinct+roles+for+RNA+interference+and+gut+microbiota.">Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota.</a> PNAS. 112 (2), E176-E185 (2015).</li><li>Christensen, B. M., Li, J., Chen, C. -. C., Nappi, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanization+immune+responses+in+mosquito+vectors.">Melanization immune responses in mosquito vectors.</a> Trends parasito. 21 (4), 192-199 (2005).</li><li>Collins, F., Sakai, R., 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=Genetic+selection+of+a+Plasmodium-refractory+strain+of+the+malaria+vector+Anopheles+gambiae.">Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae.</a> Science. 234 (4776), 607-610 (1986).</li><li>Warr, E., Lambrechts, L., Koella, J. C., Bourgouin, C., Dimopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anopheles+gambiae+immune+responses+to+Sephadex+beads:+Involvement+of+anti-Plasmodium+factors+in+regulating+melanization.">Anopheles gambiae immune responses to Sephadex beads: Involvement of anti-Plasmodium factors in regulating melanization.</a> Insect Biochem Molec. 36 (10), 769-778 (2006).</li><li>Fuchs, S., Behrends, V., Bundy, J. G., Crisanti, A., Nolan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenylalanine+metabolism+regulates+reproduction+and+parasite+melanization+in+the+malaria+mosquito.">Phenylalanine metabolism regulates reproduction and parasite melanization in the malaria mosquito.</a> PloS one. 9 (1), e84865 (2014).</li><li>Cerenius, L., S&#246;derh&#228;ll, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prophenoloxidase-activating+system+in+invertebrates.">The prophenoloxidase-activating system in invertebrates.</a> Immuno Rev. 198, 116-126 (2004).</li><li>Cerenius, L., Lee, B. L., S&#246;derh&#228;ll, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proPO-system:+pros+and+cons+for+its+role+in+invertebrate+immunity.">The proPO-system: pros and cons for its role in invertebrate immunity.</a> Trend Immuno. 29 (6), 263-271 (2008).</li><li>Moret, Y., Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Survival for immunity: the price of immune system activation for bumblebee workers. , 1166-1168 (2000).</li><li>Suwanchaichinda, C., Paskewitz, S. M. <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+Larval+Nutrition,+Adult+Body+Size,+and+Adult+Temperature+on+the+Ability+of+Anopheles+gambiae(Diptera:+Culicidae)+to+Melanize+Sephadex+Beads.">Effects of Larval Nutrition, Adult Body Size, and Adult Temperature on the Ability of Anopheles gambiae(Diptera: Culicidae) to Melanize Sephadex Beads.</a> J Med Entomol. 35 (2), 157-161 (1998).</li><li>Chun, J., Riehle, M., Paskewitz, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Mosquito+Age+and+Reproductive+Status+on+Melanization+of+Sephadex+Beads+in+Plasmodium-Refractory+and+-Susceptible+Strains+of+Anopheles+gambiae.">Effect of Mosquito Age and Reproductive Status on Melanization of Sephadex Beads in Plasmodium-Refractory and -Susceptible Strains of Anopheles gambiae.</a> J Invertebr Pathol. 66 (1), 11-17 (1995).</li><li>Schwartz, A., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanization+of+Plasmodium+falciparum+and+C-25+Sephadex+Beads+by+Field-Caught+Anopheles+gambiae+(Diptera:+Culicidae)+from+Southern+Tanzania.">Melanization of Plasmodium falciparum and C-25 Sephadex Beads by Field-Caught Anopheles gambiae (Diptera: Culicidae) from Southern Tanzania.</a> J Med Entomol. 39 (1), 84-88 (2002).</li><li>Zahedi, M., Denham, D. A., Ham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+and+melanization+responses+of+Armigeres+subalbatus+against+inoculated+Sephadex+beads.">Encapsulation and melanization responses of Armigeres subalbatus against inoculated Sephadex beads.</a> J Invertebr Pathol. 59 (3), 258-263 (1992).</li><li>Laughton, A. M., Garcia, J. R., Altincicek, B., Strand, M. R., Gerardo, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+immune+responses+in+the+pea+aphid,+Acyrthosiphon+pisum.">Characterisation of immune responses in the pea aphid, Acyrthosiphon pisum.</a> J insect physiol. 57 (6), 830-839 (2011).</li><li>Barreaux, A. M. G., Barreaux, P., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overloading+the+immunity+of+the+mosquito+Anopheles+gambiae+with+multiple+immune+challenges.">Overloading the immunity of the mosquito Anopheles gambiae with multiple immune challenges.</a> Parasite Vector. 9 (1), 210 (2016).</li><li>Lazzaro, B. P., Rolff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Danger,+Microbes,+and+Homeostasis.">Danger, Microbes, and Homeostasis.</a> Science. 332 (6025), 43-44 (2011).</li><li>Arrighi, R. B. G., Faye, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+GPI+toxin:+a+common+foe+for+man+and+mosquito.">Plasmodium falciparum GPI toxin: a common foe for man and mosquito.</a> Acta trop. 114 (3), 162-165 (2010).</li><li>Michel, 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=Mosquito+immunity+against+Plasmodium.">Mosquito immunity against Plasmodium.</a> Insect Biochem Molec. 35 (7), 677-689 (2005).</li><li>Osta, M. A., Christophides, G. K., Vlachou, D., 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=Innate+immunity+in+the+malaria+vector+Anopheles+gambiae:+comparative+and+functional+genomics.">Innate immunity in the malaria vector Anopheles gambiae: comparative and functional genomics.</a> J Exp Biol. 207 (15), 2551-2563 (2004).</li><li>Christophides, G. K., Vlachou, D., 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=Comparative+and+functional+genomics+of+the+innate+immune+system+in+the+malaria+vector+Anopheles+gambiae.">Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae.</a> Immunol Rev. 198 (1), 127-148 (2004).</li><li>Blair, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+RNAi+is+the+major+innate+immune+pathway+controlling+arbovirus+infection+and+transmission.">Mosquito RNAi is the major innate immune pathway controlling arbovirus infection and transmission.</a> Future microbiol. 6 (3), 265-277 (2011).</li><li>Fragkoudis, R., Attarzadeh-Yazdi, G., Nash, A. A., Fazakerley, J. K., Kohl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+dissecting+mosquito+innate+immune+responses+to+arbovirus+infection.">Advances in dissecting mosquito innate immune responses to arbovirus infection.</a> J Gen Virol. , (2009).</li><li>Schwartz, A., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cost+of+immunity+in+the+yellow+fever+mosquito,+Aedes+aegypti+depends+on+immune+activation.">The cost of immunity in the yellow fever mosquito, Aedes aegypti depends on immune activation.</a> J evol biol. 17 (4), 834-840 (2004).</li><li>Lambrechts, L., Vulule, J. M., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+correlation+between+melanization+and+antibaterial+immune+responses+in+a+natural+population+of+the+malaria+vector+Anopheles+gambiae.">Genetic correlation between melanization and antibaterial immune responses in a natural population of the malaria vector Anopheles gambiae.</a> Evolution. 58 (10), 2377 (2004).</li><li>Boete, C., Paul, R. E. L., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+indirect+immunosuppression+by+a+malaria+parasite+in+its+mosquito+vector.">Direct and indirect immunosuppression by a malaria parasite in its mosquito vector.</a> P Roy Soc B-Biol Sci. 271 (1548), 1611-1615 (2004).</li><li>Sacks, D., Sher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evasion+of+innate+immunity+by+parasitic+protozoa.">Evasion of innate immunity by parasitic protozoa.</a> Nat immunol. 3 (11), 1041-1047 (2002).</li><li>Zambrano-Villa, S., Rosales-Borjas, D., Carrero, J. C., Ortiz-Ortiz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+protozoan+parasites+evade+the+immune+response.">How protozoan parasites evade the immune response.</a> Trend Parasito. 18 (6), 272-278 (2002).</li><li>Damian, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite+immune+evasion+and+exploitation:+reflections+and+projections.">Parasite immune evasion and exploitation: reflections and projections.</a> Parasitology. 115, S169-S175 (1997).</li><li>Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasite+immune+evasion:+a+momentous+molecular+war.">Parasite immune evasion: a momentous molecular war.</a> Trend ecol evol. 23 (6), 318-326 (2008).</li><li>Schmid-Hempel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+defence,+parasite+evasion+strategies+and+their+relevance+for+&amp;#34;macroscopic+phenomena&amp;#34;+such+as+virulence.">Immune defence, parasite evasion strategies and their relevance for &amp;#34;macroscopic phenomena&amp;#34; such as virulence.</a> P Roy Soc B-Biol Sci. 364 (1513), 85-98 (2009).</li><li>Stearns, S. C., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+phenotypic+plasticity+in+life+history+traits-+predictions+of+reaction+norms+for+age+and+size+at+maturity.">The evolution of phenotypic plasticity in life history traits- predictions of reaction norms for age and size at maturity.</a> Evolution. 40 (5), 893-913 (1986).</li><li>Stearns, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life-history+tactics:+a+review+of+the+ideas.">Life-history tactics: a review of the ideas.</a> Q rev biol. 51 (1), 3-47 (1976).</li><li>Valtonen, T. M., Kleino, A., Ramet, M., Rantala, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starvation+Reveals+Maintenance+Cost+of+Humoral+Immunity.">Starvation Reveals Maintenance Cost of Humoral Immunity.</a> Evol Biol. 37 (1), 49-57 (2010).</li><li>Sheldon, B. C., Verhulst, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+immunology:+costly+parasite+defences+and+trade-offs+in+evolutionary+ecology.">Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology.</a> Trend Ecol Evo. 11 (8), 317-321 (1996).</li><li>Graham, A. L., Allen, J. E., Read, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+causes+and+consequences+of+immunopathology.">Evolutionary causes and consequences of immunopathology.</a> Annu Rev Ecol Evol S. 36, 373-397 (2005).</li><li>Best, A., Long, G., White, A., Boots, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+implications+of+immunopathology+for+parasite+evolution.">The implications of immunopathology for parasite evolution.</a> P Roy Soc B-Biol Sci. 279 (1741), 3234-3240 (2012).</li><li>Cator, L. 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.

Diese Injektionstechnik ist nützlich, um die melanization Reaktion in Mücken zu stimulieren und zu studieren. Zum Beispiel, hier untersuchten wir die Wirkung der Immunstimuli Last. Der entscheidende Schritt in diesem Verfahren ist, um richtig die Mücke impfen. Jede übermäßige Schäden an den Flugmuskeln oder die Mücke selbst kann die Mücke von der Fütterung zu verhindern oder sie kann vor der Dissektion töten. Ein zweiter wichtiger Schritt ist, um die Mücken auf Eis lange genug zu...

Insekten verlassen sich auf Immunreaktionen , sich gegen Parasiten zu schützen und Krankheitserreger von 1 bis 3 , die 4 durch ihre Kutikula oder ihre midgut Epithel verletzen. In Mücken, sind diese Reaktionen wirksam gegen Bakterien 5, Viren 6, Filariosen 7 und Malariaparasiten 1,8,9. In Mücken, ein Schlüsselimmunantwort ist die Verkapselung von Fremdpartikeln mit Melanin 10. - 12.. Diese Einkapselung kann in der Mitteldarms oder in der Hämolymphe passieren System zirkulierenden 10 bis 12. Diese melanization Antwort ist das Ergebnis der pro-Phenoloxidase 10 kaskadieren - 12, und es kann zum Tod der Parasiten oder deren Phagozytose führen. Bei erwachsenen Mücken, wo die Anzahl der Hämozyten Zellen beschränkt ist, ist Melanisierung eine humorale Antwort, wie gegen Plasmodium Parasiten oder Filariosen 7.In einigen anderen Insekten ist es direkt die hemocytes Zellen, die sich um den Parasiten sammelt sie 7 bis melanize. Außerdem ist Melanin auch wichtig für mehrere andere physiologische Prozess , wie die Eierproduktion und Hautwunden 7 heilen. Die Stimulation von Immunantworten wird als Werkzeug benutzt , um studieren Insekten Immunität in mehreren landwirtschaftlichen und öffentlichen Gesundheit Modellsysteme 13-18. 16,19 - Es ist in Anopheles gambiae Mücken, die Hauptvektor von Malaria in Afrika, zu studieren Wirt-Parasit - Interaktionen 14 verwendet. Diese Techniken basieren auf der Fähigkeit der Insekten - Parasiten mit ihren Mustererkennungsrezeptoren (PRR) 2 zu erkennen. Stechmücken können auch andere Moleküle mit ihren biology wie pathogen-associated molecular Muster (PAMPs) oder erkennen ihre eigenen geschädigter Zellen durch die Freisetzung von Kollagen und Nukleinsäuren stören detektieren. Die Mücke Immunzelle23 - s wie die Hämocyten werden zur Detektion 20 verwendet. Die wichtigsten Immunsignalwege sind Imd, Toll, JAK / STAT - 24 und Ribonukleinsäure - Interferenz (RNAi) 25,26. Sowohl Toll und Imd Wege beeinflussen die melanization Reaktion und Interaktion mit der pro-Phenoloxidase Kaskade 10 bis 12. Das Standardwerkzeug verwendet, um die melanization Antwort stimulieren ist die Impfung einer Mücke mit einer kleinen Perle in die Hämolymphe der Brusthöhle. Der Grad der Melanin - Verkapselung kann dann 19 gemessen werden , nachdem der Wulst durch die Dissektion der Mücke abruft. In den meisten Studien wurde nur eine Perle pro Mücke injiziert 15,16,27, aber mehr Perlen Injektion möglich ist, um 19 die Grenzen der melanization Antwort zu studieren. Diese Kügelchen injiziert unter Verwendung einer Injektionslösung (physiologische Serum) Störung der Moskito Physiologie zu begrenzen unddie Austrocknung der Mücke 15,16,27. Ein Farbstoff wird zu dieser Lösung gegeben bead Auswahl zu erleichtern. Es ist das gleiche für die Dissektion Lösung verwendet , um den Wulst 15,16,27 abzurufen. Der Vorteil von Insekten mit nicht pathogenen Stimuli Inokulieren ist die Fähigkeit, auf die direkte Wirkung auf die Immunantwort zu konzentrieren. Es gibt keine komplizieren Effekte aufgrund von Parasiten Pathogenität 28, Immunsuppression 29-31 oder Immunevasion 31-34. Außerdem werden die Folgen der Stimulationen auf andere Lebensgeschichte Züge, wie Langlebigkeit oder Fruchtbarkeit, ebenfalls untersucht. So Forscher evolutionäre Ökologie studieren können solche Werkzeuge 2,35,36 erfordern. Zum Beispiel Immunherausgefordert Hummeln haben eine verkürzte Lebensdauer unter Hunger. Ähnliche negative Auswirkungen von Immun Stimulationen und Einsätze haben in verschiedenen Invertebraten Modellen beobachtet, die häufig in einer kurzen ergeber Lebensdauer oder weniger Fortpflanzungserfolg 13,27,37. Solche Studien können in unterschiedlichen Umgebungen 2,4,38 durchgeführt werden. Immunität Anregende ist auch für diejenigen von Interesse direkt auf Immunopathologie 39,40 konzentriert. Dieses Protokoll basiert auf der Inokulation von Kügelchen mit Moskitos die Melanisierung Antwort zu stimulieren und direkt die Menge an Melanin zu messen. Dies ermöglicht eine quantitative und qualitative Untersuchung der melanization Reaktion in verschiedenen experimentellen Bedingungen. Solch ein Werkzeug zur Stimulation anderer Immunreaktionen, wie die antibakterielle Reaktion Bakterien verlängert werden 41 Hitze abgetöteten. Es kann auch in vielen ökologischen Einstellungen durchgeführt werden.

<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

Die Stimulation der Immunantwort ist ein übliches Werkzeug in Invertebraten-Studien, die die Wirksamkeit und die Mechanismen der Immunität zu untersuchen. Diese Stimulation basiert auf der Injektion von nicht-pathogene Partikel in Insekten, da die Partikel durch das Immunsystem erkannt werden, und wird die Produktion von Immun Effektoren induzieren. Wir konzentrieren uns hier auf die Stimulation der Melanisierung Reaktion in der Mücke Anopheles gambiae. Die melanization Reaktion führt zur Verkapselung von Fremdpartikeln und Parasiten mit einer dunklen Schicht von Melanin. mit Mikrokapillare Glasröhren Um diese Reaktion zu stimulieren, Mücken sind mit Perlen in der Brusthöhle impft. Dann, nach 24 Stunden werden die Mücken seziert die Perlen zu holen. Der Grad der Melanisierung des Wulstes gemessen Bildanalyse-Software. Beads nicht die pathogene Wirkung von Parasiten haben oder ihre Fähigkeit, die Immunantwort zu entziehen oder zu unterdrücken. Diese Injektionen sind ein Weg, um Measure Immun Wirksamkeit und die Auswirkungen der Immunstimulationen auf andere Lebensgeschichte Züge, wie Fruchtbarkeit oder Langlebigkeit. Es ist nicht genau das gleiche wie direkt Wirt-Parasit-Wechselwirkungen zu studieren, aber es ist ein interessantes Tool, Immunität und seine evolutionäre Ökologie zu studieren.

<html> Stechmücken nicht alle melanize die Perlen in der gleichen Weise, wie einige Perlen weniger mit Melanin bedeckt waren als andere (Abbildung 1). Tatsächlich blieben einige Perlen wegen eines Mangels an melanization blau, während andere völlig dunkel waren (Abbildung 1). Der Melanisierung Wert wurde durch lineare Interpolation auf einen Wert zwischen 0 standardisiert (was zu einem blauen und unmelanized bead entsprach) und 100 (entsprechend einer...

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.

Impfen<em&gt; Anopheles gambiae</em&gt; Mosquitoes mit Perlen der Melanisierung Immunantwort zu induzieren und Messen

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 Aufrufe.  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.

Serie: 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.

Injektion eines. stephensi Embryonen zu generieren Malaria-resistenter Moskitos

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

Forschung

Biologie

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.

Ortsgerichtete φC31-vermittelte Integration und Kassettenaustausch in Anopheles-Vektoren der Malaria

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

Genetik

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.

Mikroinjektionsmethode für Anopheles gambiae Embryonen

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.

Ein<em&gt; In-vitro-</em&gt; Modell zur Messung der Immunantwort auf Malaria im Kontext der HIV-Koinfektion

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

Immunologie und Infektion

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.

Myeloischen Zellisolierung von Mäusehaut und ableitenden Lymphknoten Nach intradermale Impfung mit attenuierten Lebend<em&gt; Plasmodium</em&gt; Sporozoiten

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.

In vivo Verfolgung von Ödementwicklung und mikrovaskulärer Pathologie in einem Modell der experimentellen zerebralen Malaria mit Magnetresonanztomographie

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 ...

Medizin

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.

Standard-Membran-Fütterungstest zum Nachweis von Plasmodium falciparum-Infektionen in Anopheles-Mückenvektoren

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.

Gentechnisch veränderte Plasmodium berghei Sporozoiten

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 Lieferung in<em&gt; Anopheles gambiae</em&gt; Pupae

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.

Impfen

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Injektion eines. stephensi Embryonen zu generieren Malaria-resistenter Moskitos

Ortsgerichtete φC31-vermittelte Integration und Kassettenaustausch in Anopheles-Vektoren der Malaria

Mikroinjektionsmethode für Anopheles gambiae Embryonen

Ein

Myeloischen Zellisolierung von Mäusehaut und ableitenden Lymphknoten Nach intradermale Impfung mit attenuierten Lebend

In vivo Verfolgung von Ödementwicklung und mikrovaskulärer Pathologie in einem Modell der experimentellen zerebralen Malaria mit Magnetresonanztomographie

Standard-Membran-Fütterungstest zum Nachweis von Plasmodium falciparum-Infektionen in Anopheles-Mückenvektoren

Gentechnisch veränderte Plasmodium berghei Sporozoiten

RNAi-Trigger Lieferung in

Effektive orale RNA-Interferenz (RNAi) Verabreichung an erwachsene Anopheles gambiae-Mücken