Name: physiological recordings and rna sequencing of the gustatory appendages of the yellow-fever mosquito aedes aegypti
Uploaded: 2014-12-30T14:00:00.000+00:00
Duration: 9 min 9 s
Description: Watch this Scientific Journal Video about Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti at JoVE.com

The authors thank Bryan T. Vinyard of the USDA, Agricultural Research Service, Henry A. Wallace Beltsville Agricultural Research Center, Biometrical Consulting Service, Beltsville, MD for statistical analyses. This work was supported in part by a grant to J.C.D. from the Deployed War Fighter Protection (DWFP) Research Program funded by the Department of Defense through the Armed Forces Pest Management Board (AFPMB).

1. Cria Ae. adultos aegypti <ol><li> Ovos eclodem em cerca de ¾ de polegada de água na bandeja rasa. A superlotação irá reduzir o tamanho dos adultos. </li><li> Larvas Rear a 25 ° C (12-HL: 12-HD) e se alimentam com comida de peixe chão. NOTA: super-alimentação pode reduzir as taxas de sobrevivência. </li><li> Remover pupas individualmente por Pasteur pipeta diária e transferir para pratos de plástico (9 cm × 5,5 cm) dentro de pequenos baldes de confinamento, com tampas de malha fina, estabelecendo, assim, 24 grupos etários hr. </li><li> Mosquitos adultos alimentar 10% de solução de sacarose por bola de algodão colocado nas paredes de tela de uma habitação gaiola os mosquitos em uma câmara a 27 ° C e 70% de umidade relativa do ar sob o mesmo fotoperíodo como larvas. </li><li> Para eletrofisiologia, use 5-10 dias animais velhos. Em geral, os animais maiores irão produzir melhores resultados. Para o isolamento do RNA, usar animais que estão dentro de um único agrupamento idade 24 hr no intervalo de 5-10 dias de idade. </li></ol><ol><li> Determinar a concentração adequada de eletrólitos (por exemplo, 1-10 mM) que provoca atividade mínima de neurônios sensoriais selecionadas. NaCl 10 mM ou 1 mM KCl são eletrólitos razoáveis ​​para testar actividade de base ao começar um experimento. </li><li> Dissolver cada produto químico experimental em solvente apropriado (se não a água, o uso de etanol ou DMSO) que tem pouco ou nenhum efeito na actividade de pico quando comparadas aos electrólito sozinho. As concentrações finais de etanol a 10% ou 1% de DMSO são concentrações de partida razoáveis. </li><li> Selecione adequado (por exemplo quinina por amargo ou sacarose para doces) de controle de produtos químicos que são bem descritos impedimentos alimentação ou estimulantes em insetos. </li></ol> 3. Eletrofisiologia (gravação ponta 8; Figura 1) <ol><li> Eletrodos de vidro Form puxando mecanicamente capilares de vidro. Otimizar calor e puxar configurações para puxar vidro para formar recordi forma apropriadaeletrodos GN. Quebre cuidadosamente off final do eletrodo de vidro usando uma pinça sob um microscópio de dissecação para criar ponta do eletrodo de abertura que é grande o suficiente para envolver sensillum alvo, mas pequeno o suficiente para evitar o contacto com estruturas vizinhas. </li><li> Sob um microscópio, identificar visualmente pela morfologia e posicionar o alvo sensilla / sensillum para garantir a repetibilidade de gravação ponta. Animais da preparação para permitir o acesso livre para sensilla de ângulo de entrada do eletrodo. </li><li> Anesthetize insetos adultos frias no congelador a -20 ° C. Evite danos aos neurônios periféricos por exposição excessiva ao frio. Corte tiras estreitas de fita adesiva com lâmina de barbear em lâmina de vidro. Imobilizar todo inseto em uma lâmina de vidro usando tiras estreitas. </li><li> Insira um fio de tungstênio quimicamente afiada no tórax dorsal ou olho para servir como o indiferente (terra) eletrodo. </li><li> Preencha eletrodo de vidro feita no passo 3.1 com uma solução estimulante de interesse. Usando uma seringa inserido, preencher capIllary de ponta puxado para blunt end. Deitar fora todas as bolhas de ar, segurando fim puxou para baixo. Coloque um fio de prata (chloriding opcional) para o eletrodo de vidro feita no passo 3.1 para servir como gravação e eletrodo estimulante. </li><li> Conecte-se eletrodos para um pré-amplificador que é projetado para gravações de contato chemoreceptive sensilla em insetos. Este pré-amplificador (300 Hz-3 kHz filtro de banda) irá reduzir o tempo de estabilização para capturar a atividade neuronal tão perto de estímulo introdução possível. Coletar, armazenar e analisar sinais elétricos usando um microcomputador equipado com software de gravação de pico. NOTA: O aterramento da configuração de gravação corretamente pode melhorar drasticamente a relação sinal-ruído. </li><li> Estimular sensilas com uma solução de controlo (apenas electrólito) para garantir a funcionalidade. Contagem de pontos a partir de 200 ms após o artefato estímulo para todas as preparações. </li><li> Aleatória a fim de produtos químicos de teste para todas as experiências, excepto estudos de dose-resposta,para eliminar o preconceito. Para os estudos de dose-resposta, para expor sensilas concentrações crescentes da substância química experimental. Permitir que um mínimo de 3 min entre as estimulações para garantir a recuperação adequada. Nota: Esta exigência pode variar dependendo do inseto e sensilla. O limiar é definido como a concentração para a qual o erro padrão não se sobrepõe com o erro padrão para a concentração mais baixa testada. </li></ol> 4. Isolamento de ARN e Sequenciação (Figura 2) <ol><li> Prepare os pilões livre de RNAse bem ajustados pelo arrefecimento los em gelo seco antes de tecido disruption.Prepare acompanham pilões livre de RNase que se encaixam tubos de tampa de pressão com força. Manter tubos de coleta fechados a menos que dissecando ativamente para evitar o excesso de condensação. </li><li> Usando aspirador filtrada, coloque 30 a 40 mosquitos frio anestesiados (15 seg em freezer -20 ° C) na fase fria e ordenar por sexo. Coloque animais dorsal lado para baixo, segurando asas para não danificar apêndices gustativas.</li><li> O uso de dois pares de uma pinça fina, dissecar labella pareado ou tarsos de machos ou fêmeas sob um microscópio de dissecação e limitar a inclusão dos tecidos adjacentes. NOTA: 500 labella emparelhado rende aproximadamente 800-1.000 nanogramas de ARNm total. 400 tarsos indivíduo (5 cada) produzem segmentos 1,200-1,800 nanogramas de ARNm total. </li><li> Com um par de ofegantes-fórceps, levemente agarrar tromba mosquito apenas proximais para labella expondo estiletes interiores. Usando outro par de recolha-pinça, retire labella e adicionar o tecido para o lado do tubo de coleta de frio. </li><li> Com um par de fórceps, levemente agarrar mosquito perna no cruzamento da tíbia e do primeiro segmento tarsal. Usando outro par de fórceps, retire tarsos e adicionar o tecido para o lado do tubo de coleta de frio. Nota: Tenha em conta os tipos de células na interface do tecido queria e indesejado. Embora seja importante para limitar a inclusão de tecidos vizinhos, é igualmente importante para não omitir as porções de tecido desejado. A final amostra deve representar fielmente todo o órgão de interesse. Criar um limite preciso com a pinça de apreensão de tal forma que uma pinça de coleta pode precisamente libertar por raspagem ou pegar apenas o tecido desejado. </li><li> Periodicamente, girar tubo de coleta brevemente em 0 ° C centrifugar a 9000 xg para manter o tecido no fundo do tubo. Se a umidade se acumula nos tubos de coleta durante a dissecção, adicione 50uL de reagente Trizol fria para cobrir o tecido coletado. </li><li> Usando marcador laboratório anti-manchas, identifique claramente um livre de RNase 1,5 ml encaixe tubo de tampa para cada tipo de tecido para a coleção. Usando 1,5 ml suporte de tubos e nitrogênio líquido, congelada em seguida, moer tecido em pó fino (pedaços finos se em Trizol). Repetir uma vez. Levar o volume total de 1 ml de Trizol para ressuspender e tecido triturado por agitação em vórtex. </li><li> Adicionar 200 ul de clorofórmio e misture bem. Depois de 5 min à temperatura ambiente, rotação para baixo numa centrífuga a 4 ° C e 11.000 xg durante 15 min. Cuidadosamenteadicionar fase aquosa directamente numa coluna de filtro de DNA genómico. Girar a 11.000 x g durante 5 min. Adicionar um volume igual de RNase etanol a 70% a fluir-through e misture por pipeta. Transferir líquido a uma coluna de recolha de RNA e continuar a extracção de RNA por as instruções fornecidas. </li><li> Quantificar e avaliar a pureza do RNA total em um espectrofotômetro de precisão e prosseguir com qualquer método RNA seqüenciamento padrão. </li></ol> 5. quantitativa de RT-PCR de RNA Sequenciação de validação (Figura 3) <ol><li> Seleccione tantos genes quanto possível comparar os níveis de expressão de genes relativos ao longo de um intervalo dinâmico, tanto em abundância previsto sequência e função do gene chemosensory presumido. </li><li> Pares de criação de iniciadores para cada gene alvo para amplificar um produto de PCR específico 100-180 pares de bases. Excluir amplificação gDNA não específica, garantindo pelo menos um iniciador por conjunto se estende por uma fronteira intron. Como alternativa, use o tratamento DNase para reduzir a contaminação genômica. </li><li> Coletartecidos de insecto, tal como descrito na secção anterior. Calcule quanto tecido é obrigada a fornecer RNA suficiente para completar todas as reações qRT-PCR. NOTA: triplicado Biológica e reações triplicado técnicos irá proporcionar o máximo de confiança nos resultados. </li><li> Calcule quantificação gene relativo como alvo X - (Ct [alvo] -Ct [referência]) para cada gene alvo e médio para cada réplica, tanto biológica quanto técnico. Use gene (s) serviço de limpeza para normalizar valores Ct entre repetições biológicas e para erradicar a escala do eixo Y em comparações de expressão relativa. </li><li> Avaliar a semelhança de RNA-seq e conjuntos de dados de qRT-PCR utilizando o, procedimento de bootstrap equivalência com base em regressão 9, aplicada iterativamente, para os dados a partir de cada tecido. NOTA: Este procedimento identifica a mais pequena &quot;região de semelhança&quot; que pode ser construído em torno dos dados de RNA-seq e qRT-PCR observado, e permitir a expressão do gene como medido pela relação qRT-PCR para ser considerado estatisticamente equivalente à medição da expressão do gene em relação ao ARN-seq. </li></ol>

<ol>
	<li>Klun, J. A., Khrimian, A., Debboun, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repellent+and+deterrent+effects+of+SS220,+picaridin,+and+DEET+suppress+human+blood+feeding+by+Aedes+aegypti,+Anopheles+stephensi,+and+Phlebotomus+papatasi.">Repellent and deterrent effects of SS220, picaridin, and DEET suppress human blood feeding by Aedes aegypti, Anopheles stephensi, and Phlebotomus papatasi.</a> J. Med. Entomol. 43 (1), 34-39 (2006).</li><li>Dickens, J. C., Bohbot, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini+review:+Mode+of+action+of+mosquito+repellents.Pestic.">Mini review: Mode of action of mosquito repellents.Pestic.</a> Biochem. Physiol. 106 (3), 149-155 (2013).</li><li>Pitts, R. J., Rinker, D. C., Jones, P. L., Rokas, A., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+chemosensory+appendages+in+the+malaria+vector+Anopheles+gambiae+reveals+tissue-+and+sex-specific+signatures+of+odor+coding.">Transcriptome profiling of chemosensory appendages in the malaria vector Anopheles gambiae reveals tissue- and sex-specific signatures of odor coding.</a> BMC Genomics. 12 (271), (2011).</li><li>Zhou, X., Slone, J. D., Rokas, A., Berger, S. L., Liebig, , 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=Phylogenetic+and+transcriptomic+analysis+of+chemosensory+receptors+in+a+pair+of+divergent+ant+species+reveals+sex-specific+signatures+of+odor+coding.">Phylogenetic and transcriptomic analysis of chemosensory receptors in a pair of divergent ant species reveals sex-specific signatures of odor coding.</a> PLOS Genet. 8 (8), e1002930 (2012).</li><li>Shiao, M., Fan, W., Fang, S., Lu, M. J., Kondo, R., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+adult+Drosophila+antenna+by+high-throughput+sequencing.">Transcriptional profiling of adult Drosophila antenna by high-throughput sequencing.</a> Zoological Studies. 52 (42), (2013).</li><li>Bohbot, J., Pitts, R. J., Kwon, H. W., R&#252;tzler, M., Robertson, H. M., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+Aedes+aegypti+odorant+receptor+gene+family.">Molecular characterization of the Aedes aegypti odorant receptor gene family.</a> Insect Mol. Biol. 16 (5), 525-537 (2007).</li><li>Kain, P., Boyle, S. M., Tharadra, S. K., Guda, T., Pham, C., 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=Odour+receptors+and+neurons+for+DEET+and+new+insect+repellents.">Odour receptors and neurons for DEET and new insect repellents.</a> Nature. 502 (7472), 507-512 (2013).</li><li>Hodgson, E. S., Lettvin, J. Y., Roeder, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+a+primary+chemoreceptor+unit.">The physiology of a primary chemoreceptor unit.</a> Science. 122 (3166), 417-418 (1955).</li><li>Robinson, A. P., Duursma, R. A., Marshall, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regression-based+equivalence+test+for+model+validation:+shifting+the+burden+of+proof.">A regression-based equivalence test for model validation: shifting the burden of proof.</a> Tree Physiol. 25 (7), 903-913 (2005).</li><li>Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+quantifying+mammalian+transcriptomes+by+RNA-seq.">Mapping and quantifying mammalian transcriptomes by RNA-seq.</a> Nat. Methods. 5 (7), 621-628 (2008).</li><li>Trapnell, C., Williams, B. A., Pertea, G., Mortazavi, A., Kwan, G., 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=Transcript+assembly+and+quantification+by+RNA-seq+reveals+unannotated+transcripts+and+isoform+switching+during+cell+differentiation.">Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation.</a> Nat. Biotechnol. 28 (5), 511-515 (2010).</li><li>Sanford, J. L., Shields, V. D. C., Dickens, 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=Gustatory+receptor+neuron+responds+to+DEET+and+other+insect+repellents+in+the+yellow-fever+mosquito,+Aedes+aegypti.">Gustatory receptor neuron responds to DEET and other insect repellents in the yellow-fever mosquito, Aedes aegypti.</a> Naturwiss. 100 (3), 269-273 (2013).</li><li>Sparks, J. T., Vinyard, B. T., Dickens, 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=Gustatory+receptor+expression+in+the+labella+and+tarsi+of+Aedes+aegypti.">Gustatory receptor expression in the labella and tarsi of Aedes aegypti.</a> Insect Biochem. Mol. Biol. 43 (12), 1161-1171 (2013).</li><li>Kent, L. B., Walden, K. K. O., Robertson, H. 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+Gr+family+of+candidate+gustatory+and+olfactory+receptors+in+the+yellow-fever+mosquito+Aedes+aegypti.">The Gr family of candidate gustatory and olfactory receptors in the yellow-fever mosquito Aedes aegypti.</a> Chem. Senses. 33 (1), 79-93 (2008).</li><li>Ramsk&#246;ld, D., Wang, E. T., Burge, C. B., Sandberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+abundance+of+ubiquitously+expressed+genes+revealed+by+tissue+transcriptome+sequence+data.">An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data.</a> PLoS Comput. Biol. 5 (12), e1000598 (2009).</li><li>Wagner, G. P., Kin, K., Lynch, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+based+criterion+for+gene+expression+calls+using+RNA-seq+data.">A model based criterion for gene expression calls using RNA-seq data.</a> Theory Biosci. 132 (3), 159-164 (2013).</li><li>Hart, T., Komori, H. K., LaMere, S., Podshivalova, K., Salomon, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+active+genes+in+deep+RNA-seq+gene+expression+studies.">Finding active genes in deep RNA-seq gene expression studies.</a> BMC Genomics. 14 (778), (2013).</li><li>Isono, K., Morita, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+designs+of+insect+taste+receptor+system.">Molecular and cellular designs of insect taste receptor system.</a> Front. Cellu. Neurosci. 4 (20), 1-16 (2010).</li></ol>

The authors have nothing to disclose.

O aspecto mais desafiador de potenciais de ação de gravação de sensilla gustativa é decidir quais as respostas são &quot;normais&quot;. Quando se emprega única ponta sensillum gustativa gravação pela primeira vez para uma determinada espécie de inseto, o número total e sensibilidade dos neurônios dos receptores gustativos (GRNs) são provavelmente desconhecido. Muitas gravações preliminares são primeiros obrigados a decidir o alcance e as concentrações de produtos químicos para testar. Neste exemplo, q...

Compostos como DEET, Picaridin, Citronelal e IR3535 foram mostrados para repelir os mosquitos de forma eficaz, incluindo os importante vetor da doença Aedes aegypti 1,2. Registramos potenciais de ação de neurônios sensoriais associados com sensilla gustativa específica para determinar as células envolvidas com mosquito repelência. Juntamente com a jusante sequenciação de genes expressos nestes tecidos, que pode identificar os genes mais provável que medeiam as respostas destas células, a fim de rastrear novos compostos para melhorar a capacidade de repelência. RNA-seq é uma ferramenta poderosa, rapidamente se tornando padrão para controlar alterações temporais e espaciais na expressão gênica. Analisa RNA-seq de apêndices quimio insectos e órgãos foram usadas para descobrir receptores moleculares em várias espécies de insectos 3-5, melhorando consideravelmente em pesquisas de genes baseados em PCR convencional pelo gene 6. Insetos representar a classe animal mais diverso, prétando muitas oportunidades para estudar a relação entre os genes e os fenótipos únicos. Tecnologia de RNA-seq pode ser empregue em qualquer tecido de insectos vivos. Da mesma forma, a gravação electrofisiológica de células sensoriais dentro sensilas gustativa uniporous pode ser conseguido de muitas diferentes espécies de insectos. O emparelhamento das duas técnicas, permite que os pesquisadores para reduzir os genes envolvidos no conjunto de um fenótipo chemosensory observado. Diferentes espécies apresentará desafios específicos, mas podem informar a conexão entre genes de receptores de quimio e uma adaptação chemosensory. O tamanho ea morfologia dos chemosensory sensilla é variável e poderá exigir solução de problemas durante a gravação de potenciais de ação para reduzir o ruído e identificar sinais repetíveis. As dissecções de órgãos quimio pode ser trivial ou delicado e demorado, dependendo da morfologia e tamanho do insecto. Recuperação de RNA de alta qualidade pode exigir alguma resolução de problemas, bem como, tais como evitar certos pigmentos durantecoleta de tecido. Ao demonstrar os efeitos dos compostos repelentes através de ensaios de comportamento é direta e informativa, esta abordagem é demorada e ampla em relação ao mecanismo de ação. Eletrofisiologia juntamente com RNA-seq permite análises mais específicas do que impulsiona comportamentos de esquiva em insetos. Uma vez que o &quot;kit&quot; de discriminação produtos químicos foi identificado em uma espécie de insecto, tentativas mais específicos para melhorar repelentes conhecidos são possíveis. Receptores e proteínas associadas em células sensoriais responsáveis ​​por estes comportamentos podem ser expressas de forma heteróloga para o rastreio química directa. Além disso, modelagem molecular pode prever quais os produtos químicos que induzem respostas fortes de estes receptores 7. O instantâneo de todos os genes activos num conjunto restrito de tecidos quimio pode também ser útil na identificação de genes semelhantes noutras espécies. Usando homologia de sequência e de expressão de similarities, os investigadores podem formar conjuntos de receptores moleculares mais provável que medeiam respostas aos repelentes que são amplamente eficaz de insectos. Nós apresentamos o seguinte protocolo para auxiliar os pesquisadores na desconstrução vias quimio insetos e persuadir mais se aprofundar no neuroethology de não-modelo e insetos de importância econômica.

<table><tbody><tr><td>Glass capillary</td><td>A-M Systems</td><td>628000</td><td>Standard, 1.5 mm X 0.86 mm, 4&quot;</td></tr><tr><td>Silver wire</td><td>A-M Systems</td><td>7875</td><td>.015&quot; bare</td></tr><tr><td>Tungsten wire</td><td>Alfa Products</td><td>369</td><td>0.127 mm diameter</td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td>11252</td><td>#5SF Inox</td></tr><tr><td>Refridgerated stage</td><td>BioQuip Products</td><td>1429</td><td>Chill Table</td></tr><tr><td>Preamplifier</td><td>Syntech</td><td>Taste Probe</td><td>preamplifier</td></tr><tr><td>Software for electrophysiology</td><td>Syntech</td><td>Autospike</td><td>software for electrophysiology</td></tr><tr><td>TetraMin fish food</td><td>Tetra</td><td>Tropical fish food granules</td><td>fish food ground to fine powder</td></tr><tr><td>TRIzol</td><td>Life Technologies</td><td>15596-026</td><td>RNA isolation reagent</td></tr><tr><td>RNeasy Plus Mini Kit</td><td>Qiagen</td><td>74136</td><td>includes gDNA eliminator and RNeasy spin columns</td></tr><tr><td>Nanodrop spectrophotometer</td><td>Nano Drop Products</td><td>ND-1000</td><td>tabletop spectrometer</td></tr><tr><td>R statistics</td><td>freeware (created by Robert Gentleman and Ross Ihaka)</td><td>www.r-project.org</td><td>Use the lm function of the stats package and the equiv.boot function of the equivalence package in the R computing environment.</td></tr><tr><td>1.5 ml tube rack</td><td>Evergreen</td><td>240-6388-030</td><td>Pour liquid nitrogen into a few empty wells to freeze and grind tissue.</td></tr><tr><td>1.5 ml collection tubes with pestle</td><td>Grainger</td><td>6HAX6</td><td>RNase free</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>11178160</td><td>Spin down frozen tissue to keep at bottom of 1.5 ml tube.</td></tr><tr><td>Primer-BLAST Primer Designing tool</td><td>NCBI</td><td>n/a</td><td></td></tr></tbody></table>

physiological recordings and rna sequencing of the gustatory appendages of the yellow-fever mosquito aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

As gravações de traço de potenciais de ação de Ae. sensilas gustativa aegypti (Figura 1) demonstrar a eficácia da estimulação directa com uma gama de produtos químicos. Esta técnica pode ser usada para quantificar respostas a qualquer químico estimulador contando picos de uma determinada amplitude e duração ao longo de um intervalo de tempo razoável (geralmente menos do que 500 ms). Gravações de rastreamento deve ser facilmente reproduzíveis sob um dado conjunto de con...

Watch this Scientific Journal Video about Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti at JoVE.com

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

O uso de dois métodos para estimar a expressão do gene nos principais apêndices gustativas de Aedes aegypti, identificamos o conjunto de genes putativamente subjacentes às respostas neuronais para compostos amargos e repulsivas, conforme determinado pelo exame eletrofisiológico.

Fisiológicas Recordings e RNA Sequenciação dos gustativas Appendages do Mosquito-febre amarela<em&gt; Aedes aegypti</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

physiological-recordings-rna-sequencing-gustatory-appendages-yellow

Research

JoVE Journal

Biology

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

8.0K visualizações.  United States Department of Agriculture.  O uso de dois métodos para estimar a expressão do gene nos principais apêndices gustativas de Aedes aegypti, identificamos o conjunto de genes putativamente subjacentes às respostas neuronais para compostos amargos e repulsivas, conforme determinado pelo exame eletrofisiológico. 

Vídeo: Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Único Recordings Sensillum na Insetos Drosophila melanogaster E Anopheles gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Neurociência

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

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


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

Dissecção das glândulas salivares e intestino médio de Ae. aegypti mosquitos

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

Biologia

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

Um Protocolo de Coleta e coloração Hemócitos do mosquito da febre amarela Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Imunologia e Infecção

Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.

Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology.

Edição do genoma no mosquito da febre amarela Aedes aegypti usando CRISPR-Cas9

The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering ...

Genética

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Alimentação e quantificação de sangue derivados de animais e refeições artificiais em mosquitos Aedes aegypti

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong preference for human hosts over other warm-blooded animals for blood meals. Olfactory cues play a critical role for mosquitoes as they explore their environment and seek a human host to obtain blood meals, thus transmitting human diseases. Odorant receptors (ORs) expressed in the olfactory sensory neurons are known to be responsible for the interaction of mosquito vectors with human odors. To gain deeper insights into Ae. aegypti&#8217;s olfactory physiology and investigate their interactions with humans at the molecular level, we used an optimized protocol of Xenopus Oocytes heterologous expression to functionally analyze Ae. aegypti odorant receptors in response to human odors. Three example experiments are presented: 1) Cloning and synthesizing cRNAs of ORs and odorant receptor co-receptor&#160;(Orco) from four to six days old Ae. aegypti antennae; 2) Microinjection and expression of ORs and Orco in Xenopus oocytes; and 3) Whole-cell current recording from Xenopus oocytes expressing mosquito ORs/Orco with a two-electrode voltage-clamp. These optimized procedures provide a new way for researchers to investigate human odor reception in Aedes mosquitoes and reveal the underlying mechanisms governing their host-seeking activity at a molecular level.

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

A protocol is presented that functionally characterizes mosquito ORs in response to human odors using a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a powerful new technique for exploring the responses of mosquitoes ORs to exposure to human odors.

The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Uma Experimental e Bioinformática Protocolo de Análises de RNA-seq de fotoperiódicas diapausa no Tigre Asiático Mosquito,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

Sistema de cultura de órgão de gordura corporal em Aedes Aegypti, um vetor de vírus Zika

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

Um protocolo de extração de DNA de mosquito baseado em contas magnéticas para sequenciamento de próxima geração

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

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.

Fisiológicas Recordings e RNA Sequenciação dos gustativas Appendages do Mosquito-febre amarela

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Único Recordings Sensillum na Insetos Drosophila melanogaster E Anopheles gambiae

Dissecção das glândulas salivares e intestino médio de Ae. aegypti mosquitos

Um Protocolo de Coleta e coloração Hemócitos do mosquito da febre amarela Aedes aegypti

Edição do genoma no mosquito da febre amarela Aedes aegypti usando CRISPR-Cas9

Alimentação e quantificação de sangue derivados de animais e refeições artificiais em mosquitos Aedes aegypti

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

Uma Experimental e Bioinformática Protocolo de Análises de RNA-seq de fotoperiódicas diapausa no Tigre Asiático Mosquito,

Sistema de cultura de órgão de gordura corporal em Aedes Aegypti, um vetor de vírus Zika

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