Name: fat body organ culture system in aedes aegypti, a vector of zika virus
Uploaded: 2017-08-19T13:00:00.000+00:00
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Esta investigación fue apoyada por subvenciones de NIH #SC1AI109055, el 2014 NMSU HHMI grant #52008103 y PGR NSF grant #1238731. Agradecemos a los participantes de la clase de NMSU primavera 2015 BIOL302 Molecular métodos y Lavesh Bhatia por su apoyo técnico con los experimentos de la cultura de la grasa corporal.

1. preparación de las soluciones y reactivos <ol><li>solución madre de ácido Amino <ol><li>preparar 4 X aminoácidos solución 23 , 24 por peso y agregar los 20 aminoácidos diferentes a un matraz de Erlenmeyer según las concentraciones dadas en la tabla 1. 
		Nota: En algunos casos, amino acid(s) puede ser excluido de la solución y sustituido por cantidades molares iguales de manitol para mantener una concentración igual de osmolitos.</li>
		<li>Añadir la cantidad apropiada de ddH 2 O para producir el volumen deseado de solución.</li>
		<li>Para asegurarse de que los aminoácidos han disuelto completamente, calentar suavemente agitando continuamente hasta que el líquido quede totalmente claro, tendrá un leve tono amarillo. 
		Nota: Puede ser necesario reducir el pH a 6 mediante la adición de ácido clorhídrico para permitir que algunos de los aminoácidos más hidrofóbicos para disolver.</li>
		<li>Alicuotar la solución y filtro estéril usando un filtro de jeringa con un 0.2 μm tamaño de poro.</li>
		<li>Almacén en un envase sellado a-20 ° C hasta por 6 meses.</li>
	</ol></li> <li>APS Nota: ver 25.
	<ol><li>Para el 20 X sal solución, combinar las sales enumeradas en la tabla 2 en 50 mL de agua. Mezclar hasta que se haya disuelto por completo. 
		Nota: Puede ser necesario calentar ligeramente la solución para disolver completamente las sales.</li>
		<li>Filtro estéril usando un filtro de jeringa con un 0.2 μm tamaño de poro y guarde la solución en un tubo de 50 mL a -20 ° C.</li>
	</ol></li> <li>Solución calcio <ol><li>para 50 X solución calcio, añadir a 100 mL de agua (tabla 3) 0,90 g de cloruro de calcio, mezclar hasta que se disuelva completamente.</li>
		<li>Utilizando una jeringa estéril-filtro filtro con un tamaño de poro de 0.2 μm, alícuota de la solución en tubos de 50 mL y almacenar a -20 ° C.</li>
	</ol></li> <li>Buffer Tris (pH 7.4) <ol><li>para el Tris almacenador intermediario, combinar las soluciones enumeradas en la tabla 4 y sal y añadir ddH 2 O hasta 100 mL.</li>
		<li>Utilizando una jeringa estéril-filtro filtro con un tamaño de poro de 0,2 μm y una parte alícuota de la solución en tubos de 50 mL, almacenar a temperatura ambiente.</li>
	</ol></li> <li>Grasa corporal medio de cultivo <ol><li>para preparar el medio de cultivo de la grasa corporal, preparar todas las soluciones mencionadas.</li>
		<li>Combinar según los volúmenes en la tabla 5 para hacer 200 mL de medio de cultivo de la grasa corporal.</li>
		<li>Ajustar el pH a 7.2 con NaOH o HCl.</li>
		<li>Filtro estéril la solución utilizando un filtro de jeringa con un tamaño de poro de 0.2 μm.</li>
		<li>Dividir la solución en alícuotas de 15 mL y almacenar a-20 ° C hasta por 6 meses.</li>
	</ol></li></ol> 2. Preparación para la disección <ol><li>preparar un estereomicroscopio (aumento de x 10-20) con la iluminación y colocar dos pinzas muy finas y un par de tijeras de disección micro.</li>
	<li>Preparar todas las soluciones necesarias para el experimento. Descongelar el medio de cultivo de la grasa corporal a temperatura ambiente. 
	Nota: Los precipitados pueden disolver calentando suavemente la solución a no superior a 30 ° C.</li>
	<li>Con un aspirador, recoger los mosquitos hembras adultos (3-7 días después de la emergencia) y anestesiarlos usando dióxido de carbono o hielo. 
	Nota: Una vez anestesiados, los mosquitos deben guardarse bajo CO 2 para el menor tiempo posibles, no superior a 20 minutos o bien, los mosquitos pueden ser anestesiados en hielo y durante aproximadamente 30 minutos</li>
	<li>Prepare una placa de la pozo de 6 con 3 mL de APS por bien.</li>
</ol> 3. Disección de la grasa corporal <ol><li>enfocar la disección Estereomicroscopio y ajustar la silla a una altura cómoda.</li>
	<li>Colocar un portaobjetos cóncavo en la superficie de microscopio y añadir dos gotas de APS para el centro.</li>
	<li>Recoger un mosquito por una pierna usando un fórceps y transferirla a la superficie de la APS en el portaobjetos.</li>
	<li>Cuidadosamente y agarre el mosquito por el tórax, con las pinzas en la mano izquierda y gire el mosquito para que el lado ventral hacia arriba.</li>
	<li>Sosteniendo el cuerpo constante por el tórax, sujete los dos últimos segmentos abdominales, con las pinzas en la mano derecha y tire suavemente de. 
	Nota: Los dos últimos segmentos abdominales va a separar del abdomen y los ovarios adjuntos, Malpighi túbulos, hindgut y midgut; a veces el cultivo se deslizará fuera del abdomen. Si no han sido removidos, inserte las puntas cerradas de las pinzas en el pequeño orificio creado y remover el tejido restante.</li>
	<li>Apartar la pinza derecha y coger las tijeras de la primavera. Deslice una hoja en el orificio en el abdomen hasta el segmento a continuación del tórax. Suavemente y rápidamente cortar el abdomen longitudinalmente. 
	Nota: Una vez realizado el corte, el abdomen debe comenzar a expandirse hacia el exterior, aunque esto puede no ocurrir inmediatamente.</li>
	<li>Proceder a realizar el segundo corte, que será un corte lateral por debajo del tórax y ligeramente por debajo de donde terminó el primer corte. 
	Nota: El abdomen debe abrir y disociar el tórax, con la cutícula hacia arriba y los órganos de grasa lado inmerso dentro de la APS. Este cuerpo de la pared abdominal es la preparación de la grasa corporal en vitro cultura.</li>
	<li>Levante los cuerpos grasos de la solución APS con la punta de un par de pinzas y transferirlas desde la solución a la placa de 6 pozos con APS a temperatura ambiente. Permitir que el tejido reposar aproximadamente 0,5 h antes de avanzar a la cultura de la grasa corporal.</li>
</ol> 4. Grasa corporal cultura <ol><li>incubar los cuerpos grasos en APS a temperatura ambiente durante por lo menos 0.5 h a que se equilibren los.</li>
	<li>Transferir los órganos de grasa utilizando la punta de las pinzas y con cuidado saque la solución APS. Bajar la punta en el medio de cultivo de grasa corporal. 
	Nota: La grasa corporal debe abrir en la superficie del medio y flotar libremente. La cultura de la grasa corporal se realiza generalmente en placas de 96 pocillos, con 150-200 μL de medio en cada pozo. Uno bien puede caber hasta tres cuerpos individuales de la grasa, y son viables por más de 12 h (no publicados resultados personales).</li>
	<li>Después de la incubación, transferir los órganos de grasa del medio en tubos de centrífuga de 1,5 mL que contienen los reactivos adecuados para el procesamiento de abajo. 
	Nota: Los análisis típicos incluyen qPCR 16 , 17 , 18 , 19, Western Blot 18 , 20, 26 de la transcriptómica, proteómica 21 o metabolómica 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>

No tenemos nada que revelar.

Cultivo insectos se utiliza ampliamente para el estudio de Endocrinología de insectos, el desarrollo y metabolismo, así como a investigar la interacción entre órganos específicos y simbiontes bacterianos29,30,31, 32,33,34. Cultivo in vitro grasa corporal se utilizó específicamente para estudiar el transporte de aminoácidos y la regulación de la producción de proteínas de yema de huevo en los mosquitos y otros dípteros16,17,35 , 36. durante el proceso de vitelogénesis, el mosquito grasa corporal utiliza una gran variedad de transportadores de aminoácidos de alta especificidad para importar los aminoácidos derivados de la harina de sangre de la hemolinfa para sintetizar grandes cantidades de proteínas de yema de huevo12 ,19,35,36. Grasa corporal cultura contribuyó a la determinación de los requerimientos nutricionales de la grasa corporal en este contexto18.
La calidad del material de partida, los mosquitos hembra, es fundamental para el éxito de estos experimentos. Larvas de mosquitos criados en condiciones de hacinamiento bajo y alimentados con dietas altas en nutrientes generalmente producen los mejores resultados. Existen algunas variables importantes a tener en cuenta al establecer las condiciones de cultivo de mosquitos grasa corporal en el laboratorio en cuanto al diseño experimental. Hemos mostrado en estudios anteriores que la expresión de genes de la grasa corporal varía significativamente dependiendo de la historia de la vida individual y el estado nutricional de los mosquitos11,22. Las condiciones de cultivo del mosquito deben ser uniformes y para reducir la variabilidad en el tamaño y las reservas nutricionales de los mosquitos experimentales. Además, personal que realiza las disecciones debería ser entrenado para disecciones rápidas y exactas con resultados consistentes. Viabilidad celular en cuerpos aislados de grasa puede ser comprobado utilizando de37,de métodos de tinción diferentes38.
El diseño experimental de un experimento de cultivo de grasa corporal debe tener en cuenta el número de disecciones posible en un determinado período de tiempo. Cuando grandes cantidades de cuerpos grasos son necesarias, pueden ser necesarios varias sesiones de disección o disectores múltiples. Hay una amplia gama de aplicaciones futuras en vitro cultura de grasa corporal de los mosquitos y otros insectos. Será especialmente útil para probar posibles candidatos de medicamentos para control de insectos. El uso de técnicas transgénicas en insectos para expresar proteínas específicas reportero en trophocytes grasa corporal abrirá nuevos métodos para el desarrollo de pruebas biológicas de gran alcance para el estudio de la fisiología de la grasa corporal.

Los mosquitos son vectores de enfermedades humanas devastadoras, incluyendo la malaria, dengue, Fiebre chikungunya y Zika1,2,3. A pesar de intensos esfuerzos internacionales para frenar estas enfermedades y para controlar las poblaciones de mosquitos transmisores de enfermedades, brotes epidémicos de enfermedades transmitidas por el mosquito son aún campo común, especialmente en los países en desarrollo. Vacunas efectivas contra muchas de estas enfermedades son de carácter o de eficacia limitada4,5. La forma más efectiva para prevenir brotes es controlar las poblaciones de mosquitos, principalmente mediante el uso de tratamientos insecticidas. Sin embargo, la resistencia a los insecticidas ha desarrollado en muchas poblaciones de mosquitos y convertido en un problema común en el mundo6,7,8. El estudio de la fisiología del mosquito es esencial para el desarrollo de nuevas herramientas y estrategias para el control de la enfermedad.
El mosquito grasa corporal desempeña un papel central en el almacenamiento de nutrientes, la homeostasis metabólica, reproducción y xenobióticos catabolismo9,10,11,12. Es el órgano de almacenamiento principal para triglicéridos, glucógeno y aminoácidos en forma de proteínas de almacenamiento de información. También funciona como lugar de síntesis para la mayoría de las proteínas de la hemolinfa y metabolitos. En los mosquitos, la grasa corporal es la única fuente de producción de proteínas de yema de huevo que se produce en las mujeres después de que una comida de sangre13,14.
El tipo de célula principal de la grasa corporal es la trophocyte grande, poliploide o adipocito3,9,10,12. Tejido de la grasa corporal se organiza en lóbulos o vainas y puede encontrarse en todas partes del cuerpo del mosquito, con la porción más grande situada en el abdomen, donde grandes lóbulos de grasa corporal se adhieren a la pared abdominal del cuerpo.
El sistema de cultivo de mosquitos grasa corporal presentado aquí fue desarrollado en los años 70 y sigue siendo una poderosa herramienta para el estudio de grasa corporal fisiología10, especialmente en combinación con las tecnologías actuales de análisis. El fundamento de esta técnica se basa en el aislamiento de las paredes abdominales del cuerpo y el tejido de grasa corporal asociada. La naturaleza hidrofóbica de la cutícula abdominal hace que flote en la superficie del medio de cultivo, con los lóbulos conectados de abdominal grasa corporal sumergido. Los espiráculos y la estructura tracheolar se mantienen, asegurando la oxigenación de los tejidos cultivados. En adelante, nos referiremos a estas preparaciones como "cuerpos grasos". Cuerpos aislados de grasa permanecen viables durante más de 12 horas cuando se incuban en medio apropiado (resultados no publicados). Cultura de la grasa corporal es una herramienta valiosa que ha abordado una variedad de preguntas sobre grasa corporal Endocrinología y fisiología9,10,12,15,16, 17.
Cuerpos grasos cultivados puede someterse a varios experimentales y control de tratamientos, el momento en que puede ser decidido por el investigador. Al final de la incubación, los órganos de grasa puede recabados y tratados para los análisis posteriores, incluyendo qPCR16,17,18,19, Western Blot18 ,20,21de la proteómica o metabolómica22. Experimentos pueden realizarse en diferentes escalas, desde cuerpos de grasa individuales a grupos de cientos que pueden ser cultivadas juntas.
Los resultados representativos incluidos aquí fueron derivados de cuerpos grasos, cultivados en presencia de los aminoácidos y la ecdisona 20-hidroxi de la hormona esteroide para simular la activación de la harina de sangre de vitelogénesis16,17, 23,24. Había analizado y había comparado la expresión génica diferencial de no activado versus activados cuerpos grasos mediante análisis de secuenciación de próxima generación.

<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

El insecto grasa corporal desempeña un papel central en el metabolismo de insectos y almacenamiento de nutrientes, reflejo de las funciones del hígado y del tejido graso en los vertebrados. Insectos cuerpo grasa tejido generalmente se distribuye en todo el cuerpo del insecto. Sin embargo, a menudo es concentrada en el abdomen y atado a la pared abdominal del cuerpo.
El mosquito grasa corporal es la única fuente de proteínas de la yema de huevo, que son críticos para la producción de huevo. Por lo tanto, el cultivo en vitro de los tejidos de grasa corporal de mosquito representa un sistema importante para el estudio de la fisiología del mosquito, metabolismo y, en última instancia, la producción de huevos. El proceso de cultura de la grasa corporal comienza con la preparación de soluciones y reactivos, incluyendo soluciones del aminoácido, Aedes suero fisiológico solución stock (APS), solución madre de calcio y grasa corporal medio de cultivo. El proceso continúa con la disección de la grasa corporal, seguida de un tratamiento experimental. Después del tratamiento, puede realizarse una variedad de diferentes análisis, incluyendo RNA (RNA-Seq) de la secuencia, qPCR, Western Blot, proteómica y metabolómica.
En nuestro experimento ejemplo, demostramos el protocolo a través de la supresión y la cultura de cuerpos grasos del mosquito de la fiebre amarilla, Aedes aegypti, principal vector de arbovirus incluyendo Zika, dengue y chikungunya. ARN de cuerpos grasos cultivadas bajo una condición fisiológica conocida a proteínas de yema de huevo alza versus el control fueron sometidos a análisis de RNA-Seq para demostrar la potencial utilidad de este procedimiento para las investigaciones de la expresión génica.

Como ejemplo, realizamos un experimento de cultivo de la grasa corporal y estimulados cuerpos aislados de grasa por la incubación en una solución que contiene una mezcla equilibrada de los veinte aminoácidos que ocurren naturalmente y la ecdisona 20-hidroxi de insectos hormona esteroide (10 μm) durante 6 h . Como control, cuerpos grasos se incubaron en APS para la misma cantidad de tiempo.
Después de la incubación, el RNA total fue aislado usando un tri-reactivo27 siguiendo las instrucciones del fabricante. La calidad y cantidad de muestras de RNA extraídas se evaluaron utilizando un espectrofotómetro, cuantificación fluorométrica y electroforesis en gel de agarosa. Las bibliotecas de la secuencia de RNA se generaron usando 4 μg de ARN total y se cuantificaron usando dos técnicas diferentes. Posteriormente, las bibliotecas fueron enviadas a un proveedor comercial de secuenciación del extremo apareado.
Los resultados de este experimento se muestran en la tabla 6. Genes que muestran la más fuerte respuesta transcripcional a aminoácidos y 20-hydroxyecdysone fueron principalmente yema proteína los genes, que está de acuerdo con anteriores resultados11.
La figura 1 muestra un mapa que indica los niveles de expresión de gene de 1.256 diferencialmente expresados genes de cuerpos grasos cultivados después de dos tratamientos diferentes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55508/55508fig1.jpg"> 
Figura 1 . Mapa de genes expresados en la cultura de la grasa corporal. El mapa de calor se calculó con base en el número de las transcripciones específicas para cada gen en las diferentes bibliotecas con el mapa de calor paquete28 que es parte del entorno de software de R. La sombra más oscura representa la mayor expresión de genes. 1.256 genes con variación estadísticamente significativa en la expresión (valores de Q &#60; 0.05) se muestran. Los genes se ordenan según su nivel de expresión promedio, indicada por el dendrograma de la izquierda (no según sus relaciones filogenéticas). Tenga en cuenta el elevado número de genes con expresión regulada hacia arriba o abajo después del estímulo con aminoácidos (AA) y 20-hydroxyecdysone (20E). APS = Aedes fisiológico. Ver archivo suplementario 1 para una lista de genes y su expresión relativa. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55508/55508fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Del aminoácido</td>
			<td>Peso molecular g/mol</td>
			<td>m concentración</td>
			<td>mg por litro</td>
			<td>mg de volumen 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>Asparragina</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 glutámico</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>Triptófano</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>
Tabla 1. 4 solución madre de ácido Amino.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Componente</td>
			<td>Peso en gramos a 50 ml ddH2O</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>8.0 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>g 0,074</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>
Tabla 2. 20 X sal solución.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Componente</td>
			<td>Peso en gramos agregados a 100 ml ddH2O</td>
		</tr>
		<tr>
			<td>CaCl2-2 H2O</td>
			<td>g 0,90</td>
		</tr>
	</tbody>
</table>
<stron></stron>g &gt; tabla 3. 50 x solución calcio.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Componente</td>
			<td>Concentración de la solución madre</td>
			<td>Valores de volumen de 100 ml de tampón</td>
		</tr>
		<tr>
			<td>PH8.0 Tris</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>NA</td>
			<td>0.3 g</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>NA</td>
			<td>a 100 mL (~ 93 mL)</td>
		</tr>
	</tbody>
</table>
Cuadro 4. Tampón Tris.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Componente</td>
			<td>Valores de volumen de 200 ml</td>
		</tr>
		<tr>
			<td>Solución madre de ácido amino</td>
			<td>150 mL</td>
		</tr>
		<tr>
			<td>Solución de sal común</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Calcio solución</td>
			<td>4 mL</td>
		</tr>
		<tr>
			<td>TES Buffer</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>26 mL</td>
		</tr>
	</tbody>
</table>
Tabla 5. Medio de cultivo de la grasa corporal.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anotación</td>
			<td>Descripción gene</td>
			<td>Doble cambio</td>
			<td>Valor de P</td>
		</tr>
		<tr>
			<td>AAEL006138</td>
			<td>Vitelogenina-B</td>
			<td>3443</td>
			<td>2.52E-112</td>
		</tr>
		<tr>
			<td>AAEL006126</td>
			<td>Vitelogenina-C</td>
			<td>2795</td>
			<td>8.64E-91</td>
		</tr>
		<tr>
			<td>AAEL006563</td>
			<td>Carboxipeptidasa vitelogénicos</td>
			<td>1002</td>
			<td>2.17E-119</td>
		</tr>
		<tr>
			<td>AAEL010434</td>
			<td>Vitelogenina-A</td>
			<td>220</td>
			<td>1.14E-27</td>
		</tr>
		<tr>
			<td>AAEL006542</td>
			<td>Carboxipeptidasa vitelogénicos</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>Vitelógenas 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>
Tabla 6. Experimentales resultados.
Archivo suplementario 1. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55508/Table_of_genes_for_figure_1.xlsx" target="_blank">Haga clic aquí para descargar este archivo.</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

La grasa corporal es el órgano metabólico central en insectos. Presentamos un sistema de cultivo de órgano vivo que permite al usuario estudiar las respuestas del tejido corporal de grasa aislados a varios estímulos.

Sistema de cultivo de órganos de grasa corporal en Aedes Aegypti, Vector del Virus 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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Nanyang Technological University

Research

JoVE Journal

Biology

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

9.4K vistas.  New Mexico State University. La grasa corporal es el órgano metabólico central en insectos. Presentamos un sistema de cultivo de órgano vivo que permite al usuario estudiar las respuestas del tejido corporal de grasa aislados a varios estímulos.

Video: 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 de alfavirus Transducción: Herramientas para la Visualización de la infección en los mosquitos vectores

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

Investigación

Inmunología e Infección

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 las infecciones de dengue en mosquitos (Aedes aegypti) y determinación de fenotipo infección

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 ...

Biología

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 infecciosa cultivo celular y la<em&gt; In Vitro</em&gt; Profiláctica Efecto de los interferones

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.

Análisis de competencia vectorial en mosquitos Aedes aegypti con el virus Zika

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.

Propagación del parásito microsporidiano Edhazardia aedis en 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.

Alimentación y cuantificación de sangre de origen animal y comidas artificiales en 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.

Infección viral experimental en mosquitos adultos por alimentación oral y microinyección

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.

Mantener los mosquitos Aedes aegypti infectados con 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.

Forzado de la salivación como un método para analizar la competencia de vectores 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.

Ensayo mejorado de fecundidad y fertilidad para Aedes aegypti utilizando 24 placas de cultivo de tejidos de pozos (placas EAgaL)

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