Name: a bacterial oral feeding assay with antibiotic-treated mosquitoes
Uploaded: 2020-09-12T14:00:00
Description: Watch this Scientific Journal Video about A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes at JoVE.com

Este trabalho foi apoiado pela Fundação Nacional de Ciência Natural da China (Grant No. 81902094, 81600497), e pelo Projeto do Plano de Ciência e Tecnologia da Província de Hunan (2019RS1036).

1. Dissecção midgut e isolamento de bactérias cultivais
<ol><li>Prepare o mosquito para dissecção.<ol>
<li>Recolher os mosquitos de 7 a 9 dias após o surgimento com um aspirador. Anestesiar os mosquitos coletados submetendo-os a uma temperatura de 4 °C por 3-5 min e manter os mosquitos anestesiados em uma placa de Petri gelada até a dissecção.</li>
	</ol></li>
	<li>Esterilizar instrumentos de laboratório e a superfície do mosquito.<ol>
<li>Esterilize o banco de experimentos...

<ol>
	<li>Tolle, 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=Mosquito-borne+diseases.">Mosquito-borne diseases.</a> Current Problems in Pediatric and Adolescent Health Care. 39 (4), 97-140 (2009).</li><li>Wu, P., Yu, X., Wang, P., Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arbovirus+lifecycle+in+mosquito:+acquisition,+propagation+and+transmission.">Arbovirus lifecycle in mosquito: acquisition, propagation and transmission.</a> Expert Reviews in Molecular Medicine. 21, 1 (2019).</li><li>Jayakrishnan, L., Sudhikumar, A. V., Aneesh, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+gut+inhabitants+on+vectorial+capacity+of+mosquitoes.">Role of gut inhabitants on vectorial capacity of mosquitoes.</a> Journal of Vector Borne Diseases. 55 (2), 69 (2018).</li><li>Jupatanakul, N., Sim, S., 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=The+insect+microbiome+modulates+vector+competence+for+arboviruses.">The insect microbiome modulates vector competence for arboviruses.</a> Viruses. 6 (11), 4294-4313 (2014).</li><li>Moro, C. V., Tran, F. H., Raharimalala, F. N., Ravelonandro, P., Mavingui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+culturable+bacteria+including+Pantoea+in+wild+mosquito+Aedes+albopictus.">Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus.</a> BMC Microbiology. 13 (1), 70 (2013).</li><li>Chouaia, B., 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=Molecular+evidence+for+multiple+infections+as+revealed+by+typing+of+Asaia+bacterial+symbionts+of+four+mosquito+species.">Molecular evidence for multiple infections as revealed by typing of Asaia bacterial symbionts of four mosquito species.</a> Applied and Environmental Microbiology. 76 (22), 7444-7450 (2010).</li><li>Terenius, O., 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=Midgut+bacterial+dynamics+in+Aedes+aegypti.">Midgut bacterial dynamics in Aedes aegypti.</a> FEMS Microbiology Ecology. 80 (3), 556-565 (2012).</li><li>Bando, H., 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=Intra-specific+diversity+of+Serratia+marcescens+in+Anopheles+mosquito+midgut+defines+Plasmodium+transmission+capacity.">Intra-specific diversity of Serratia marcescens in Anopheles mosquito midgut defines Plasmodium transmission capacity.</a> Scientific Reports. 3, 1641 (2013).</li><li>Telang, A., Skinner, J., Nemitz, R. Z., McClure, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenome+and+culture-based+methods+reveal+candidate+bacterial+mutualists+in+the+Southern+house+mosquito+(Diptera:+Culicidae).">Metagenome and culture-based methods reveal candidate bacterial mutualists in the Southern house mosquito (Diptera: Culicidae).</a> Journal of Medical Entomology. 55 (5), 1170-1181 (2018).</li><li>Wang, Y., Gilbreath, T. M., Kukutla, P., Yan, G., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+gut+microbiome+across+life+history+of+the+malaria+mosquito+Anopheles+gambiae+in+Kenya.">Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya.</a> PloS One. 6 (9), (2011).</li><li>Xiao, X., 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=A+Mesh-Duox+pathway+regulates+homeostasis+in+the+insect+gut.">A Mesh-Duox pathway regulates homeostasis in the insect gut.</a> Nature Microbiology. 2 (5), 17020 (2017).</li><li>Gu&#233;gan, M., 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=Short-term+impacts+of+anthropogenic+stressors+on+Aedes+albopictus+mosquito+vector+microbiota.">Short-term impacts of anthropogenic stressors on Aedes albopictus mosquito vector microbiota.</a> FEMS Microbiology Ecology. 94 (12), 188 (2018).</li><li>Valzania, L., Coon, K. L., Vogel, K. J., Brown, M. R., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-induced+transcription+factor+signaling+is+essential+for+larval+growth+of+the+mosquito+Aedes+aegypti.">Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquito Aedes aegypti.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (3), 457-465 (2018).</li><li>Coon, K. L., Vogel, K. J., Brown, M. R., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquitoes+rely+on+their+gut+microbiota+for+development.">Mosquitoes rely on their gut microbiota for development.</a> Molecular Ecology. 23 (11), 2727-2739 (2014).</li><li>de O Gaio, A., 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=Contribution+of+midgut+bacteria+to+blood+digestion+and+egg+production+in+Aedes+aegypti+(diptera:+culicidae)(L).">Contribution of midgut bacteria to blood digestion and egg production in Aedes aegypti (diptera: culicidae)(L).</a> Parasites &amp; Vectors. 4 (1), 105 (2011).</li><li>Ramirez, J. L., 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=Reciprocal+tripartite+interactions+between+the+Aedes+aegypti+midgut+microbiota,+innate+immune+system+and+dengue+virus+influences+vector+competence.">Reciprocal tripartite interactions between the Aedes aegypti midgut microbiota, innate immune system and dengue virus influences vector competence.</a> PLoS Neglected Tropical Diseases. 6 (3), 1561 (2012).</li><li>Dong, Y., Morton, J. C., Ramirez, J. L., Souza-Neto, J. A., 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=The+entomopathogenic+fungus+Beauveria+bassiana+activate+toll+and+JAK-STAT+pathway-controlled+effector+genes+and+anti-dengue+activity+in+Aedes+aegypti.">The entomopathogenic fungus Beauveria bassiana activate toll and JAK-STAT pathway-controlled effector genes and anti-dengue activity in Aedes aegypti.</a> Insect Biochemistry and Molecular Biology. 42 (2), 126-132 (2012).</li><li>Anglero-Rodriguez, Y. I., 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=An+Aedes+aegypti-associated+fungus+increases+susceptibility+to+dengue+virus+by+modulating+gut+trypsin+activity.">An Aedes aegypti-associated fungus increases susceptibility to dengue virus by modulating gut trypsin activity.</a> Elife. 6, 28844 (2017).</li><li>Wu, P., 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=A+gut+commensal+bacterium+promotes+mosquito+permissiveness+to+arboviruses.">A gut commensal bacterium promotes mosquito permissiveness to arboviruses.</a> Cell Host &amp; Microbe. 25 (1), 101-112 (2019).</li><li>M&#246;hlmann, T. W., 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=Impact+of+gut+bacteria+on+the+infection+and+transmission+of+pathogenic+arboviruses+by+biting+midges+and+mosquitoes.">Impact of gut bacteria on the infection and transmission of pathogenic arboviruses by biting midges and mosquitoes.</a> Microbial Ecology. , (2020).</li><li>Llorca, M., Gros, M., Rodr&#237;guez-Mozaz, S., Barcel&#243;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preservation+for+the+analysis+of+antibiotics+in+water.">Sample preservation for the analysis of antibiotics in water.</a> Journal of Chromatography. A. 1369, 43-51 (2014).</li><li>Berendsen, B., Elbers, I., Stolker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+stability+of+antibiotics+in+matrix+and+reference+solutions+using+a+straightforward+procedure+applying+mass+spectrometric+detection.">Determination of the stability of antibiotics in matrix and reference solutions using a straightforward procedure applying mass spectrometric detection.</a> Food Additives &amp; Contaminants: Part A, Chemistry, Analysis, Control, Exposure &amp; Risk Assessment. 28 (12), 1657-1666 (2011).</li><li>Hill, C. L., Sharma, A., Shouche, Y., Severson, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+midgut+microflora+and+dengue+virus+impact+on+life+history+traits+in+Aedes+aegypti.">Dynamics of midgut microflora and dengue virus impact on life history traits in Aedes aegypti.</a> Acta Tropica. 140, 151-157 (2014).</li><li>Eng, M. W., 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=Multifaceted+functional+implications+of+an+endogenously+expressed+tRNA+fragment+in+the+vector+mosquito+Aedes+aegypti.">Multifaceted functional implications of an endogenously expressed tRNA fragment in the vector mosquito Aedes aegypti.</a> PLoS Neglected Tropical Diseases. 12 (1), 0006186 (2018).</li><li>Kajla, M. K., Barrett-Wilt, G. A., 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=Bacteria:+A+novel+source+for+potent+mosquito+feeding-deterrents.">Bacteria: A novel source for potent mosquito feeding-deterrents.</a> Science Advances. 5 (1), 6141 (2019).</li><li>Gon&#231;alves, G. G. A., 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=Use+of+MALDI-TOF+MS+to+identify+the+culturable+midgut+microbiota+of+laboratory+and+wild+mosquitoes.">Use of MALDI-TOF MS to identify the culturable midgut microbiota of laboratory and wild mosquitoes.</a> Acta Tropica. 200, 105174 (2019).</li><li>Kuss, S. K., 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=Intestinal+microbiota+promote+enteric+virus+replication+and+systemic+pathogenesis.">Intestinal microbiota promote enteric virus replication and systemic pathogenesis.</a> Science. 334 (6053), 249-252 (2011).</li><li>Rani, A., Sharma, A., Rajagopal, R., Adak, T., Bhatnagar, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+diversity+analysis+of+larvae+and+adult+midgut+microflora+using+culture-dependent+and+culture-independent+methods+in+lab-reared+and+field-collected+Anopheles+stephensi-an+Asian+malarial+vector.">Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector.</a> BMC Microbiology. 9 (1), (2009).</li><li>Apte-Deshpande, A., Paingankar, M., Gokhale, M. D., Deobagkar, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serratia+odorifera+a+midgut+inhabitant+of+Aedes+aegypti+mosquito+enhances+its+susceptibility+to+dengue-2+virus.">Serratia odorifera a midgut inhabitant of Aedes aegypti mosquito enhances its susceptibility to dengue-2 virus.</a> PLoS One. 7 (7), 40401 (2012).</li><li>Behura, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+microbiota+and+metagenomics,+and+its+relevance+to+disease+transmission.">Mosquito microbiota and metagenomics, and its relevance to disease transmission.</a> Nature. 436, 257-260 (2013).</li><li>Dickson, L. 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Pesquisas sobre interações hospedeiras-micróbios descobriram que diferentes micróbios intestinais afetam sua fisiologia hospedeira através de mecanismos divergentes. Este artigo introduz o método para investigar o respectivo papel do micróbio intestinal do mosquito, incluindo dissecação de mosquito midgut, cultivo de bactérias intestinais cultivable, tratamento antibiótico e reintrodução das bactérias de interesse.
Para o tratamento com antibióticos bem-sucedido, os seguintes de...

Os mosquitos são considerados os vetores mais importantes das doenças patogênicas humanas, transmitindo mais de cem patógenos, incluindo o vírus Zika, o vírus da dengue e os parasitas do Plasmodium 1. Quando os mosquitos fazem uma refeição sanguínea para adquirir nutrientes para oviposição, eles podem acidentalmente ingerir patógenos de um hospedeiro infectado através do trato digestivo2. É importante ressaltar que o mosquito midgut, que desempenha um papel fundamental tanto na digestão da refeição sanguínea quanto na entrada do patógeno, abriga um microbioma altamente dinâmico3.
Vários estudos caracterizaram microbiota de mosquitos criados em laboratório e coletados em campo usando um método dependente da cultura ou um ensaio de sequenciamento de bactérias4,,5,,6. Espécies como Pantoea, Serratia, Klebsiella, Elizabethkingiae Enterococcus são comumente isoladas de mosquitos em diversos estudos5,,7,,8,9. Curiosamente, a microbiota intestinal do mosquito flutua dinamicamente tanto na diversidade comunitária quanto na quantidade de espécies de bactérias, afetadas pelo estágio de desenvolvimento, espécies, origem geográfica e comportamento alimentar4. Estudos mostram que a alimentação sanguínea aumenta drasticamente a carga bacteriana total com rápida expansão de espécies de Enterobacteriaceae e redução da diversidade global10,11. Além disso, a microbiota intestinal do mosquito do estágio larval é geralmente erradicada quando o inseto sofre metamorfose durante a pupação e a eclosão; assim, os mosquitos adultos recém-emergidos precisam repovoar sua microbiota4.
Microbiota intestinal modula a fisiologia de insetos em vários aspectos, incluindo absorção de nutrientes, imunidade, desenvolvimento, reprodução e competência vetorial12. As larvas de mosquitos axenic não se desenvolvem além da primeira instar enquanto uma bactéria de suprimento oral resgata o desenvolvimento, indicando que o micróbio intestinal do mosquito é essencial para o desenvolvimento larval13,14. Além disso, o esgotamento das bactérias intestinais retarda a digestão da farinha de sangue e a absorção de nutrientes, afeta a maturação do oócito e diminui a oviposição15. Além disso, mosquitos com microflora intestinal provocam maiores respostas imunes em comparação com mosquitos tratados com antibióticos, com expressão de peptídeo antimicrobiano constantemente elevada contra outros patógenos para infectar16. Antibióticos são geralmente administrados oralmente para remover bactérias intestinais pan nesses estudos, e então experimentos são realizados para comparar a diferença entre mosquitos machados e mosquitos com micróbios commensais. No entanto, o mosquito midgut abriga uma comunidade diversificada de micróbios, e cada espécie de bactéria poderia exercer um efeito distinto em relação à fisiologia hospedeira.
Microbiota de mosquitos regula a competência vetorial com efeitos divergentes. Colonização por Proteus isolada de mosquitos derivados de campo de áreas endêmicas da dengue confere expressão de peptídeo antimicrobiano regulamentada e resistência contra a infecção pelo vírus da dengue16. O fungo entomopatômico Beauveria bassiana ativa a via imunológica Toll e JAK-STAT contra a infecção por arboviroses17. Em contrapartida, o fungo Talaromyces isolado do Aedes aegypti midgut facilita a infecção pelo vírus da dengue através da modulação da atividade de trippsina intestinal18. Além disso, Serratia marcescens promove a transmissão de arboviroses através de uma proteína secreta chamada SmEnhancin, que digere a camada de mucina no epitélio intestinal dos mosquitos19.,
Este procedimento fornece um método sistemático e intuitivo para dissecção do mosquito midgut, isolamento de colônias de bactérias cultivavel, identificação das espécies de bactérias e reintrodução via alimentação oral. Fornece resultados representativos de alimentação sanguínea com uma bactéria commensal, Chryseobacterium meningosepticum,no desenvolvimento do ovário do mosquito e oviposição.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td >Sigma</td>
			<td >A2383</td>
			<td >Adenosine 5&#8242;-triphosphate disodium salt hydrate has been used to prepare adenosine triphosphate (ATP) standard solutions</td>
		</tr>
		<tr >
			<td >Aedes aegypti</td>
			<td></td>
			<td></td>
			<td>Female mosquitoes</td>
		</tr>
		<tr >
			<td >Anticoagulant tube</td>
			<td>BD Vacutainer</td>
			<td>363095</td>
			<td>Collect fresh blood</td>
		</tr>
		<tr >
			<td >Centrifuge tube</td>
			<td>Sangon Biotech</td>
			<td>F601620-0010</td>
			<td>1.5 ml, Natural, Graduated, Sterile</td>
		</tr>
		<tr >
			<td >Cotton balls</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Disposable Tissue Grinding Pestle</td>
			<td>Sangon Biotech</td>
			<td>F619072-0001</td>
			<td>70 mm Long, Conical, Blue, Sterile</td>
		</tr>
		<tr >
			<td >Ethanol absolute</td>
			<td>Paini</td>
			<td></td>
			<td>Dilute it to 75% ethanol</td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td>RWD</td>
			<td>F11029</td>
			<td>Dissection</td>
		</tr>
		<tr >
			<td >Hemotek Membrane Feeding System</td>
			<td>Hemotek</td>
			<td></td>
			<td>Components of the feeding system, including&#160; Hemotek temperature controller, feeder-housing assembly, metal feeder assembled.</td>
		</tr>
		<tr >
			<td >Incubator shaker</td>
			<td></td>
			<td>ZQZY-78AN</td>
			<td></td>
		</tr>
		<tr >
			<td >Inoculation Loops</td>
			<td>Sangon Biotech</td>
			<td>F619312-0001</td>
			<td>10 &#956;l, Yellow</td>
		</tr>
		<tr >
			<td >LB Agar Powder</td>
			<td>Sangon Biotech</td>
			<td>A507003</td>
			<td>Tryptone 10.0 g&#65307; Yeast Extract 5.0 g&#65307; NaCl 10.0 g&#65307; Agar 15.0 g.</td>
		</tr>
		<tr >
			<td >LB Broth Powder</td>
			<td>Sangon Biotech</td>
			<td>A507002</td>
			<td>Tryptone 10.0 g&#65307; Yeast Extract 5.0 g&#65307; NaCl 10.0 g.</td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td>Zeiss</td>
			<td>Stemi508</td>
			<td></td>
		</tr>
		<tr >
			<td >Paper cup</td>
			<td></td>
			<td></td>
			<td>Place mosquito</td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td>Sangon Biotech</td>
			<td>F104002</td>
			<td>4 inx 125 ft</td>
		</tr>
		<tr >
			<td >Petri dish</td>
			<td>Sangon Biotech</td>
			<td>F611203</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin G procaine salt hydrate</td>
			<td>Sangon Biotech</td>
			<td>A606248</td>
			<td>White powder. Soluble in water, soluble in methanol, slightly soluble in water, ethanol</td>
		</tr>
		<tr >
			<td >Single Channal Pipettor</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Streptomycin sulfate</td>
			<td>Sangon Biotech</td>
			<td>A610494</td>
			<td>Streptomycin sulfate is a glucosamine antibiotic that interferes with the synthesis of prokaryotic proteins.</td>
		</tr>
		<tr >
			<td >Sucrose</td>
			<td>Sangon Biotech</td>
			<td>A502792</td>
			<td>Soluble in water, ethanol and methanol, slightly soluble in glycerol and pyridine.</td>
		</tr>
		<tr >
			<td >TIANamp Bacteria DNA Kit</td>
			<td>TIANGEN</td>
			<td>DP302</td>
			<td>Extract DNA&#160;</td>
		</tr>
		<tr >
			<td >Utility Fabric-Mosquito Netting White</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vortex mixer</td>
			<td>Scintic Industries</td>
			<td>S1-0246</td>
			<td></td>
		</tr>
		<tr >
			<td >1.5ml EP tube</td>
			<td>Sangon Biotech</td>
			<td>F600620</td>
			<td></td>
		</tr>
		<tr >
			<td >10X PBS buffer</td>
			<td>Sangon Biotech</td>
			<td>E607016</td>
			<td>This product is a 10X solution. Please dilute it 10 times before use. The pH value is 7.4.</td>
		</tr>
	</tbody>
</table>

a bacterial oral feeding assay with antibiotic-treated mosquitoes

O mosquito midgut abriga um microbioma altamente dinâmico que afeta o metabolismo hospedeiro, reprodução, aptidão e competência vetorial. Estudos têm sido realizados para investigar o efeito de micróbios intestinais como um todo; no entanto, diferentes micróbios poderiam exercer efeitos distintos em relação ao hospedeiro. Este artigo fornece a metodologia para estudar o efeito de cada micróbio intestinal específico do mosquito e o mecanismo potencial.
Este protocolo contém duas partes. A primeira parte introduz como dissecar o mosquito midgut, isolar colônias de bactérias cultivaveles e identificar espécies de bactérias. A segunda parte fornece o procedimento para gerar mosquitos tratados com antibióticos e reintroduzir uma espécie específica de bactérias.

Os midguts de mosquitos tratados com antibióticos e sem antibióticos foram retirados para extração de DNA, e qPCR foi realizado com primers bacterianos universais. A Figura 1 mostra a expressão de rRNA bacteriana 16S no grupo controle e grupo de tratamento de antibióticos. Os resultados mostram que cerca de 98% das bactérias intestinais foram removidas, e a esterilização intestinal de penicilina e estreptomicina foi bem sucedida.
Com os métodos descritos, as cepas de...

Watch this Scientific Journal Video about A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes at JoVE.com

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

Um ensaio de alimentação oral bacteriana com mosquitos tratados com antibióticos

Este artigo apresenta um protocolo para investigar o efeito de bactérias intestinais individuais do mosquito, incluindo isolamento e identificação de micróbios cultivados de mosquitos midgut, esgotamento de antibióticos de bactérias intestinais de mosquitos e reintroduzir uma espécie específica de bactérias.

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Mosquito midgut abriga uma comunidade complexa de micróbios que afetam o metabolismo hospedeiro, reprodução, aptidão e competência veterinária. Com cada micróbio intestinal é um efeito diferente. Este vídeo introduz o procedimento para estudar o respectivo papel de cada cepa de bactérias ou fisiologia hospedeira.Para adquirir uma cultura de colônias bacterianas intestinais, disseque o mosquito midgut sob condição estéril. Aterrar o meio-boca dissecado, serotologinado, e espalhe sobre a placa de ágar. Escolha colônias únicas da placa, e identifique as espécies de bactérias através de sua sequência genética de 16 SRNA.Alimente os mosquitos com COD Emboss contendo antibióticos por três dias consecutivos para remover microbiota intestinal. A eficácia do tratamento com antibióticos pode ser confirmada dissecando o midgut do mosquito asséptico para ensaio da unidade de formação de colônias antes dos extremistas subsequentes. Em seguida, reintroduzir espécies de bactérias específicas que anteriormente se isolaram do intestino do mosquito através do ensaio de alimentação oral.O efeito das bactérias na fisiologia hospedeira poderia ser avaliado comparando os mosquitos não tratados, mosquitos tratados com antibióticos e os mosquitos reintroduzidos com restrição específica. Dissecção midgut e o isolamento de bactérias cultivadas. Para começar, recolher os mosquitos na gaiola com um aspirador e célula dos mosquitos na caixa de coleta.Anestesiar os mosquitos submetendo-se a uma temperatura de 4 graus por três a cinco minutos. Mantenha os mosquitos anestesiados colocando-os em uma placa de Petri gelada. Desinfetar o banco, dissecando microscópio e fórceps pulverizando 75% de etanol para evitar contaminação por bactérias do meio ambiente.A superfície esteriliza o mosquito gravando e sacudindo-os em 75% de etanol por três minutos e enxágue duas vezes com tampão PBS. Bissecte o mosquito separadamente em uma lâmina de vidro estéril contendo sua gota de PBS. Remova cuidadosamente as pernas, asas e a cabeça do mosquito usando fórceps.Prender o peito do mosquito e a última seção do abdômen foi fórceps e se separar para isolar o trato digestivo. Use fórceps para remover a cultura e o tubo malpighiano do trato digestivo. A cultura é posicionada no pnterior de midgut e protuberâncias após a ingestão de água de açúcar.Tubos malpighianos são um grupo de tubos excretórios finos na junção de midgut e hindgut. Coloque a maquiagem dissecada no tubo EP com 200 microliters estéreis tampão PBS e moer com o pastor de moagem estéril no banco limpo. Cereal para carregar o homogeneizar para três delírios tempo em 50 microliters de cada ilusão para a placa de ágar LB e placa desesperada.Incubar a placa como 37 graus por um a dois dias até que colônias únicas sejam viáveis. Escolha colônias simples em um frasco cônico de 150 mililitros contendo 50 mililitros de sutiãs lb. agitar a bactéria a 37 graus durante a noite.Identificação de espécies. Extrair DNA TOTO por kit de construção de DNA genômico bacteriano e amplificar 16 SRDNA por PCR. Recuperação e purificação de fragmentos de DNA de produtos PCR por eletroforese de gel ambrose e o kit de recuperação de dirigir.Realizou sequenciamento de DNA nos fragmentos de DNA purificados para ter uma sequência de cadeia de bactérias alcançadas. Execute a pesquisa de explosão para uma consulta a sequência genética de 16 SRNA de identificar as bactérias contra as bactérias e o archaea e banco de dados. A identificação ao nível da espécie foi definida como maior ou igual a 99%16 de sequência SRDNA similaridade à entrada de banco de equipe mais próxima.O isolado foi atribuído ao gênero correspondente, quando é 16 sequência SRDNA, a semelhança foi inferior a 99% e maior ou igual a 95% de tratamento antibiótico e reintrodução de bactérias. pesar a quantidade necessária de penicilina de sacarose e estreptomicina para nos preparar solução de 10% de sacarose, incluindo tentar 20 unidades de penicilina e 20 microgramas de estreptomicina por mililitro. Infiltrar as bolas de algodão na solução de sacarose contendo antibióticos e colocá-lo sobre o topo do copo de papel contendo mosquito.Cubra a bola de algodão com uma placa de Petri de 10 centímetros para evitar que uma orla evapore. Substitua as bolas de algodão duas vezes por dia e alimente o mosquito por três dias consecutivos. Os mosquitos são divididos em dois grupos.O primeiro grupo foi colocado em um copo de mosquito sem qualquer tratamento, qualquer médico de bolas de algodão com 10% Então, a cruz era dada diariamente. O segundo grupo foi tratado com antibióticos e serviu por três dias. Em cada grupo, os mosquitos Sergei foram dissecados.O Mika foi extraído para extração total de DNA e o QPCR foi realizado utilizando primers universais de bactérias, figura 1 mostra a expressão de 16 SRNA no grupo controle e grupo de tratamento de antibióticos. Os resultados mostram que o custo ou realização da penicilina e estreptomicina foi bem sucedido. preparar solução bacteriana, mede uma solução de bactérias no vírus com um espectrofotômetro, adicione uma suspensão de bactérias OT no tubo EP.Centrifugar a suspensão a 5.000 RCA por cinco minutos a quatro graus Descarte o supernascedor, lave a paleta bacteriana com tampão PBS estéril. Suspenda a palete bacteriana com 200 microliters tampão PBS estéril Adicione 600 microliter, solução de 10% de sacarose, 200 microliter ATP e 200 microliter suspensão bacteriana em um novo tubo AP e misture bem. Alternativamente, as bactérias podem ser re-suspensas no calor para ativar seu sangue.Preparação de aquecimento ativou sangue. coágulo sangue fresco com tubo anticoagulante, tome de dois a três mililitros de sangue fresco. centrífuga a 1000 por 10 minutos a 4 graus para separar plasma e células sanguíneas.Colete o plasma em um novo tubo EP e aqui ativar ativado a 56 graus por uma hora. Lave a casa de explosão através de seus tempos com tampão PBS estéril. Enxágüe a casa de explosão suspensa com plasma ativado de aquecimento.Montar membrana e sistema de alimentação. Puxe a doença do filme ao máximo. fix com o anel de plástico e parafilm local de trabalho para evitar o vazamento lentamente adicione a solução bacteriana preparada à unidade alimentadora, ao máximo da carteira.Para evitar que a bolha caísse de uma direção e selasse a unidade alimentador. Conecte a fonte de alimentação. Aguarde até que a temperatura atinja 37 graus Instale a unidade alimentador com solução de bactérias no alimentador.Antes de alimentar os mosquitos das bactérias deve ser interrompido por 24 horas para metabolizar antibióticos. Para a unidade de alimentação no copo de papel com mosquitos, em seguida, deixe a alimentação por 90 minutos. Mosquitos totalmente encorajados poderiam ser escolhidos para estudos posteriores.Resultados representativos. A figura dois nos mostra o reconhecimento médio por mosquito. Após a in loção de antibiótico tratado os mosquitos vemos meningosépticos.A Figura Três mostra a expressão de uma variante de desenvolvimento de genes relacionados 24 horas após a refeição sanguínea contendo o meningosséptico C. Os resultados mostram que não há mudança significativa na produção de ovos do grupo controle e do grupo de desbotamento. Obrigado por seu interesse por bactérias ou uma redação de alimentação com mosquitos tratados com antibióticos.

a-bacterial-oral-feeding-assay-with-antibiotic-treated-mosquitoes

Research

JoVE Journal

Biology

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.

Uma injeção de. Embriões stephensi para gerar malária resistente a mosquitos

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

Biologia

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

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

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

Impedir a propagação da malária e dengue por meio de mosquitos geneticamente modificados

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

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

Protocolos para a Infecção Oral de lagartas de Lepidoptera com Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be ...

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

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

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

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

RNAi mediada knockdown do gene e<em&gt; In Vivo</em&gt; Ensaio diurese no Feminino Adulto<em&gt; Aedes aegypti</em&gt; Os mosquitos

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

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

Um ensaio para detecção multi-malária mosquitos transmissores

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

Alto teor de gordura Alimentação Paradigma para larval Zebrafish: Alimentação, Imagens ao vivo, e quantificação da ingestão de alimentos

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

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

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

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

Manter o Aedes aegypti , mosquitos infectados com Wolbachia

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

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Infecção bacteriana e oral Shedding em Drosophila melanogaster.

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

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

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

Protocolos para testar a toxicidade de romance químicas inseticidas para mosquitos

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

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.