Name: fat body organ culture system in aedes aegypti, a vector of zika virus
Uploaded: 2017-08-19T13:00:00.000+00:00
Duration: 6 min 18 s
Description: Watch this Scientific Journal Video about Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus at JoVE.com

Esta pesquisa foi apoiada pela concessão de NIH #SC1AI109055, o 2014 NMSU HHMI grant #52008103 e NSF PGR concedem #1238731. Agradecemos aos participantes da classe NMSU Primavera 2015 BIOL302 Molecular métodos e Lavesh Bhatia seu apoio técnico com os experimentos de cultura de gordura corporal.

1. preparar as soluções e reagentes <ol><li>solução de aminoácidos <ol><li>Prepare um 4 X aminoácidos solução 23 , 24 por pesagem e adicionando os 20 aminoácidos diferentes para um Erlenmeyer de acordo com as concentrações dadas na tabela 1. 
		Nota: Em alguns casos, amino acid(s) podem ser excluídos a solução e substituído por igual quantidade molar de manitol para manter uma concentração igual de osmólitos.</li>
		<li>Adicionar a quantidade apropriada de DDQ 2 O para produzir o volume desejado da solução.</li>
		<li>Para garantir que os aminoácidos têm completamente dissolvido, aquecer suavemente agitando continuamente até que o líquido é completamente desobstruído; tomará em um ligeiro matiz amarelo. 
		Nota: Pode ser necessário reduzir o pH para 6 adicionando HCl para permitir que alguns dos aminoácidos hidrofóbicos mais dissolver-se.</li>
		<li>Alíquota da solução e estéril-filtro usando um filtro de seringa com um 0,2 µm-pore tamanho.</li>
		<li>Loja em um recipiente selado a-20 ° C por até 6 meses.</li>
	</ol></li> <li>APS Nota: ver 25.
	<ol><li>Para o 20 X sal solução, combine os sais listados na tabela 2, em 50 mL de água. Mexa até dissolver completamente. 
		Nota: Pode ser necessário aquecer ligeiramente a solução até dissolver completamente os sais.</li>
		<li>Estéril-filtro usando um filtro de seringa com um 0,2 µm-reduz o tamanho dos poros e armazenar a solução em um tubo de 50 mL em -20 ° C.</li>
	</ol></li> <li>Solução estoque de cálcio <ol><li>para o 50 X solução estoque de cálcio, adicionar 100 mL de água (tabela 3) 0,90 g de cloreto de cálcio; mexa até dissolver completamente.</li>
		<li>Usando uma seringa estéril-filtro filtro com um tamanho de poro de 0,2 µm, alíquota da solução em tubos de 50 mL e armazenar a -20 ° C.</li>
	</ol></li> <li>Tampão Tris (pH 7,4) <ol><li>para os Tris buffer, combinar as soluções listadas na tabela 4 e o sal e adicionar O ddH 2 até 100 mL.</li>
		<li>Usando uma seringa estéril-filtro filtro com um tamanho de poro de 0,2 µm e alíquota da solução em tubos de 50 mL; armazenar à temperatura ambiente.</li>
	</ol></li> <li>Meio de cultura de gordura corporal <ol><li>para preparar o meio de cultura de gordura corporal, preparar todas as soluções listadas acima.</li>
		<li>Combiná-los conforme os volumes na tabela 5 para fazer 200 mL de meio de cultura de gordura corporal.</li>
		<li>Ajustar o pH para 7,2 usando NaOH ou HCl.</li>
		<li>Estéril-filtrar a solução através de um filtro de seringa com um tamanho de poro de 0,2 µm.</li>
		<li>Dividir a solução em alíquotas de 15 mL e armazenar a-20 ° C por até 6 meses.</li>
	</ol></li></ol> 2. Preparando-se para dissecação <ol><li>preparar um estereomicroscópio (ampliação de x de 10-20), com iluminação e esquematizar duas pinças ultra fina e um par de tesouras de dissecação micro.</li>
	<li>Preparar todas as soluções necessárias para o experimento. Descongelar o meio de cultura de gordura corporal a temperatura. 
	Nota: Precipitants podem ser dissolvidos ao aquecer a solução para não superior a 30 ° C.</li>
	<li>Com um aspirador, coletar mosquitos fêmeas adultos (3-7 dias após a emergência) e anestesia usando dióxido de carbono ou gelo 
	Nota: Uma vez anestesiado, os mosquitos devem ser mantidos sob CO 2 para o menor tempo possíveis, não superior a 20 min. como alternativa, os mosquitos podem ser anestesiados em gelo e mantidos por aproximadamente 30 min.</li>
	<li>Preparar uma placa de 6 com 3 mL de APS por alvéolo.</li>
</ol> 3. Dissecação de gordura corporal <ol><li>concentrar o dissecação estereomicroscópio e ajustar a cadeira para uma altura confortável.</li>
	<li>Colocar uma lâmina de microscópio côncava na superfície de microscópio e adicionar duas gotas de APS ao centro.</li>
	<li>Pegar um mosquito por uma perna, usando uma pinça e transferi-lo para a superfície APS do slide de microscópio.</li>
	<li>Cuidadosamente pega o mosquito pelo tórax, com a pinça na mão esquerda e gire o mosquito, de modo que o lado ventral para cima é.</li>
	<li>, Mantendo o corpo firme pelo tórax, segure os dois últimos segmentos abdominais, com a pinça na mão direita e puxe delicadamente. 
	Nota: Os dois últimos segmentos abdominais irão separar o abdômen e os ovários anexados, Malpighi túbulos, intestino grosso e intestino; às vezes a colheita irá deslizar fora do abdômen. Se eles não tiverem sido removidos, inserir as pontas fechadas da pinça no pequeno orifício criado e remover os tecidos remanescentes.</li>
	<li>Anular a pinça direita e pegar a tesoura de mola. Deslize um pino no orifício no abdômen até o segmento abaixo do tórax. Suavemente e rapidamente cortar o abdômen longitudinalmente. 
	Nota: Uma vez que o corte foi feito, o abdômen deve começar a expandir para o exterior, embora isto pode não ocorrer imediatamente.</li>
	<li>Prosseguir para fazer o segundo corte, que será um corte lateral abaixo do tórax e ligeiramente abaixo de onde terminou o primeiro corte. 
	Nota: O abdome deve então abrir e dissociar-se do tórax, com a cutícula acima e os corpos de gordura lateral-imerso dentro da APS. Esta parede abdominal do corpo é a preparação de gordura corporal para cultura em vitro.</li>
	<li>Levantar os corpos de gordura a partir da solução APS usando a ponta de um par de pinças e transferi-los a partir da solução para a placa de 6 contendo APS à temperatura ambiente. Permitir que o tecido repousar antes de avançar para a cultura de gordura corporal com aproximadamente 0,5 h.</li>
</ol> 4. Gordura corporal cultura <ol><li>incubar os corpos de gordura na APS em temperatura ambiente pelo menos 0,5 h equilibrar os.</li>
	<li>Transferir os corpos de gordura com a ponta da pinça e retire-lhes fora a solução APS. Abaixe a ponta no meio de cultura de gordura corporal. 
	Nota: A gordura corporal deve abrir na superfície do meio e flutuar livremente. A cultura de gordura corporal é normalmente realizada em placas de 96 poços, com 150-200 µ l de meio em cada poço. Um bem pode caber até três corpos individuais de gordura, e são viáveis por mais de 12 h (resultados pessoais inéditos).</li>
	<li>Após o período de incubação, transferir os corpos de gordura do meio em tubos de centrífuga de 1,5 mL contendo os reagentes apropriados para processamento a jusante. 
	Nota: As análises típicas incluem qPCR 16 , 17 , 18 , 19, 18 , de mancha ocidental 20, transcriptomics 26, proteomics 21 ou metabolomics 22.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+to+determine+cell+viability+by+simultaneous+staining+with+fluorescein+diacetate-propidium+iodide.">An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide.</a> J Histochem Cytochem. 33 (1), 77-79 (1985).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Curr Protoc Immunol. A3B, (2001).</li></ol>

Nós não temos nada para divulgar.

Cultura de inseto órgão foi amplamente utilizada para estudar o inseto Endocrinologia e metabolismo, bem como para investigar a interação entre órgãos específicos e bactérias simbiontes29,30,31, desenvolvimento 32,33,34. In vitro cultura órgão de gordura corporal foi utilizada especificamente para estudar o transporte de aminoácidos e a regulação da produção de proteína de gema em mosquitos e outros Diptera16,17,35 , 36. durante o processo de vitelogênese, o mosquito gordura corporal usa uma matriz de transportadores de aminoácidos de alta especificidade para importar aminoácidos derivados de farinha de sangue da hemolinfa para sintetizar grandes quantidades de proteínas de gema12 ,19,35,36. Cultura de gordura corporal foi fundamental para a delimitação dos requisitos nutricionais gordura corporal neste contexto18.
A qualidade das matérias-primas, mosquitos fêmeas, é crítica para o sucesso dessas experiências. Larvas de mosquito, erguido em condições sob-lotado e alimentados com dietas de alta-nutriente geralmente produzem os melhores resultados. Existem algumas variáveis importantes a serem considerados ao estabelecer as condições de cultura de gordura corporal de mosquito no laboratório em termos de design experimental. Mostramos em estudos anteriores que a expressão do gene de gordura corporal varia significativamente dependendo da história de vida individual e o estado nutricional do mosquito11,22. As condições de cultura mosquito devem ser uniformes para reduzir a variabilidade no tamanho e reservas nutricionais dos mosquitos experimentais. Além disso, pessoal, realizando as dissecações deve ser treinado para garantir a dissecações rápidas e exatas com resultados consistentes. Viabilidade celular em corpos isolados de gordura pode ser verificada utilizando diferentes métodos coloração37,38.
O delineamento experimental de um experimento de cultura de gordura corporal deve levar em consideração o número de dissecações possível em um determinado período de tempo. Quando grandes quantidades de gordura corpos são necessárias, várias sessões de dissecação ou dissectors múltiplo podem ser necessários. Há uma vasta gama de aplicações futuras em vitro cultura de gordura corporal em mosquitos e outros insetos. Será especialmente útil para testar potenciais candidatos a fármacos para controle de insetos. A utilização das técnicas transgênicas em insetos para expressar proteínas específicas repórter na gordura corporal trophocytes abrirá novos métodos para desenvolver os bioensaios poderosos para o estudo da fisiologia de gordura corporal.

Os mosquitos são vetores de doenças humanas devastadoras, incluindo a febre do dengue, malária, chikungunya e Zika1,2,3. Apesar de intensos esforços internacionais para travar estas doenças e controlar populações de mosquitos transmissores de doenças, surtos epidêmicos de doenças transmitidas por mosquitos são ainda comuns, especialmente nos países em desenvolvimento. Vacinas eficazes contra muitas dessas doenças são indisponíveis ou de eficácia limitada,4,5. A maneira mais eficaz para prevenir surtos é controlar as populações de mosquito, principalmente através do uso de tratamentos insecticidas. No entanto, resistência inseticida tem desenvolvido em muitas populações de mosquito e tornar-se um problema comum em torno do mundo6,7,8. O estudo da fisiologia do mosquito é essencial para o desenvolvimento de novas ferramentas e estratégias para controlar a doença.
O mosquito gordura corporal desempenha um papel central no armazenamento de nutrientes, homeostase metabólica, reprodução e catabolismo xenobióticos9,10,11,12. É o órgão principal de armazenamento de glicogênio, triglicerídeos e aminoácidos em forma de proteínas de armazenamento. Ele também funciona como o local de síntese para a maioria das proteínas hemolinfa e metabólitos. Em mosquitos, a gordura corporal é a única fonte de produção de proteína de gema que ocorre em mulheres, depois que eles tomam uma refeição de sangue13,14.
O tipo de célula principal da gordura corporal é o trophocyte grande, poliploide ou adipócito3,9,10,12. Tecido de gordura corporal é organizado em lóbulos ou casacos de pele e pode ser encontrado em todas as partes do corpo do mosquito, com a maior parte localizada no abdômen, onde grandes lóbulos de gordura corporal são anexados à parede abdominal do corpo.
O sistema de cultura de gordura corporal de mosquito aqui apresentado foi desenvolvido nos anos 70 e continua a ser uma poderosa ferramenta para estudar a fisiologia gordura corporal10, especialmente em combinação com as tecnologias atuais de análise. A fundação desta técnica baseia-se o isolamento das paredes abdominais corpo e o tecido associado gordura corporal. A natureza hidrofóbica da cutícula abdominal faz com que ele flutue na superfície do meio de cultura, com os lóbulos anexados de abdominal gordura corporal imergido. A estrutura tracheolar e espiráculos são mantidas, assegurando a oxigenação do tecido culta. De agora em diante, nos referiremos a estas preparações como "corpos de gordura". Isolada de gordura de corpos permanecem viáveis por mais de 12 h, quando incubados no meio adequado (resultados não publicados). Cultura de gordura corporal é uma ferramenta valiosa que abordou uma variedade de perguntas sobre a gordura corporal Endocrinologia e fisiologia9,10,12,15,16, 17.
Corpos de gordura culturas pode ser submetidos a vários experimental e controlar tratamentos, o sincronismo dos quais pode ser decidido pelo investigador. No final do período de incubação, os corpos de gordura podem ser coletados e processados para análises a jusante, incluindo qPCR16,17,18,19, mancha18 ocidental ,20, proteomics21ou metabolomics22. Experiências podem ser executadas em diferentes escalas, de corpos de gordura individuais para grupos de centenas que podem ser cultivados juntos.
Os resultados representativos aqui incluídos foram derivados de gordura organismos cultivados na presença de aminoácidos e o isoinokosterone 20-hidroxi hormônio esteroide para simular a ativação de refeição de sangue de vitelogênese16,17, 23,24. Analisamos e comparado a expressão diferencial do gene de não-ativado contra corpos de gordura ativados através de análise de sequenciamento de próxima geração.

<table><tbody><tr><td>Scissors&amp;nbsp;</td><td>Fiskars</td><td>83872</td><td></td></tr><tr><td>Fly pad</td><td>Genesee Scientific</td><td>789060</td><td></td></tr><tr><td>Battery-powered aspirator w/ collection vial</td><td>Hausherrs Machine Works, Inc.</td><td>3740-01-210-2368</td><td></td></tr><tr><td>Fine tip forceps</td><td>World Precision Instruments, Inc.&amp;nbsp;</td><td>500085</td><td></td></tr><tr><td>Light microscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>96 well plate&amp;nbsp;</td><td>Sigma</td><td>CL S3383</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>Alanine</td><td>Sigma</td><td>A7627</td><td></td></tr><tr><td>Arginine</td><td>Sigma</td><td>A5006</td><td></td></tr><tr><td>Asparagine</td><td>Sigma</td><td>A0884</td><td></td></tr><tr><td>Aspartic Acid</td><td>Sigma</td><td>A9256</td><td></td></tr><tr><td>Cysteine</td><td>Sigma</td><td>W326305</td><td></td></tr><tr><td>Glutamic Acid</td><td>Sigma</td><td>G1251</td><td></td></tr><tr><td>Glutamine</td><td>Sigma</td><td>G3126</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G2879</td><td></td></tr><tr><td>Histidine</td><td>Sigma</td><td>H6034</td><td></td></tr><tr><td>Isoleucine</td><td>Sigma</td><td>I2752</td><td></td></tr><tr><td>Lysine</td><td>Sigma</td><td>L5501</td><td></td></tr><tr><td>Leucine</td><td>Sigma</td><td>L8000</td><td></td></tr><tr><td>Phenylalanine</td><td>Sigma</td><td>P2126</td><td></td></tr><tr><td>Proline</td><td>Sigma</td><td>P0380</td><td></td></tr><tr><td>Serine</td><td>Sigma</td><td>S4500</td><td></td></tr><tr><td>Threonine</td><td>Sigma</td><td>T8625</td><td></td></tr><tr><td>Tryptophan</td><td>Sigma</td><td>T0254</td><td></td></tr><tr><td>Tyrosine</td><td>Sigma</td><td>T3754</td><td></td></tr><tr><td>Valine</td><td>Sigma</td><td>V0500</td><td></td></tr><tr><td>Methionine</td><td>Sigma</td><td>M9625</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;-6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>M2670</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>C8106</td><td></td></tr><tr><td>Tris pH8.0&amp;nbsp;</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>W4502</td><td></td></tr></tbody></table>

fat body organ culture system in aedes aegypti, a vector of zika virus

O inseto gordura corporal desempenha um papel central no metabolismo do inseto e armazenamento de nutrientes, funções do fígado e tecido adiposo em vertebrados de espelhamento. Tecido de inseto gordura corporal normalmente é distribuído por todo o corpo do inseto. No entanto, muitas vezes concentrada no abdômen e está ligado à parede abdominal do corpo.
O mosquito gordura corporal é a única fonte de proteínas da gema, que são críticas para a produção de ovos. Portanto, a cultura em vitro de tecidos de gordura corporal de mosquito representa um sistema importante para o estudo da fisiologia do mosquito, metabolismo e, finalmente, a produção de ovos. O processo de cultura de gordura corporal começa com a preparação de soluções e reagentes, incluindo as soluções de aminoácidos, Aedes sal estoque fisiológico (APS), solução estoque de cálcio e gordura corporal meio de cultura. O processo continua com a dissecação de gordura corporal, seguida por um tratamento experimental. Após o tratamento, uma variedade de diferentes análises podem ser executados, incluindo RNA (RNA-Seq) de sequenciamento, qPCR, borrões ocidentais, proteómica e metabolómica.
Na nossa experiência de exemplo, demonstramos o protocolo através da excisão e cultura dos corpos de gordura com o febre amarela mosquito, Aedes aegypti, um vetor principal de arbovírus incluindo dengue e chikungunya Zika. RNA de gordura corpos cultivadas sob uma condição fisiológica conhecida por proteínas de gema upregulate versus o controle foram objecto de análise de RNA-Seq para demonstrar o potencial utilitário deste procedimento para investigações de expressão gênica.

Como exemplo, realizamos um experimento de cultura de gordura corporal e estimulou a corpos de gordura isolados incubando-os em uma solução contendo uma mistura equilibrada de todos os vinte aminoácidos naturais e a isoinokosterone 20-hidroxi inseto hormônio esteroide (10 µM) para 6 h . Como um controle, corpos de gordura foram incubados na APS por uma quantidade igual de tempo.
Após a incubação, o RNA total foi isolado usando um reagente tri27 seguindo as instruções do fabricante. A qualidade e a quantidade de amostras de RNA extraídas foram avaliados usando um espectrofotômetro, fluorométrica quantificação e eletroforese em gel de agarose. Bibliotecas de sequenciamento de RNA foram geradas usando 4 µ g de RNA total e foram quantificadas usando duas técnicas diferentes. Posteriormente, as bibliotecas foram enviadas para um provedor comercial para final-sequenciamento emparelhado.
Os resultados deste experimento são mostrados na tabela 6. Genes, mostrando a mais forte resposta transcricional de aminoácidos e 20-hidroxiecdisona foram principalmente gema proteína genes, que está de acordo com anterior resultados11.
A Figura 1 mostra um mapa de calor, indicando os níveis de expressão de gene de 1.256 diferencialmente expressos genes de corpos de gordura cultivados após dois tratamentos diferentes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55508/55508fig1.jpg"> 
Figura 1 . Mapa de calor dos genes expressado na cultura de gordura corporal. O mapa de calor foi calculado com base no número de transcrições específicos para cada gene nas diferentes bibliotecas usando o pacote heatmap28 que faz parte do ambiente de software a R. O tom mais escuro representa a maior expressão do gene. 1.256 genes com variação estatisticamente significativa na expressão (Q-valores &#60; 0.05) são mostrados. Os genes estão ordenados de acordo com seus níveis de expressão média, indicados pela dendrograma à esquerda (não de acordo com suas relações filogenéticas). Observe o elevado número de genes com expressão de cima ou para baixo-regulado após estimulação com aminoácidos (AA) e 20-hidroxiecdisona (20E). APS = soro fisiológico de Aedes . Ver arquivo suplementar 1 para obter uma lista de genes e seus níveis de expressão relativa. <a href="//ecsource.jove.com/files/ftp_upload/55508/55508fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amino ácido</td>
			<td>Peso molecular g/mol</td>
			<td>mM concentração</td>
			<td>mg por litro</td>
			<td>mg para o volume de 300 mL</td>
		</tr>
		<tr>
			<td>Alanina</td>
			<td>89.1</td>
			<td>26.68</td>
			<td>2377.19</td>
			<td>713.16</td>
		</tr>
		<tr>
			<td>Arginina</td>
			<td>174.2</td>
			<td>26.68</td>
			<td>4647.66</td>
			<td>1394.3</td>
		</tr>
		<tr>
			<td>Asparagina</td>
			<td>150.1</td>
			<td>26.68</td>
			<td>4004.67</td>
			<td>1201.4</td>
		</tr>
		<tr>
			<td>Ácido aspártico</td>
			<td>133</td>
			<td>26.68</td>
			<td>3548.44</td>
			<td>1064.53</td>
		</tr>
		<tr>
			<td>Cisteína</td>
			<td>121.16</td>
			<td>10,68</td>
			<td>1293.99</td>
			<td>388.2</td>
		</tr>
		<tr>
			<td>Ácido glutâmico</td>
			<td>147.1</td>
			<td>26.68</td>
			<td>3924.63</td>
			<td>1177.39</td>
		</tr>
		<tr>
			<td>Glutamina</td>
			<td>146</td>
			<td>26.68</td>
			<td>3895.28</td>
			<td>1168.58</td>
		</tr>
		<tr>
			<td>Glicina</td>
			<td>75</td>
			<td>53.32</td>
			<td>3999</td>
			<td>1199.7</td>
		</tr>
		<tr>
			<td>Histidina</td>
			<td>155,16</td>
			<td>80</td>
			<td>12412.8</td>
			<td>3723.84</td>
		</tr>
		<tr>
			<td>Isoleucina</td>
			<td>131</td>
			<td>10,68</td>
			<td>1399.08</td>
			<td>419.72</td>
		</tr>
		<tr>
			<td>Lycine</td>
			<td>183</td>
			<td>26.68</td>
			<td>4882.44</td>
			<td>1464.73</td>
		</tr>
		<tr>
			<td>Leucina</td>
			<td>131</td>
			<td>26.68</td>
			<td>3495.08</td>
			<td>1048.52</td>
		</tr>
		<tr>
			<td>Fenilalanina</td>
			<td>165</td>
			<td>10,68</td>
			<td>1762.2</td>
			<td>528.66</td>
		</tr>
		<tr>
			<td>Prolina</td>
			<td>115</td>
			<td>26.68</td>
			<td>3068.2</td>
			<td>920.46</td>
		</tr>
		<tr>
			<td>Serina</td>
			<td>105</td>
			<td>53.32</td>
			<td>5598.6</td>
			<td>1679.58</td>
		</tr>
		<tr>
			<td>Treonina</td>
			<td>119</td>
			<td>10,68</td>
			<td>1270.92</td>
			<td>381.28</td>
		</tr>
		<tr>
			<td>Triptofano</td>
			<td>204</td>
			<td>10,68</td>
			<td>2178.72</td>
			<td>653.62</td>
		</tr>
		<tr>
			<td>Tirosina</td>
			<td>181</td>
			<td>5.32</td>
			<td>962.92</td>
			<td>288.88</td>
		</tr>
		<tr>
			<td>Valina</td>
			<td>117</td>
			<td>10,68</td>
			<td>1249.56</td>
			<td>374.87</td>
		</tr>
		<tr>
			<td>Metionina</td>
			<td>149</td>
			<td>10,68</td>
			<td>1591.32</td>
			<td>477.4</td>
		</tr>
	</tbody>
</table>
Tabela 1. 4 x solução estoque de ácido aminado.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Componente de</td>
			<td>Peso em gramas adicionadas ao 50ml ddH2O</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>8,0 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>0,074 g</td>
		</tr>
		<tr>
			<td>MgCl2-6 H2O</td>
			<td>0,120 g</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>0,0250 g</td>
		</tr>
	</tbody>
</table>
Tabela 2. Solução de sal de 20 X.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Componente de</td>
			<td>Peso em gramas adicionadas ao ddH2O 100ml</td>
		</tr>
		<tr>
			<td>CaCl2-2 H2O</td>
			<td>0,90 g</td>
		</tr>
	</tbody>
</table>
<stron></stron>g > tabela 3. 50 x solução cálcio.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Componente de</td>
			<td>Concentração da solução-mãe</td>
			<td>Estoque de volume para 100 ml de tampão</td>
		</tr>
		<tr>
			<td>Tris pH 8.0</td>
			<td>1 M</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>0,25 M</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>AT</td>
			<td>0,3 g</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>AT</td>
			<td>a 100 mL (~ 93 mL)</td>
		</tr>
	</tbody>
</table>
Tabela 4. Tampão Tris.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Componente de</td>
			<td>Estoque de volume para 200 ml</td>
		</tr>
		<tr>
			<td>Solução de aminoácidos</td>
			<td>150 mL</td>
		</tr>
		<tr>
			<td>Solução-mãe de sal</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Solução de reserva de cálcio</td>
			<td>4 mL</td>
		</tr>
		<tr>
			<td>TES de Buffer</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>26 mL</td>
		</tr>
	</tbody>
</table>
Tabela 5. Meio de cultura de gordura corporal.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anotação</td>
			<td>Descrição do gene</td>
			<td>Dobre a mudança</td>
			<td>P-valor</td>
		</tr>
		<tr>
			<td>AAEL006138</td>
			<td>Vitellogenin-B</td>
			<td>3443</td>
			<td>2.52E-112</td>
		</tr>
		<tr>
			<td>AAEL006126</td>
			<td>Vitellogenin-C</td>
			<td>2795</td>
			<td>8.64E-91</td>
		</tr>
		<tr>
			<td>AAEL006563</td>
			<td>carboxipeptidase vitelogênicos</td>
			<td>1002</td>
			<td>2.17E-119</td>
		</tr>
		<tr>
			<td>AAEL010434</td>
			<td>Vitellogenin-A</td>
			<td>220</td>
			<td>1.14E-27</td>
		</tr>
		<tr>
			<td>AAEL006542</td>
			<td>carboxipeptidase vitelogênicos</td>
			<td>185</td>
			<td>2.14E-65</td>
		</tr>
		<tr>
			<td>AAEL012678</td>
			<td>AAEL003006-PA [Aedes aegypti](65%)</td>
			<td>96</td>
			<td>4.00E-70</td>
		</tr>
		<tr>
			<td>AAEL000080</td>
			<td>proteína hipotética</td>
			<td>82</td>
			<td>6.69E-188</td>
		</tr>
		<tr>
			<td>AAEL015312</td>
			<td>Vitelogênicos catepsina B</td>
			<td>77</td>
			<td>1.27E-15</td>
		</tr>
		<tr>
			<td>AAEL009588</td>
			<td>receptor nuclear 3</td>
			<td>75</td>
			<td>4.58E-56</td>
		</tr>
		<tr>
			<td>AAEL010529</td>
			<td>proteína hipotética</td>
			<td>66</td>
			<td>1.32E-29</td>
		</tr>
	</tbody>
</table>
Tabela 6. Resultados experimentais.
Arquivo suplementar 1. <a href="//ecsource.jove.com/files/ftp_upload/55508/Table_of_genes_for_figure_1.xlsx" target="_blank">Clique aqui para baixar este arquivo.</a>

Watch this Scientific Journal Video about Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus at JoVE.com

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

A gordura corporal é o órgão central metabólico em insetos. Apresentamos um sistema de cultura de órgãos vivos que permite ao usuário estudar as respostas do tecido isolado gordura corporal a vários estímulos.

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 ...

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Biology

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

9.4K visualizações.  New Mexico State University. A gordura corporal é o órgão central metabólico em insetos. Apresentamos um sistema de cultura de órgãos vivos que permite ao usuário estudar as respostas do tecido isolado gordura corporal a vários estímulos.

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

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Sistema Alphavirus Transdução: ferramentas para visualização de infecção em mosquitos vetores

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

Imunologia e Infecção

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Protocolo para infecções por dengue em mosquitos (A. aegypti) e Determinação Fenótipo Infecção

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

Biologia

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Virus Zika sistema de cultura celular Infecciosas e do<em&gt; In Vitro</em&gt; Efeito profilático de interferões

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

Análises de competência vetorial sobre mosquitos Aedes aegypti que usam zika vírus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Propagação do Parasita Microsporidiano Edhazardia aedis em Mosquitos Aedes aegypti

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

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 ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Infecção viral experimental em mosquitos adultos por alimentação oral e microinjeção

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

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, ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

Forçado a salivação como um método para analisar a competência vetorial de mosquitos

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

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 ...