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>

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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 border="0" cellpadding="0" cellspacing="0" width="1272">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Name of Material/ Equipment</td>
			<td style="width:367px;">Company</td>
			<td style="width:189px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glass capillary</td>
			<td style="width:367px;">A-M Systems</td>
			<td style="width:189px;">628000</td>
			<td>Standard, 1.5mm X 0.86mm, 4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Silver wire</td>
			<td style="width:367px;">A-M Systems</td>
			<td style="width:189px;">7875</td>
			<td>.015&#34; bare</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tungsten wire</td>
			<td style="width:367px;">Alfa Products</td>
			<td style="width:189px;">369</td>
			<td>0.127mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Fine forceps</td>
			<td style="width:367px;">Fine Science Tools</td>
			<td style="width:189px;">11252</td>
			<td>#5SF Inox</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Refridgerated stage</td>
			<td style="width:367px;">BioQuip Products</td>
			<td style="width:189px;">1429</td>
			<td>Chill Table</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Preamplifier</td>
			<td style="width:367px;">Syntech</td>
			<td style="width:189px;">Taste Probe</td>
			<td style="width:467px;">preamplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Software for electrophysiology</td>
			<td style="width:367px;">Syntech</td>
			<td style="width:189px;">Autospike</td>
			<td style="width:467px;">software for electrophysiology</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">TetraMin fish food</td>
			<td style="width:367px;">Tetra</td>
			<td style="width:189px;">Tropical fish food granules</td>
			<td>fish food ground to fine powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy Plus Mini Kit</td>
			<td>Qiagen</td>
			<td style="width:189px;">74136</td>
			<td style="width:467px;">includes gDNA eliminator and RNeasy spin columns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop spectrophotometer</td>
			<td>Nano Drop Products</td>
			<td style="width:189px;">ND-1000</td>
			<td>tabletop spectrometer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:251px;">R statistics</td>
			<td style="width:367px;">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 height="21">
			<td height="21" style="height:21px;width:251px;">1.5ml tube rack</td>
			<td style="width:367px;">Evergreen</td>
			<td style="width:189px;">240-6388-030</td>
			<td>Pour liquid nitrogen into a few empty wells to freeze and grind tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">1.5mL collection tubes with pestle</td>
			<td style="width:367px;">Grainger</td>
			<td style="width:189px;">6HAX6</td>
			<td>RNase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Centrifuge</td>
			<td style="width:367px;">Thermo Scientific</td>
			<td style="width:189px;">11178160</td>
			<td>Spin down frozen tissue to keep at bottom of 1.5 mL tube.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primer-BLAST Primer Designing tool</td>
			<td style="width:367px;">NCBI</td>
			<td style="width:189px;">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

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

Construção de uma melhor Mosquito: identificar os genes habilitação Malária e Dengue Febre Resistência em A. gambiae e A. aegypti mosquitos

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

Biologia

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 ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

Esgotamento de RNA ribossômico para metagenomico Gut Mosquito RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

O sequenciamento de próxima geração de 16S RNA ribossomal Gene amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

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, ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Identificação dos fatores chave que regula a auto-renovação e diferenciação em células precursoras hematopoéticas EML por Análise RNA-seqüenciamento

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

Targeted RNA Sequenciação Ensaio para Caracterizar expressão gênica e genômica Alterações

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

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 ...

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, ...

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness parameters, in particular egg production and hatch rates at the individual level, conventional methods have put a substantial burden on investigators due to high labor intensity and laboratory space requirements. Described is a simple method using 24 well tissue culture plate with agarose in each well and digital imaging of each well to determine egg numbers and hatch rates at an individual level with substantially reduced time and space requirements.

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates

Described is a time and space-saving method to count eggs and determine hatch rates of individual mosquitoes using 24 well tissue culture plates, which can substantially increase the scale and speed of fecundity and fertility assays.

Ensaio melhorado de fecundidade e fertilidade para a Aedes aegypti usando 24 placas de cultura de tecido poço (placas EAgaL)

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...