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. Saline Solution for Injection and Dissection
<ol>
	<li>Prepare the saline solution by adding NaCl, KCl, and CaCl2 to distilled water to obtain 1.3 mM NaCl, 0.5 mM KCl, and 0.2 mM CaCl2 at pH = 6.8.</li>
	<li>Add 1 ml of 0.1% methyl green solution to 99 milliliters of the saline solution to color the transparent beads. This is the 0.001% methyl green &#8220;injection solution&#34;.</li>
	<li>Then, add 5 ml of 0.1% methyl green solution to 45 ml of the saline solution. This 0.01% methyl green &#8220;dissection solution&#8221; is 10 times more concentrated in methyl green to facilitate the coloration of the beads and their observation during the dissection. 
	NOTE: Filter sterilize the buffer if necessary.</li>
</ol>
2. Capillary Preparation
<ol>
	<li>Heat-pull microcapillary glass tubes to obtain a very fine tip slightly bigger than the smallest beads (&#966; = 40 &#181;m)42.</li>
	<li>Open and adjust each capillary by breaking the tip with tweezers or by sawing the tip off.</li>
</ol>
3. Mosquito Rearing
<ol>
	<li>Rear A. gambiae at 26 &#177; 1 &#176;C, 70 &#177; 5% relative humidity, and a 12:12 hr light:dark cycle. Keep adult females and give them access to a 10% sugar solution.</li>
</ol>
4. Bead Selections with the Capillary
<ol>
	<li>Pour 0.009 g of negatively charged beads (40-120 &#181;m in diameter) in a 5 cm Petri dish and add 5 ml of injection solution 30 min in advance. 
	NOTE: The goal is to let the dye color the beads to make bead selection for injection easier. Autoclave the beads if they don&#39;t come in a sterile jar.</li>
	<li>Using a capillary mounted in a capillary pipette bulb, visually select the smallest bead in 0.1 &#181;l of the saline solution under a binocular microscope. 
	NOTE: When inoculating several beads simultaneously, select three beads from a total volume of 0.1 &#181;l.</li>
</ol>
5. Mosquito Handling and Inoculation
<ol>
	<li>Using an insect aspirator, place each mosquito in a 50 ml tube.</li>
	<li>Chill each mosquito briefly by placing the tube in crushed ice (2-5 min).</li>
	<li>Place a mosquito on its right side under the binocular microscope.</li>
	<li>Under a stereomicroscope, inoculate the mosquito with the capillary by firmly piercing through the left side of the thoracic cavity cuticle and then injecting the liquid and the bead. 
	NOTE: Take care not to damage the flight muscles by holding the capillary as perpendicularly to the mosquito as possible.</li>
	<li>Remove the capillary.</li>
	<li>Using featherweight entomology forceps, place each female individually in a 180 ml plastic cup covered with mosquito netting; give them access to a 10% sugar solution. 
	NOTE: Caution is required with the sugar solution, as the mosquito may get stuck on sugar solution drops.</li>
</ol>
6. Mosquito Dissection and Bead Photography
<ol>
	<li>Check if the mosquitoes are alive 24 hr after the inoculation. If so, check their condition and keep only the mosquitoes that are able to fly.</li>
	<li>Freeze these mosquitoes at -20 &#176;C. 
	NOTE: They can be kept several days to months before dissection, if needed.</li>
	<li>Remove the mosquitoes&#39; wings using tweezers under a binocular microscope at 32X magnification. Fix the wings onto glass slides with transparent tape. Write down the code used for each individual mosquito.</li>
	<li>Put the glass slides in a scanner and scan them at 1,200 dpi. 
	NOTE: Check that the code is readable for each individual mosquito.</li>
	<li>On a glass microscope slide and in dissection solution, use forceps to separate the thorax from the head and abdomen.</li>
	<li>Open the thorax with forceps to retrieve the beads. 
	NOTE: Beads that do not move to the abdomen, are not in contact with each other and most of them float freely in the hemolymph. Be careful not to break the bead with the forceps, as a non-melanized bead may be tough to find. Wait for 1 to 5 min for beads to obtain color.</li>
	<li>Transfer the beads in a droplet of dissection solution on a glass microscope slide, before taking the picture. 
	NOTE: No need to wash the beads.</li>
	<li>Using a microscope equipped with a camera, take a digital image of each bead at a standard lighting setting and 400X magnification. 
	NOTE: Do not change the illumination between the different pictures to enable their comparison.</li>
</ol>
7. Picture Analysis: Bead Melanization
<ol>
	<li>Open the image analysis software. Click on &#34;file&#34; and then on &#34;open&#34; to find a bead image.</li>
	<li>Click on &#34;analyze.&#34; Click on &#34;set measurements.&#34; Tick the &#34;mean grey value&#34; box in the &#34;set measurements&#34; window.</li>
	<li>Click on the circle selection tool icon. Select the bead perimeter; remain inside the bead and avoid the luminous halo around it.</li>
	<li>Click on the &#34;analyze&#34; menu. Click on &#34;measure.&#34; Obtain the mean grey value and size of the cross-sectional area of the bead.
	<ol>
		<li>To obtain the melanization value of each bead, perform the following operation using a calculator or calculator software: &#34;256 - mean grey value.&#34; The melanization measure is a value between 0 and 256, with 0 representing a totally white image.</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.

This injection technique is useful to stimulate and study the melanization response in mosquitoes. For example, here we studied the effect of the immune stimuli load.
The critical step in this procedure is to properly inoculate the mosquito. Any excessive damage to the flight muscles or to the mosquito itself may prevent the mosquito from feeding or may kill it before the dissection. A second key step is to keep the mosquitoes on ice long enough to knock them out without killing them. A small ...

Insects rely on immune responses to protect themselves against parasites and pathogens1-3 that breach through their cuticle or their midgut epithelium4. In mosquitoes, these responses are efficient against bacteria5, viruses6, filarial nematodes7, and malaria parasites1,8,9. In mosquitoes, a key immune response is the encapsulation of foreign particles with melanin10-12. This encapsulation may happen in the midgut or in the hemolymph circulating system10-12. This melanization response is the result of the pro-phenoloxidase cascade10-12, and it can lead to the death of the parasites or to their phagocytosis. In adult mosquitoes, where the number of hemocytes cells is limited, melanization is a humoral response, like against plasmodium parasites or filarial nematodes 7. In some other insects, it is directly the hemocytes cells that gather around the parasite to melanize them7. Besides, melanin is also essential for several other physiological process like egg production and cuticle wounds healing7.
The stimulation of immune responses is used as a tool to study insect immunity in several agricultural and public health model systems13-18. It is used in Anopheles gambiae mosquitoes, the major vector of malaria in Africa, to study host-parasite interactions14-16,19. These techniques are based on the capacity of insects to detect parasites with their pattern recognition receptors (PRR)2. Mosquitoes may also detect other molecules interfering with their biology such as pathogen-associated molecular patterns (PAMPs), or detect their own damaged cells due to the release of collagen and nucleic acids. The mosquito immune cells such as the hemocytes are used for detection20-23. The main immune signaling pathways are Imd, Toll, JAK/STAT24, and ribonucleic acid interference (RNAi)25,26. Both Toll and Imd pathways influence the melanization response and interact with the pro-phenoloxidase cascade10-12.
The standard tool used to stimulate the melanization response is the inoculation of a mosquito with a small bead into the hemolymph of the thoracic cavity. The degree of melanin encapsulation can then be measured19 after retrieving the bead through the dissection of the mosquito. In most studies, only one bead was injected per mosquito15,16,27, but injecting more beads is possible in order to study the limits of the melanization response19. These beads are injected using an injection solution (physiological serum) to limit disturbance of the mosquito physiology and the desiccation of the mosquito15,16,27. A dye is added to this solution to ease bead selection. It is the same for the dissection solution used to retrieve the bead15,16,27.
The advantage of inoculating insects with non-pathogenic stimuli is the ability to focus on the direct effect on the immune response. There are no complicating effects due to parasite pathogenicity28, immunosuppression29-31, or immune evasion31-34. Besides, the consequences of the stimulations on other life history traits, such as longevity or fecundity, can also be studied. Thus, researchers studying evolutionary ecology may require such tools2,35,36. For example, immuno-challenged bumblebees have a shortened life span under starvation. Similar negative effects of immune stimulations and deployments have been observed in different invertebrate models, often resulting in a shorter lifespan or less reproductive success13,27,37. Such studies can be conducted in varying environments2,4,38. Stimulating immunity is also of interest to those focusing directly on immunopathology39,40.
This protocol is based on the inoculation of beads with mosquitoes to stimulate the melanization response and directly measure the amount of melanin. This enables quantitative and qualitative study of the melanization response in different experimental settings. Such a tool can be extended to the stimulation of other immune responses, such as the antibacterial response to heat-killed bacteria41. It can also be conducted in many ecological settings.

<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

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

Mosquitoes did not all melanize the beads in the same way, as some beads were less covered with melanin than others (Figure 1). Indeed, some beads remained blue because of a lack of melanization, whereas others were completely dark (Figure 1). The melanization value was standardized by linear interpolation to a value between 0 (which corresponded to a blue and unmelanized bead) and 100 (corresponding to a dark and heavily melanized bead) (Figure 2...

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.

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

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

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.

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

Biology

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.

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

Genetics

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.

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.

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

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.

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.

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 ...

Medicine

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.

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.

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.

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.

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

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

Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Microinjection Method for Anopheles gambiae Embryos

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

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

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

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

Generating Genetically Modified Plasmodium berghei Sporozoites

RNAi Trigger Delivery into Anopheles gambiae Pupae

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