Esta investigación fue financiada por el Fondo de Ciencias de Austria (FMF): P23681-B16.

1. Preparación de materiales vegetales <ol><li> Utilice cualquiera de las hojas o plantas enteras de material de la asignatura planta (Hosta spp. O Phaseolus vulgaris). </li></ol> 2. Preparación de las soluciones de agua que contienen hielo nucleación activos (INA) Bacterias <ol><li> Cultura la bacteria INA, Pseudomonas syringae (Strain Cit-7) en placas de Petri a 25 ° C en Pseudomonas Agar F preparada con 10 g / L de 100% de glicerol por la dirección del fabricante. </li><li> Después de culturas han crecido lo suficiente, a 4 ° C hasta que sea necesario, pero mantener a 4 ° C durante dos días previos para asegurar un alto nivel de actividad de nucleación de hielo. </li><li> Raspar las bacterias de una sola placa de la superficie del agar con un plástico, desechable o reutilizable espátula de metal en el momento de uso y lugar en 10-15 ml de agua desionizada en una cubeta desechable de 25 ml. La concentración debe estar en el rango de 1 × 10 7 hasta 1 x 10 9 · </sup&gt; Ml -1. La solución aparecerá nublado. No hay necesidad de confirmar la concentración usando un hemocitómetro o espectrofotómetro, como concentración sólo necesita ser aproximada. </li><li> Vórtex con la cubeta durante un mínimo de 10 seg para distribuir las bacterias. Nota: La concentración específica de la mezcla de INA resultante no es importante y el protocolo descrito proporcionará más de un nivel adecuado de actividad de nucleación de hielo. Esta mezcla de bacterias INA y el agua se utilizará más adelante en los experimentos de nucleación. </li></ol> 3. Configuración de un experimento de congelación <ol><li> Coloque la cámara de infrarrojos de alta resolución (SC-660) dentro de la caja protectora de acrílico para que los proyectos lente a través de la abertura en el frente de la caja y los cables que conectan la cámara a una salida del ordenador portátil o dispositivo de grabación a través de la abertura posterior de la caja . Asegure la tapa de la caja y colocar la caja dentro de la cámara ambiental o congelador en un lugar que lo hará todoflujo del material objeto planta para ser visto. <ol><li> Proporcionar un fondo oscuro en todo el material de la planta por el forro las paredes de la cámara con papel de construcción negro para evitar la interferencia de la energía infrarroja reflejada. </li><li> Coloque la cámara con iluminación LED para minimizar el calentamiento de la fuente de luz cuando se requiere la grabación de imágenes en longitudes de onda visibles. Sólo un mínimo de iluminación, tal como una luz armario funciona con batería u otro dispositivo LED pequeña, se requiere para que las plantas sean visibles por la cámara. <ol><li> Una vez que se toman imágenes visibles de la materia objeto de la planta, apagar la iluminación LED. Distribuya todas las conexiones cableadas externos (conexión FireWire a la computadora, cable de alimentación, etc.) a la cámara a través de un puerto u otra abertura en la cámara. </li></ol></li><li> Rellene cualquier espacio extra en el puerto o abertura con material aislante de espuma para evitar o reducir los gradientes de temperatura dentro de la cámara. Ajuste la temperatura inicial de la cámara en 1 ° C. </li></ol></li><li> Alinear las plantas o partes de plantas de manera que el material de la planta se encuentra en el campo de visión de la cámara y el material vegetal es visible en la pantalla de visualización remota o dentro del software elegido. </li><li> Permitir que las plantas se equilibre a 1 ° C durante 30 min a 1 hr, dependiendo del tamaño del material de planta, antes de iniciar un experimento de congelación controlada. Esto asegura que la temperatura de la planta no se quedará atrás temperatura del aire en muchos grados una vez que se inicia el experimento de congelación. La equilibración se logra cuando la temperatura del material vegetal está dentro de 0,5 ° C de temperatura del aire. <ol><li> Coloque una capa de aislamiento de espuma de poliestireno en la parte superior del suelo de las plantas en macetas si se utilizan las plantas en maceta. Una vez que las plantas hayan equilibrado, iniciar el enfriamiento de la cámara. Nota: La capa de aislamiento sobre la superficie del suelo de la olla reduce la cantidad de pérdida de calor continuación de la olla para el aire que rodea la planta, y evita que las raíces de freezing, ya que esto no sería típicamente ocurrir durante un evento de helada en la naturaleza debido al depósito masivo de calor residual presente en el suelo. </li></ol></li><li> Establezca los parámetros de la cámara que desee (paleta de colores, rango de temperatura, áreas específicas de interés, etc.), como se comenta en 3.4.1-3.4.4. <ol><li> Seleccione la paleta del arco iris para mostrar las variaciones de temperatura mientras se visualiza la imagen en directo. </li><li> Ajuste el intervalo de temperatura de 5 ° C ajustando la barra de temperatura situado justo debajo de la imagen en el software. </li><li> Elija la escala lineal (algoritmo) para convertir los datos infrarrojos en la imagen en color falso como se define en la paleta seleccionada (arco iris) y establecer el rango de temperatura de 5 ° C y para realizar un seguimiento de forma automática basándose en la imagen. Alternativamente, ajuste el rango establecido manualmente mientras que la realización del experimento. <ol><li> Utilice la temperatura de un punto específico o una temperatura promedio dentro del área definida de interés proporcionada por el softwson. Recuperar los datos de temperatura de todos los píxeles de la secuencia de vídeo grabado o de la información incrustada en el archivo de imagen. La figura 3 muestra una captura de pantalla típico desde dentro software ResearchIR. </li></ol></li><li> Colocar un cursor en una ubicación en el tejido de la planta que representa un punto específico de interés. Definir el área de interés como puntos (1 -3 píxeles de tamaño), cajas, líneas, elipses o círculos. Múltiples combinaciones de puntos o formas se pueden situar sobre la imagen. </li></ol></li><li> Grabación de una secuencia de vídeo <ol><li> Ajuste la cámara al grabar a 60 Hz y para la grabación se detenga manualmente. </li><li> Indique la ubicación en el ordenador o disco duro externo donde se colocará el archivo de vídeo grabado. </li><li> Comenzar la grabación. Nota: Grabación en un disco duro externo es muy recomendable ya que se generarán grandes archivos de video. Archivos de vídeo grabados se pueden editar más tarde para contener sólo la parte que contiene la neinformación nece-. Esto reducirá en gran medida el tamaño del archivo. </li><li> Baje la temperatura de la cámara de forma incremental en un 0,5 -1,0 ° C. Espere hasta que la temperatura de la planta se equilibra con la temperatura del aire y luego bajar la temperatura de nuevo por 0,5 a 1,0 ° C. Dependiendo de la masa del tejido de la planta siendo observado y su morfología, de equilibrio puede tomar de 10 a 15 min. Por lo tanto, dando una tasa de enfriamiento de aproximadamente 4 ° C / hr. </li><li> Continúe de esta manera hasta que la planta se congela y observaciones se han completado. Finalice la grabación cuando el proceso de congelación se ha completado. Nota: El tejido de la planta se ha equilibrado con la temperatura del aire cuando el material vegetal y el fondo son del mismo color, ya que son a la misma temperatura. Puesto que la temperatura de fondo y la temperatura del tejido de la planta son los mismos, puede ser difícil de visualizar el material vegetal hasta que de nuevo más baja la temperatura y no hay diferencial de temperatura entre el tejido de la planta y unatemperatura IR. </li></ol></li></ol>

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Los autores no tienen intereses financieros en competencia o conflictos de intereses.

El agua tiene la capacidad de superenfriar a temperaturas muy por debajo de 0 ° C y la temperatura a la que el agua se congele puede ser bastante variable. 36 El límite de temperatura para el subenfriamiento del agua pura es de aproximadamente -40 ° C y se define como el punto de nucleación homogénea. Cuando el agua se congela a temperaturas más cálidas a -40 ° C, se produce por la presencia de nucleantes heterogéneos que permiten a los pequeños embriones de hielo para formar y que constituirán un ...

Congelación temperaturas que se producen cuando las plantas están creciendo activamente puede ser letal, particularmente si la planta tiene tolerancia a la congelación poco o no. Tales heladas suelen tener efectos devastadores en la producción agrícola y también pueden desempeñar un papel importante en la conformación de la estructura de la comunidad en las poblaciones naturales de las plantas, sobre todo en los Alpes, los ecosistemas sub-árticas y árticas 1-6. Los episodios de fuertes heladas de primavera han tenido un gran impacto en la producción de fruta en los EE.UU. y América del Sur en los últimos años 7-9 y se han agravado por la aparición temprana de clima cálido, seguido por las bajas temperaturas más típicas medias. El clima cálido temprana induce brotes de romper, activando el crecimiento de nuevos brotes, hojas y flores de todos los cuales tienen muy poca o ninguna tolerancia a las heladas 1,3,10-12. Tales patrones climáticos erráticos se han notificado a ser un reflejo directo del cambio climático en curso y se espera que sea un patrón de tiempo común para los foresfuturo eeable 13. Los esfuerzos para proporcionar técnicas de gestión económicos, eficaces y respetuosas del medio ambiente o agroquímicos que pueden proporcionar una mayor tolerancia a las heladas han tenido un éxito limitado por una serie de razones, pero esto puede ser parcialmente atribuido a la naturaleza compleja de la tolerancia a la congelación y congelación mecanismos de evasión en las plantas. 14 Los mecanismos adaptativos asociados con la supervivencia heladas en las plantas se han dividido tradicionalmente en dos categorías, tolerancia a la congelación y la congelación de evitación. La primera categoría se asocia con mecanismos bioquímicos regulados por un conjunto específico de genes que permiten a las plantas para tolerar las tensiones asociadas con la presencia y el efecto deshidratante de hielo en sus tejidos. Si bien esta última categoría es normalmente, pero no exclusivamente, asociado con aspectos estructurales de una planta que determinan si, cuándo, y dónde se forma hielo en una planta 14. A pesar de la prevalencia de la evitación de la congelación como un anunciomecanismo aptive, poca investigación se ha dedicado en los últimos tiempos a la comprensión de los mecanismos y regulación de evitar la congelación subyacentes. Se remite al lector a una revisión reciente 15 para mayores detalles sobre este tema. Mientras que la formación de hielo a temperaturas bajas puede parecer un proceso simple, muchos factores contribuyen a la determinación de la temperatura a la que el hielo se nuclea en los tejidos vegetales y cómo se propaga dentro de la planta. Parámetros como la presencia de extrínseca e intrínseca nucleadores de hielo, frente a eventos de nucleación homogénea heterogéneos, térmica-histéresis (anticongelante) proteínas, la presencia de azúcares específicos y otros osmolitos, y una serie de aspectos estructurales de la planta todos pueden jugar un importante papel en el proceso de congelación en las plantas. Colectivamente, estos parámetros influyen en la temperatura a la que una planta se congela, donde se inicia hielo y cómo crece. También pueden afectar a la morfología de los cristales de hielo resultantes.Varios métodos han sido utilizados para estudiar el proceso de congelación en las plantas bajo condiciones de laboratorio, incluyendo espectroscopía de resonancia magnética nuclear (RMN) 16, imágenes de resonancia magnética (MRI) 17, crio-microscopía 18-19, y microscopía electrónica de barrido de baja temperatura (LTSEM ). 20 Congelación de plantas enteras en la configuración de laboratorio y de campo, sin embargo, ha sido principalmente controlarse con termopares. El uso de termopares para estudiar la congelación se basa en la liberación de calor (entalpía de fusión) cuando el agua se somete a una transición de fase de líquido a un sólido. La congelación se registra entonces como un evento exotérmico. 21-23 A pesar de que los termopares son el método típico de elección en el estudio de la congelación en plantas, su uso tiene muchas limitaciones que limitan la cantidad de información obtenida durante un suceso de congelación. Por ejemplo, con termopares es difícil casi imposible determinar donde se inicia de hielo en las plantas, la forma en que se propaga,si se propaga a una velocidad constante, y si algunos tejidos permanecen libres de hielo. Los avances en la termografía infrarroja de alta resolución (HRIT) 24-27, sin embargo, han aumentado de manera significativa la capacidad de obtener información sobre el proceso de congelación en plantas enteras, especialmente cuando se utiliza en un modo de imagen diferencial. 28-33 En el presente informe, describen el uso de esta tecnología para estudiar diversos aspectos del proceso de congelación y diversos parámetros que afectan a dónde y en qué hielo y la temperatura se inicia en las plantas. Un protocolo será presentado que demostrar la capacidad de la bacteria de hielo-nucleación-activo (INA), Pseudomonas syringae (Cit-7) para actuar como un nucleador extrínseca iniciar la congelación en una planta herbácea a una alta temperatura, a temperaturas bajo cero. De alta resolución de la cámara de infrarrojos El protocolo y ejemplos documentados en este informe utilizan una alta resolución de infrarrojosradiómetro de vídeo. El radiómetro (Figura 1) suministra una combinación de imágenes espectro de infrarrojos y visibles y datos de temperatura. La respuesta espectral de la cámara está en el rango de 7,5 a 13,5 micras y proporciona una resolución de 640 x 480 pixel. Imágenes espectro visible generados por la cámara puede ser fusionado con IR-imágenes en tiempo real, lo que facilita la interpretación de imágenes térmicas, complejos incorporado. Una gama de lentes para la cámara se puede utilizar para hacer primeros planos y observaciones microscópicas. La cámara se puede utilizar en un modo autónomo, o interconectado y controlado con un ordenador portátil usando software propietario. El software se puede utilizar para obtener una variedad de datos térmicos embebidos en los vídeos grabados. Es importante observar que una amplia variedad de radiómetros infrarrojos están disponibles comercialmente. Por lo tanto, es esencial que el investigador discutir su aplicación prevista con un ingeniero de producto bien informado y que el investigador probar la capacidad de cualquier específic radiómetro para proporcionar la información necesaria. El radiómetro de imágenes utilizado en el protocolo descrito se coloca en una caja de acrílico (Figura 2) con aislamiento de espuma de poliestireno i on el fin de disuadir a la exposición a la condensación durante los protocolos de calentamiento y enfriamiento. Esta protección no es necesaria para todas las cámaras o aplicaciones.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Infrared Camera</td>
			<td>FLIR</td>
			<td>SC-660</td>
			<td>Many models available depending on application</td>
		</tr>
		<tr>
			<td>Infrared Analytical Software</td>
			<td>FLIR</td>
			<td>ResearchIR 4.10.2.5</td>
			<td align="right">$3,500</td>
		</tr>
		<tr>
			<td rowspan="2">Pseudomonas syringae (strain Cit-7)</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2"><a href="mailto:icelab@berkeley.edu">Kindly provided by Dr. Steven Lindow, University of California&#160; Berkeley icelab@berkeley.edu</a></td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Pseudomonas Agar F</td>
			<td>Fisher Scientific</td>
			<td>DF0448-17-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of high-resolution infrared thermography (hrit) for the study of ice nucleation and ice propagation in plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

Actividad Hielo-nucleación del hielo + bacteria, Pseudomonas syringae (cepa Cit-7) Una gota 10 l de agua y 10 l de agua que contienen P. syringae (Cit-7) se colocaron en la superficie abaxial de una hoja de Hosta (Hosta spp.) (Figura 4). Como se ilustra, la gota de agua que contiene las bacterias INA congeló primero y fue responsable de la inducción de la hoja para congelar, mientras que la gota de agua sobre la superficie de la hoja se mantuvo ...

Watch this Scientific Journal Video about The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants at JoVE.com

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

El uso de alta resolución Termografía Infrarroja (HRIT) para el Estudio de Hielo La nucleación y Hielo Propagación de Plantas

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

the-use-high-resolution-infrared-thermography-hrit-for-study-ice

Research

JoVE Journal

Environment

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

9.5K vistas.  Agricultural Research Service (USDA-ARS), Kearneysville, WV. Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Video: The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Expresión recombinante de celulasa de Cel5A<em&gt; Trichoderma reesei</em&gt; En plantas de tabaco

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Investigación

Medio ambiente

Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing seeds of Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum). We applied magnets under the culture dish. During the 4 days of application, we observed that the stem and root length increased. The group subjected to magnetic field treatment (n = 10) showed a 1.4 times faster rate of growth compared with the control group (n = 11) in a total of 8 days (p &#60;0.0005). This rate is 20% higher than that reported in previous studies. The tubulin complex lines did not have connecting points, but connecting points occur upon the application of magnets. This shows complete difference from the control, which means abnormal arrangements. However, the exact cause remains unclear. These results of growth enhancement of applying magnets suggest that it is possible to enhance the growth rate, increase productivity, or control the speed of germination of plants by applying static magnetic fields. Also, magnetic fields can cause physiological changes in plant cells and can induce growth. Therefore, stimulation with a magnetic field can have possible effects that are similar to those of chemical fertilizers, which means that the use of fertilizers can be avoided.

The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

Mejora de la tasa de crecimiento inicial de las plantas agrícolas mediante el uso de campos magnéticos estáticos

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

El uso de una batería de químicos y ecotoxicológicos Métodos para la Evaluación de la eficacia de procesos de tratamiento de aguas residuales para quitar potencia estrogénica

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Empírico, metagenómica y técnicas computacionales iluminarán los mecanismos por los cuales fungicidas compromiso Pathología Apícola

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Automatizado, de alta resolución Sistema de Recolección móvil para el análisis isotópico de nitrógeno NO<sub&gt; x</sub

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus area. Biomass capacitance plays an integral role in the avoidance of hydraulic impairment to transpiration. However, high temporal resolution measurements of dynamic changes in the hydraulic capacitance of large trees are rare. Here, we present procedures for the calibration and use of capacitance sensors, typically used to monitor soil water content, to measure the volumetric water content in trees in the field. Frequency domain reflectometry-style observations are sensitive to the density of the media being studied. Therefore, it is necessary to perform species-specific calibrations to convert from the sensor-reported values of dielectric permittivity to volumetric water content. Calibration is performed on a harvested branch or stem cut into segments that are dried or re-hydrated to produce a full range of water contents used to generate a best-fit regression with sensor observations. Sensors are inserted into calibration segments or installed in trees after pre-drilling holes to a tolerance fit using a fabricated template to ensure proper drill alignment. Special care is taken to ensure that sensor tines make good contact with the surrounding media, while allowing them to be inserted without excessive force. Volumetric water content dynamics observed via the presented methodology align with sap flow measurements recorded using thermal dissipation techniques and environmental forcing data. Biomass water content data can be used to observe the onset of water stress, drought response and recovery, and has the potential to be applied to the calibration and evaluation of new plant-level hydrodynamics models, as well as to the partitioning of remotely sensed moisture products into above- and belowground components.

The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees.

La calibración y uso de sensores de capacitancia para monitorear el contenido de agua del tallo en árboles

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) coupled with synchrotron X-radiation. A cube-shaped sample assembly is prepared and compressed, at room temperature, by a set of four X-ray transparent sintered diamond anvils and two tungsten carbide anvils, in the lateral and the vertical planes, respectively. All six anvils are housed within a 250-ton hydraulic press and driven inward simultaneously by two wedged guide blocks. A horizontal energy dispersive X-ray beam is projected through and diffracted by the sample assembly. The beam is commonly in the mode of either white or monochromatic X-ray. In the case of white X-ray, the diffracted X-rays are detected by a solid-state detector array that collects the resulting energy dispersive diffraction pattern. In the case of monochromatic X-ray, the diffracted pattern is recorded using a two-dimensional (2-D) detector, such as an imaging plate or a charge-coupled device (CCD) detector. The 2-D diffraction patterns are analyzed to derive lattice spacings. The elastic strains of the sample are derived from the atomic lattice spacing within grains. The stress is then calculated using the predetermined elastic modulus and the elastic strain. Furthermore, the stress distribution in two-dimensions allow for understanding how stress is distributed in different orientations. In addition, a scintillator in the X-ray path yields a visible light image of the sample environment, which allows for the precise measurement of sample length changes during the experiment, yielding a direct measurement of volume strain on the sample. This type of experiment can quantify the stress distribution within geomaterials, which can ultimately shed light on the mechanism responsible for compaction. Such knowledge has the potential to significantly improve our understanding of key processes in rock mechanics, geotechnical engineering, mineral physics, and material science applications where compactive processes are important.

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

Distribución de las tensiones durante la compresión fría de rocas y minerales agregados mediante difracción de rayos x de sincrotrón-basado

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

Protocolo para la producción de Video infrarroja tridimensional de congelación en las plantas

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

Seguimiento a largo plazo del vídeo de animales acuáticos Cohoused: un estudio de caso de la actividad locomotriz de la langosta Noruega (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.