Estamos agradecidos a Jochen Zeil, Paul Cooper y Birgit Greiner para compartir sus conocimientos en anatomía de insecto y para la demostración de varias de las técnicas que hemos descrito aquí. Agradecemos al personal de apoyo y talento en el centro de microscopía avanzada en ANU y la unidad de microscopía en MQU. Este trabajo fue financiado por una beca de posgrado a FRE y becas del Consejo australiano de investigación (DE120100019, FT140100221, DP150101172).

<ol>
	<li>Zeil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+homing:+an+insect+perspective.">Visual homing: an insect perspective.</a> Curr. Opin. Neurobiol. 22, 285-293 (2012).</li><li>Wehner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desert+ant+navigation:+how+miniature+brains+solve+complex+tasks.">Desert ant navigation: how miniature brains solve complex tasks.</a> J. Comp. Physiol. A. 189, 579-588 (2003).</li><li>Fent, K., Wehner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocelli:+a+celestial+compass+in+the+desert+ant+Cataglyphis.">Ocelli: a celestial compass in the desert ant Cataglyphis.</a> Science. 228, 192-194 (1985).</li><li>Warrant, E. J., Dacke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+navigation+in+nocturnal+Insects.">Visual navigation in nocturnal Insects.</a> Physiology. 31, 182-192 (2016).</li><li>Taylor, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dual+function+of+Orchid+bee+ocelli+as+revealed+by+x-ray+microtomography.">The dual function of Orchid bee ocelli as revealed by x-ray microtomography.</a> Curr. Biol. 26, 1-6 (2016).</li><li>H&#246;lldobler, B., Wilson, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Ants. , (1990).</li><li>Ali, T. M. M., Urbani, C. B., Billen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+jumping+behaviors+in+the+ant+Harpegnathos+saltator.">Multiple jumping behaviors in the ant Harpegnathos saltator.</a> Naturwissen. 79, 374-376 (1992).</li><li>Weiser, M. D., Kaspari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+morphospace+of+New+World+ants.">Ecological morphospace of New World ants.</a> Ecol. Entomol. 31, 131-142 (2006).</li><li>Bulova, S., Purce, K., Khodak, P., Sulger, E., O'Donnell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Into+the+black+and+back:+the+ecology+of+brain+investment+in+Neotropical+army+ants+(Formicidae:+Dorylinae).">Into the black and back: the ecology of brain investment in Neotropical army ants (Formicidae: Dorylinae).</a> Naturwissen. 103, 3-4 (2016).</li><li>Narendra, A., Reid, S. F., Hemmi, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+twilight+zone:+ambient+light+levels+trigger+activity+in+primitive+ants.">The twilight zone: ambient light levels trigger activity in primitive ants.</a> Proc. R. Soc. B. 277, 1531-1538 (2010).</li><li>Narendra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caste-specific+visual+adaptations+to+distinct+daily+activity+schedules+in+Australian+Myrmecia+ants.">Caste-specific visual adaptations to distinct daily activity schedules in Australian Myrmecia ants.</a> Proc. R. Soc. B. 278, 1141-1149 (2011).</li><li>Moser, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+size+and+behaviour+of+day-and+night-flying+leafcutting+ant+alates.">Eye size and behaviour of day-and night-flying leafcutting ant alates.</a> J. Zool. 264, 69-75 (2004).</li><li>St&#246;ckl, A. L., Ribi, W. A., Warrant, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptations+for+nocturnal+and+diurnal+vision+in+the+hawkmoth+lamina.">Adaptations for nocturnal and diurnal vision in the hawkmoth lamina.</a> J. Comp. Neurol. 524, 160-175 (2016).</li><li>Zeil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+the+visual+system+of+flies:+the+compound+eyes+and+neural+superposition+in+Bibionidae+(Diptera).">Sexual dimorphism in the visual system of flies: the compound eyes and neural superposition in Bibionidae (Diptera).</a> J. Comp. Physiol. A. 150, 379-393 (1983).</li><li>Dacke, M., Nordstr&#246;m, P., Scholtz, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twilight+orientation+to+polarised+light+in+the+crepuscular+dung+beetle+Scarabaeus+zambesianus.">Twilight orientation to polarised light in the crepuscular dung beetle Scarabaeus zambesianus.</a> J. Exp. Biol. 206, 1535-1543 (2003).</li><li>Greiner, B., Ribi, W. A., Warrant, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+and+optical+adaptations+for+nocturnal+vision+in+the+halictid+bee+Megalopta+genalis.">Retinal and optical adaptations for nocturnal vision in the halictid bee Megalopta genalis.</a> Cell Tiss Res. 316, 377-390 (2004).</li><li>Warrant, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nocturnal+vision+and+landmark+orientation+in+a+tropical+halictid+bee.">Nocturnal vision and landmark orientation in a tropical halictid bee.</a> Curr. Biol. 14, 1309-1318 (2004).</li><li>Lattke, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ants Standard Methods for Measuring and Monitoring Biodiversity. , 155-171 (2000).</li><li>Ribi, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Handbook in Biological Electron Microscopy. , 1-106 (1987).</li><li>Narendra, A., Ramirez-Esquivel, F., Ribi, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound+eye+and+ocellar+structure+for+walking+and+flying+modes+of+locomotion+in+the+Australian+ant,+Camponotus+consobrinus.">Compound eye and ocellar structure for walking and flying modes of locomotion in the Australian ant, Camponotus consobrinus.</a> Sci. Rep. 6, 22331 (2016).</li><li>Narendra, A., Greiner, B., Ribi, W. A., Zeil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+and+dark+adaptation+mechanisms+in+the+compound+eyes+of+Myrmecia+ants+that+occupy+discrete+temporal+niches.">Light and dark adaptation mechanisms in the compound eyes of Myrmecia ants that occupy discrete temporal niches.</a> J. Exp. Biol. 219, 2435-2442 (2016).</li><li>Ribi, W. A., Zeil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+visual+system+of+the+Australian+&amp;#34;Redeye&amp;#34;+cicada+(Psaltoda+moerens).">The visual system of the Australian &amp;#34;Redeye&amp;#34; cicada (Psaltoda moerens).</a> Arthr. Struct. Dev. 44, 574-586 (2015).</li><li>Ribi, W. A., Warrant, E. J., Zeil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+honeybee+ocelli:+regional+specializations+and+rhabdom+arrangements.">The organization of honeybee ocelli: regional specializations and rhabdom arrangements.</a> Arthr. Struct. Dev. 40, 509-520 (2011).</li><li>Ribi, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colour+receptors+in+the+eye+of+the+digger+wasp,+Sphex+cognatus+Smith:+evaluation+by+selective+adaptation.">Colour receptors in the eye of the digger wasp, Sphex cognatus Smith: evaluation by selective adaptation.</a> Cell Tiss. Res. 195, 471-483 (1978).</li><li>Ribi, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+and+migration+of+screening+pigments+in+the+retina+of+Pieris+rapae+L.+(Lepidoptera,+Pieridae).">Ultrastructure and migration of screening pigments in the retina of Pieris rapae L. (Lepidoptera, Pieridae).</a> Cell Tiss. Res. 191, 57-73 (1978).</li><li>Lau, T., Gross, E., Meyer-Rochow, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+and+light/dark+adaptation+in+the+compound+eyes+of+male+and+female+Acentria+ephemerella+(Lepidoptera:+Pyraloidea:+Crambidae).">Sexual dimorphism and light/dark adaptation in the compound eyes of male and female Acentria ephemerella (Lepidoptera: Pyraloidea: Crambidae).</a> Eur. J. Entomol. 104, 459-470 (2007).</li><li>Wipfler, B., Pohl, H., Yavorskaya, M. I., Beutel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+methods+for+analysing+insect+structures+-+the+role+of+morphology+in+the+age+of+phylogenomics.">A review of methods for analysing insect structures - the role of morphology in the age of phylogenomics.</a> Curr. Opin. Insect Sci. 18, 60-68 (2016).</li><li>Streinzer, M., Brockmann, A., Nagaraja, N., Spaethe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+and+caste-specific+variation+in+compound+eye+morphology+of+five+honeybee+species.">Sex and caste-specific variation in compound eye morphology of five honeybee species.</a> PLoS ONE. 8, e57702 (2013).</li><li>Somanathan, H., Warrant, E. J., Borges, R. M., Wall&#233;n, R., Kelber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+and+sensitivity+of+the+eyes+of+the+Asian+honeybees+Apis+florea,+Apis+cerana+and+Apis+dorsata.">Resolution and sensitivity of the eyes of the Asian honeybees Apis florea, Apis cerana and Apis dorsata.</a> J. Exp. Biol. 212, 2448-2453 (2009).</li></ol>

Los autores declaran a no hay intereses contrapuestos.

El conjunto de los métodos señalados anteriormente permiten una investigación efectiva sobre el sistema óptico de hormigas y otros insectos. Estas técnicas informar a nuestra comprensión de la resolución de muestreo, sensibilidad óptica y sensibilidad potencial de polarización del ojo en estudio. Este conocimiento proporciona una base importante para la investigación fisiológica y de comportamiento en sus capacidades visuales. Además, mientras que los métodos detallados aquí se han centrado en los sistemas ...

La visión es una modalidad sensorial importante para la mayoría de los animales. La visión es especialmente crucial en el contexto de navegación para objetivos de localización, establecimiento y adhiriéndose a las rutas y obtener información brújula1,2. Insectos detectan información visual utilizando un par de ojos compuestos y, en algunos casos, uno a tres ojos simples de colocado dorsalmente llaman ocelos3,4,5.
Los ojos de las hormigas son de particular interés porque, mientras que las hormigas son muy diversas, conservan algunas características claves entre especies. A pesar de la dramática variación en la anatomía, tamaño y ecología, la gran mayoría de especies es eusociales y vive en colonias; en consecuencia, diferentes especies enfrentan retos visuales similares en términos de navegación hacia adelante y hacia atrás entre un lugar central y los recursos. A través de las hormigas el mismo bauplan ojo básico puede observarse en los animales que van desde 0.5-26 mm de longitud corporal, de exclusivamente diurna a especie estrictamente nocturna y de lento caminar subterráneo a saltar los depredadores visuales6,7, 8,9,10. Todas estas diferencias asombrosas en ecología y comportamiento dan lugar a innumerables permutaciones de las mismas estructuras básicas del ojo para adaptarse a diferentes ambientes, estilos y tamaños de cuerpo11,12. En consecuencia, estudiar la ecología visual de hormigas proporciona un verdadero tesoro de posibilidades al investigador determinado.
Comprensión del sistema visual de los insectos es esencial para ganar una penetración en sus capacidades conductuales. Se trata de estudios integrados que combinan muy bien la anatomía con la ecología y comportamiento de un gran éxito en algunos grupos de insectos (p. ej., referencias13,14,15,16, 17). aunque en general el campo de la navegación de ant y ant comportamiento ha sido absolutamente acertado, ha hecho hincapié muy poca visión de hormiga fuera de unas pocas especies seleccionadas. Aquí, elaboramos las técnicas involucradas en la investigación de diseño ojo de hormigas. Si bien nos centraremos en las hormigas, estas técnicas pueden aplicarse, con ligeras modificaciones, a otros insectos, también.

<table>
	<tbody>
		<tr>
			<td>Ant</td>
			<td>Myrmecia midas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica M205 FA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Pro Sci Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica MZ6</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insect Pin</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Colourless nail polish</td>
			<td>Non branded: from any cosmetic store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foreceps</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slip</td>
			<td>ProSciTech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound microscope</td>
			<td>Leica DM5000 B</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformalydehyde</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium Hydrogen phosphate (Na2HPO4)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium di-Hydrogen Phosphate (KH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Araldite Epoxy Resin</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidie Blue</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Riechert HK120</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

techniques for investigating the anatomy of the ant visual system

Este artículo describe un conjunto de técnicas en microscopía de luz (LM) y la microscopia electrónica (EM) que puede utilizarse para estudiar la anatomía interna y externa del ojo de los insectos. Estos incluyen técnicas histológicas tradicionales optimizada para trabajo en ojos de hormiga y adaptado para trabajar en conjunto con otras técnicas como la microscopía electrónica de transmisión (TEM) y microscopía electrónica (SEM). Estas técnicas, aunque muy útil, pueden ser difíciles para el microscopista de principiante, por lo que ha hecho gran hincapié en este artículo de solución de problemas y optimización para diferentes muestras. Proporcionar información sobre las técnicas de imagen para la muestra entera (foto-microscopía y SEM) y analizar sus ventajas y desventajas. Destacar la técnica utilizada en la determinación de diámetros de lente para el ojo entero y discutir nuevas técnicas para la mejora. Por último, se discuten técnicas de preparación de muestras para LM y TEM, corte, tinción y estas muestras de la proyección de imagen. Se discuten los obstáculos que podría venir cuando preparación de muestras y la mejor manera de navegar por ellos.

Los métodos aquí descritos permiten el estudio detallado de los simples y ojos compuestos de las hormigas. Vista dorsal de la cabeza usando técnicas de microfotografía Z-stack de imágenes permite obtener una visión general de la disposición del sistema visual (figura 1). Se trata de una buena preparación para disecciones y para determinar el ángulo requerido de seccionamiento. Esta técnica también es útil para la toma de medidas tales como la anch...

Watch this Scientific Journal Video about Techniques for Investigating the Anatomy of the Ant Visual System at JoVE.com

Techniques for Investigating the Anatomy of the Ant Visual System

Este artículo describe un conjunto de técnicas de luz y microscopía electrónica para estudiar la anatomía interna y externa del ojo de los insectos. Estos incluyen diversas técnicas tradicionales optimizadas para trabajo en ojos de hormiga, detalladas de solución de problemas y sugerencias para la optimización para diferentes especies y regiones de interés.

Técnicas para investigar la anatomía del sistema Visual y

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

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13.1K vistas.  Australian National University. Este artículo describe un conjunto de técnicas de luz y microscopía electrónica para estudiar la anatomía interna y externa del ojo de los insectos. Estos incluyen diversas técnicas tradicionales optimizadas para trabajo en ojos de hormiga, detalladas de solución de problemas y sugerencias para la optimización para diferentes especies y regiones de interés.

Video: Techniques for Investigating the Anatomy of the Ant Visual System

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

Los métodos para realizar cruces en<em&gt; Setaria viridis</em&gt;, Un nuevo sistema de modelo para las gramíneas

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

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Medio ambiente

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Optimizado tinción negativa: un protocolo de alto rendimiento para el examen de Estructura Pequeña y asimétrica de proteínas por Microscopía Electrónica

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

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 ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Extracción de plaguicidas organoclorados de pellets de plástico y análisis de tipos de plástico

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

Metodología para el desarrollo de tablas de vida para los insectos sésiles en el campo de la mosca blanca, Bemisia tabaci, en algodón como un modelo de sistema de

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Procedimientos de laboratorio fumigación Control de plagas con Gas de óxido nítrico

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

Desarrollo de nuevos métodos para cuantificar la densidad de peces utilizando herramientas de estéreo y vídeo subacuáticos

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

Recolección de las toxinas del veneno de insectos asesinos y otros insectos Heteropteran

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

In Situ Técnicas de hibridación para muestras de corales adultos parafinadas

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...