Los autores desean agradecer a los miembros anteriores de la Bolsa de laboratorio: Ming-Min Lee, Kathryn Plichta, Victoria Miranda-Thompson y Sam-Kyu Kim por su contribución a la mejora de muchos de estos protocolos. Este trabajo fue financiado en parte por la subvención de la Fundación Nacional para la Ciencia IOS-0840932 y 0724978 para IOS-SP Stock

1. En vivo La cría de nematodos entomopatógenos con sus bacterias Symboitic <ol><li> Invertir un 100 x 15 mm placa Petri de plástico y colocar dos discos de papel de filtro (90 mm) en la tapa del plato. </li><li> Distribuir uniformemente 1 ml de la IJ (juveniles infectivos) suspensión de agua (a una concentración de 1,000-2,000 IJ / ml) sobre el papel de filtro. NOTA: JIs no necesitan ser esterilizados de la superficie. </li><li> Añadir 10 larvas de último instar de la mayor Galleria mellonella waxmoth al plato. El objetivo es proporcionar aproximadamente 100-200 JIs / larva. </li><li> Cubra la tapa con la parte inferior de la placa de Petri (Figura 1) y la etiqueta de la placa de Petri. Mencione la siguiente información: nombre de la especie de nematodos (si se conoce); aislar código / designación; infección fecha trampa estaba tendida. </li><li> Coloque el plato en una bolsa de plástico sin apretar sellada (para conservar la humedad) y mantenerlo en la oscuridad a temperatura ambiente o en una incubadora entre 20-25 ° C. Compruebe platos diariamente NOTA: La oscuridad facilita la infección por nematodos, ya que imita las condiciones naturales de infección en el suelo. </li><li> Después de 3-5 días, retire cadáveres con signos de infección por nematodos a una trampa Blanco modificada 7. NOTA: Si los cadáveres olor pútrido, esto puede ser una indicación de que el cultivo no tuvo éxito y / o no había contaminación. Configurar una nueva cámara de la infección con un nuevo lote de nematodos e insectos. </li></ol> 2. In vitro Crianza de nematodos entomopatógenos con sus bacterias simbióticas <ol><li> Hígado y riñón Agar Método 9,19 <ol><li> Picar el hígado y el riñón en trozos pequeños (2 cm 3 o más pequeñas) y colocar en una licuadora con NaCl, agar y 300 ml de agua y mezclar hasta que la carne se convierte en una pasta líquida, pasta espesa o puré. A continuación, transferir la mezcla a un matraz Erlenmeyer de 1 l. </li><li> Añadir los últimos 200 ml de agua al recipiente de la licuadora y decantar cualquier medio restante en laErlenmeyer. Autoclave durante 15 minutos a 121 ° C. Después de la esterilización en autoclave permite agar enfriar a tocar antes de verter. NOTA: El pipeteo de medio no es recomendable ya que este medio tiende a tener trozos de carne. </li><li> Vierta ~ 20 ml de agar a 5 (o 6) cm placas de Petri mientras se agita la mano o girando el matraz Erlenmeyer vierte entre los medios de comunicación para una más homogénea. Permitir agar para solidificar y almacenar platos a 4 ° C hasta que se necesite NOTA: Use una espátula de metal llama-esterilizados para romper los trozos grandes de carne vierte en la placa de Petri. </li></ol></li><li> Lipid Agar Método 9,19 <ol><li> Preparar medio de agar de lípidos mediante la mezcla de caldo nutriente, extracto de agar y la levadura y verter la mezcla en un matraz Erlenmeyer de 2 L. Agregar destilada H 2 O y MgCl2 • 6H 2 O y autoclave durante 15 minutos a 121 ° C. </li><li> Agregue la mezcla estéril de aceite y jarabe de maíz de mezcla de maíz y revuelva o agite vigorosamente y con frecuencia para dispersar las gotitas de aceite de manera uniforme. <br/&gt; NOTA: Esterilizar el aceite de maíz y jarabe colocando el volumen necesario en un agente de reticulación UV. El aceite no se disuelve en el líquido, pero asegúrese de que está en diminutas gotas. </li><li> Vierta ~ 20 ml de agar en un 5 (o 6) cm placas de Petri y dejar solidificar el agar y almacenar platos a 4 ° C hasta que se necesite. </li><li> La racha de bacterias simbióticas que desee en la placa de agar de lípidos e incubar las placas a 28 ° C durante 24-48 horas. NOTA: Extienda 100-200 l de la noche (12-16 horas) LB subcultura. </li><li> El día después, agregar aproximadamente 0,4 ml de superficie esterilizada suspensión de nematodos (huevos o juveniles infectivos) e incubar las placas a temperatura ambiente en la oscuridad o en una incubadora a 22 ± 3 ° C. NOTA: No agregue más de 0,4 ml de suspensión de nematodos, ya que puede crear un ambiente excesivamente húmedo que es desfavorable para el crecimiento de los nematodos. </li><li> Monitorear culturas diaria. Los cultivos deben producir una nueva generación de JIs en unos 12 días (Figura 2).</li><li> Transferencia plato inferior con agar cuando los nematodos se ven arrastrándose los lados del plato (Figura 3A) a una trampa Blanco modificado (ver Orozco et al., 12) para cosechar JIs (Figura 3B). Realice este paso en una campana de flujo laminar para reducir la contaminación de los medios de comunicación. </li><li> Recoger JIs de la trampa Blanco modificada en surgimiento y enjuague IJ suspensión para eliminar los residuos medio. Tienda IJ en agua destilada esterilizada a 10 ° C (en un incubador de temperatura fría o habitación fría) o utilizar de inmediato si es necesario. NOTA: En función del objetivo del estudio, sembrar las placas, ya sea con nematodos simbiontes-colonizado o aposymbiotic. </li></ol></li></ol> 3. In vitro Crianza de aposymbiotic (-Symbiont gratis) entomopatógenos nematodos <ol><li> Infectar 10-20 G. larvas mellonella con JIs de las especies de nematodos deseados (véase la sección 1). Después de 3 o 4 días (en este momento JIs debería haber maduradoa los adultos de la primera generación) recoger cadáveres. NOTA: El tiempo de la madurez y el número de hembras necesarios para obtener un número suficiente de huevos varía según la especie. Infectar a más G. mellonella larvas si es necesario y comenzar disecciones ya en el post-infección de 48 horas para evaluar si se encuentran en la etapa de desarrollo adecuado. </li><li> Coloque cada cadáver individualmente en una placa de Petri con solución salina (tampón M9 18). Diseccionar un cadáver tirando su cabeza con unas pinzas de punta fina. </li><li> Recoge al menos 20 grávido (con huevos dentro de) mujeres con una aguja fina (en forma de L de preferencia) y colocarlos en un vidrio de reloj que contiene 10 ml de tampón M9 (Figura 4A). </li><li> Preparar una solución axenizing considerando los siguientes ingredientes: 6,75 ml de agua destilada, 1,25 ml de NaOH 5 N, y 2,25 ml de solución de lejía disponible comercialmente diluyó a 3% de hipoclorito. NOTA: NaOH es una base de solución-guantes, gafas de protección y otra asistencia adecuada protecciónse debe usar ropa. NOTA: Preparar solución axenizing nuevo cada vez, como la lejía se degrada en la luz. Prepare varios lotes de solución axenizing y realizar la esterilización de la superficie y la recolección de huevos en varios vidrios de reloj. </li><li> Enjuague hembras al menos tres veces con tampón M9 llenado vidrio de reloj con solución tampón y absorbiendo tampón con una pipeta. Asegúrese de que las hembras permanecen en el vidrio de reloj. Repita este paso dos veces más. </li><li> Inmediatamente después, añadir la solución axenizing y permitir que las mujeres permanezcan en esta solución durante no más de 10-15 minutos. Observar los tejidos de nematodos que comienzan a desintegrarse mientras que los huevos (que son resistentes a la solución axenizing) permanecen intactos. </li><li> Interrumpir manualmente cuerpos femeninos para acelerar el proceso, cortándolos con una aguja o un bisturí (Figura 4B, C). Retire los huevos con la pipeta ellos en 1,5 ml tubos de microcentrífuga, evitando la adición de cualquier tejido sin disolver. NOTA: Uso como mcualquier tubos de microcentrífuga como sea necesario para recoger los huevos y trabajan rápidamente, ya que la viabilidad de los huevos disminuirá durante un período prolongado de exposición a la solución axenizing. </li><li> Tubos Vortex durante 15 segundos utilizando el &quot;ajuste de alta velocidad&quot; para eliminar aún más los tejidos de nematodos unidos a los huevos. Alternativamente, si un vórtice no está disponible, pipetear arriba y abajo las soluciones varias veces. </li><li> Huevos de pellets por centrifugación a aproximadamente 18.000 g durante 2 min. Aumentar la velocidad si los huevos no hacen lo suficiente pellet. Retire la solución axenizing y enjuague dos veces llenando el tubo de microcentrífuga con agua destilada estéril y se centrifuga una vez más a 900 xg durante 1 min. </li><li> Después del último lavado, resuspender huevos en 300-500 l de agua destilada estéril y colocarlos en un recipiente estéril 3 cm placa de Petri o vidrio de reloj esterilizado. Recuerde que los huevos son ahora esterilizados en superficie, a fin de utilizar pipetas estériles y / o consejos pippetter y agua para mantener la limpieza de los huevos. </li><li>Examine los huevos bajo un microscopio de disección (ampliación 30-50X) para confirmar que están intactos (Figura 4D) y transferirlos a una placa 5 cm con agar hígado-riñón (ver sección 2.1) NOTA: No agregue más de 400 l de la suspensión de huevos en las placas ya que hará que el agar excesivamente húmedo. </li><li> Las placas de identificación con la información apropiada y almacenarlos en posición vertical en la oscuridad a 28 ° C. La eclosión de los huevos debería ser evidente durante la noche. NOTA: El agua puede condensarse en la tapa del plato, pero se evaporará después de 1 ó 2 días. En este momento, colocar la cápsula en la bolsa de plástico para retener la humedad del agar y evitar su secado. </li><li> Observe platos periódicamente y deseche cualquier platos que muestran signos de hongos o la contaminación bacteriana. Una vez JIs se ven migrar hasta la pared de la cápsula de Petri, quitar la tapa y transferir el plato inferior con agar a una trampa Blanco modificada 12. Considere condiciones estériles al transferir el plato en el modified trampa blanca para evitar la contaminación de los medios expuestos. </li><li> Continuar el seguimiento de las trampas blancas y cosecha IJ según sea necesario. NOTA: los nematodos aposymbiotic seguirán migrando hacia el agua de la trampa de Blanco durante muchos días e incluso semanas. </li></ol>

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El uso de un huésped adecuado es un factor clave para el éxito en la crianza vivo de EPN. Por lo general, ambos steinernematids y heterorhabditids pueden reproducirse y completar con éxito su ciclo de vida en las larvas de la polilla mayor de la cera, Galleria mellonella (Lepidoptera: Pyralidae). Sin embargo, otras especies de insectos de diferentes familias y / o pedidos pueden ser considerados. Algunas de las especies de nematodos que se describen en la actualidad se sabe que tienen espec...

Nematodos entomopatógenos (EPN) Steinernema y Heterorhabditis spp. (Steinernematidae, Heterorhabditidae) y sus bacterias simbiontes, Xenorhabdus y Photorhabdus spp (Enterobacteriaceae) se consideran un modelo emergente de los Animales Terrestres microbica relaciones simbióticas 2-4,6,10,19. Xenorhabdus y Photorhabdus spp. se albergaron como simbiontes en el intestino de la única etapa de vida libre de los nematodos, también conocido como el infectivo juvenil (IJ) o etapa 3 rd infectivos juveniles 8,10,13. La pareja bacteria-nematodo es patógeno para una amplia gama de insectos y con éxito se ha implementado en el control biológico y los programas de manejo integrado de plagas en todo el mundo 6,8. Aquí mostramos una selección de in vivo e in vitro técnicas que se ejercen con frecuencia para la cría de EPN en condiciones de laboratorio. In métodos in vivo contemplan un insecto huésped para la cría de los nematodos. Por lo general, los estados inmaduros de diferentes órdenes de insectos (es decir, Lepidoptera, Coleoptera, Diptera, etc.) Se consideran hospedadores adecuados. In vivo métodos suelen ser considerados para el mantenimiento de cultivos de nematodos en el laboratorio. Este método puede no ser adecuado cuando se considera la producción en masa de los nematodos. Grandes cantidades de insectos hospedadores pueden ser necesarios para tal fin exigir más tiempo y costes adicionales relacionados con la cría de insectos. Nematodos entomopatógenos también pueden ser cultivadas in vitro en varios medios de comunicación. Dependiendo del objetivo del estudio; métodos in vitro o puede considerar la incorporación de las bacterias simbióticas en los medios de comunicación. En esta presentación, se describen dos métodos comúnmente usados ​​para la propagación de EPN. Los ingredientes de los medios de comunicación proporcionan una fuente de nutrientes para la bacteria simbiótica unmétodos in vitro encontrar una fuente de esteroles de los nematodos. ofrecen la ventaja de la cría de EPN sin un insecto huésped. Originalmente, muchos de los medios de comunicación in vitro desarrollado fueron utilizados para la multiplicación de EPN cuando los ejércitos de insectos adecuados no están disponibles. Sin embargo, en los últimos años, en los métodos de cría in vitro han sido ampliamente empleado en la investigación con el objetivo de entender la relación mutualista entre EPN y sus bacterias simbióticas 17,19. Las técnicas que se detallan en esta presentación corresponden a los descritos por diversos autores y refinado por la Bolsa de Laboratorio de la Universidad de Arizona (Tucson, AZ, EE.UU.). Estas técnicas son distinta del cuerpo de las técnicas que se utilizan en la producción masiva de estos organismos con fines de gestión de plagas.

<table><tbody><tr><td colspan="4">For &lt;em&gt;In vivo&lt;/em&gt; Infections</td></tr><tr><td>100 x 15 Petri dishes</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>Filter paper, 9 cm grade 1 cellulose</td><td>Whatman/VWR</td><td>28450-081</td><td>Grade 1 filter paper recommended</td></tr><tr><td>Insect hosts</td><td>Timberline</td><td>http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td><td>&lt;em&gt;Galleria mellonella&lt;/em&gt; are recommended</td></tr><tr><td colspan="4">For Liver-kidney Agar (for 500 ml)</td></tr><tr><td>60 x 15 mm Petri dishes</td><td>VWR</td><td>25384-092</td><td></td></tr><tr><td>Beef liver, 50 g</td><td>Locally sourced; butcher or supermarket</td><td></td><td>Remaining may be stored frozen and thawed for future use</td></tr><tr><td>Beef kidney, 50 g</td><td>Locally sourced; butcher or supermarket</td><td></td><td>Remaining may be stored frozen and thawed for future use</td></tr><tr><td>Sodium chloride 2.5 g (0.5% final concentration)</td><td>Acros/VWR</td><td>200002-434</td><td>any research grade NaCl can be used</td></tr><tr><td>Agar, 7.5 g (1.5% agar, final concentration)</td><td>HiMedia/VWR</td><td>95026-642</td><td>any media grade agar can be used</td></tr><tr><td>500 ml distilled H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td colspan="4">For Lipid Agar (for 1 L)</td></tr><tr><td>100 x 15 Petri dishes</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>Nutrient broth, 8 g</td><td>BD/VWR</td><td>90002-660</td><td></td></tr><tr><td>Yeast extract, 5 g</td><td>EMD/VWR</td><td>EM1.03753.0500</td><td></td></tr><tr><td>Magnesium chloride hexahydrate 10 ml (0.2 g/ml)</td><td>EMD/VWR</td><td>EM-MX0045-1</td><td></td></tr><tr><td>Corn oil, 4 ml</td><td>Any brand</td><td>Locally source; supermarket</td><td>Any brand of corn oil can be used</td></tr><tr><td>Corn syrup, 96 ml combine 7 ml corn syrup in 89 ml heated H&lt;sub&gt;2&lt;/sub&gt;O and swirl for homogeneity</td><td>Karo</td><td>Locally sourced; supermarket</td><td></td></tr><tr><td>Agar, 15 g</td><td>HiMedia/VWR</td><td>95026-642</td><td>any media grade agar can be used</td></tr><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;O, 890 ml</td><td></td><td></td><td></td></tr></tbody></table>

in vivo and in vitro rearing of entomopathogenic nematodes (steinernematidae and heterorhabditidae)

Nematodos entomopatógenos (NEP) (Steinernematidae y Heterorhabditidae) tienen una asociación mutualista con Gram-negativas Gamma-Proteobacteria en la familia Enterobacteriaceae bacterias. Xenorhabdus se asocian con steinernematids nematodos mientras Photorhabdus son simbiontes de heterorhabditids. Junto nematodos y bacterias forman un complejo insecticida potente que mata a una amplia gama de especies de insectos en una asociación íntima y específica. En la presente memoria, se demuestra in vivo e in vitro técnicas comúnmente usadas en la cría de estos nematodos en condiciones de laboratorio. Además, estas técnicas representan pasos clave para el éxito del establecimiento de cultivos de EPN y también forman la base para otros bioensayos que utilizan estos organismos para la investigación. La producción de nematodos (-simbionte libre) aposymbiotic suele ser crítico para un análisis en profundidad y enfoque multifacético para el estudio de la simbiosis. Esteprotocolo no requiere la adición de antibióticos y se puede lograr en un corto período de tiempo con el equipo de laboratorio estándar. Los nematodos producidos de esta manera son relativamente robusta, aunque su supervivencia en el almacenamiento puede variar dependiendo de las especies utilizadas. Las técnicas que se detallan en esta presentación corresponden a los descritos por diversos autores y refinado por el Laboratorio de la Universidad de Arizona (Tucson, AZ, EE.UU.) de P. Stock. Estas técnicas son distinta del cuerpo de las técnicas que se utilizan en la producción masiva de estos organismos con fines de gestión de plagas.

El método de cría in vivo utiliza insectos vivos como anfitriones para el crecimiento y la reproducción de nematodos. Cámaras de infección son un método eficaz para la exposición de insectos JIs. Esta es la única etapa del ciclo de vida de los nematodos de que los vectores de las bacterias simbiontes de un insecto huésped a otro. Figura 1 muestra la puesta a punto para una cámara de infección, así como los materiales necesarios para construir esta cámara. In vitro métodos...

Watch this Scientific Journal Video about In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae at JoVE.com

In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

El objetivo de esta presentación es demostrar en vivo y en técnicas in vitro para la crianza de los nematodos entomopatógenos. Métodos in vivo consideran la crianza de estos nematodos con un insecto huésped, mientras que los métodos in vitro utilizan rich media agar.

In vivo e in vitro Crianza de nematodos entomopatógenos (Steinernematidae y Heterorhabditidae)

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

in-vivo-vitro-rearing-entomopathogenic-nematodes-steinernematidae

Research

JoVE Journal

Bioengineering

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

20.4K vistas.  University of Arizona.  El objetivo de esta presentación es demostrar en vivo y en técnicas in vitro para la crianza de los nematodos entomopatógenos. Métodos in vivo consideran la crianza de estos nematodos con un insecto huésped, mientras que los métodos in vitro utilizan rich media agar. 

Video: In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Muestreo de suelos y aislamiento de nematodos entomopatógenos (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

Investigación

Medio ambiente

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

El nematodo Caenorhabditis Elegans - un modelo versátil en Vivo para el estudio de las interacciones huésped-microbio

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genética

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Alto y Bajo Rendimiento pantallas con nematodos- Meloidogyne spp.</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

Inmunología e Infección

Rapid Isolation of Wild Nematodes by Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

This protocol outlines a method for efficiently extracting live nematodes from natural substrates in the field.

Aislamiento rápido de nematodos silvestres por Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

Visualización de las bacterias en los nematodos mediante microscopía de fluorescencia

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Biología

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

Limpieza selectiva de nematodos silvestres de Caenorhabditis para enriquecer las bacterias del microbioma intestinal

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

Cultivo y manipulación genética de nematodos entomopatógenos

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Aislamiento y selección de hongos entomopatógenos a partir de muestras de suelo y evaluación de la virulencia fúngica contra plagas de insectos

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

Aislamiento y caracterización de la microbiota natural del nematodo modelo Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

<meta charset="utf8"></head><body>Optimización de la infección por nematodos del pino en Pinus pinaster

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily contributed to pine deforestation in Asian countries, e.g., Japan, China, and Korea. Over the last two decades, in Europe, Portugal and Spain have been greatly affected. Research on the mechanisms of PWN infection and/or PWD progression in susceptible host species relies on the controlled infection of pine seedlings under greenhouse conditions. This technique is laborious and mobilizes substantial economic and human resources. Additionally, it can be prone to variability that results from the genetic diversity associated with some pine species but also from the interference of external factors. As an alternative, in vitro co-cultures of pine with PWNs offer a more advantageous system for studying biochemical changes since they a) allow controlling single environmental or nutritional variables, b) occupy less space, c) require less time to obtain, and d) are&#160;free from contamination or from host genetic variation. The following protocol details the standard in vivo PWN infection of Pinus pinaster, the maritime pine, and the establishment of the novel in vitro co-cultures of pine shoots with the PWN as an improved methodology to study this phytoparasite influence on pine volatiles. PWN-induced volatiles are extracted from in vivo and in vitro infected pines by hydrodistillation and distillation-extraction, and the emitted volatiles are captured by solid phase microextraction (SPME), using fiber or packed column techniques.

<meta charset="utf8"></head><body>Autor destacado: Sistema de cocultivo in vitro de brotes de pino y nematodo de la madera de pino para estudiar la respuesta volátil del huésped

The protocol describes the in vivo and in vitro pinewood nematode infection of Pinus pinaster and their volatilome analysis through Gas Chromatography (GC) and GC coupled to Mass Spectrometry (GC-MS).

In vivo e in vitro Crianza de nematodos entomopatógenos (Steinernematidae y Heterorhabditidae)

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