Esta investigación fue financiada en parte por el apoyo recibido de Targeted los EE.UU. Agencia de Protección Ambiental de Cuencas Programa de Subvenciones (Proyecto # WS97704801), la Fundación Nacional de Ciencias Dinámicas de acoplados sistemas naturales y humanos (Proyecto # DEB1010259-CARD-KLIN), y el Departamento de EE.UU. de Agricultura, Instituto Nacional Coordinado de Proyecto Agrícola Foodand Agricultura (Proyecto # 20116800230190-CARD-).

<ol>
	<li>American Society of Agricultural and Biological Engineers. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+layout,+construction,+and+maintenance+of+terrace+systems.">Design, layout, construction, and maintenance of terrace systems.</a> ASAE Standard. , S268 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Raccoon River Watershed water quality master plan. , (2011).</li><li>Arabi, M., Govindaraju, R. S., Hantush, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effective+allocation+of+watershed+management+practices+using+a+genetic+algorithm.">Cost-effective allocation of watershed management practices using a genetic algorithm.</a> Water Resour. Res. 42, W10429 (2006).</li><li>Bekele, E. G., Nicklow, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiobjective+management+of+ecosystem+services+by+integrative+watershed+modeling+and+evolutionary+algorithms.">Multiobjective management of ecosystem services by integrative watershed modeling and evolutionary algorithms.</a> Water Resour. Res. 41, W10406 (2005).</li><li>Carpentier, C. L., Bosch, D. J., Batie, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+spatial+information+to+reduce+costs+of+controlling+agricultural+nonpoint+source+pollution.">Using spatial information to reduce costs of controlling agricultural nonpoint source pollution.</a> Am. J. Agr. Econ. 61, 404-413 (1998).</li><li>Gassman, P. W., Reyes, M., Green, C. H., Arnold, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Soil+and+Water+Assessment+Tool:+Historical+development,+applications,+and+future+directions.">The Soil and Water Assessment Tool: Historical development, applications, and future directions.</a> Trans. ASABE. 50 (4), 1211-1250 (2007).</li><li>Gassman, P. W., Campbell, T., Secchi, S., Jha, M., Arnold, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+i_SWAT+software+package:+a+tool+for+supporting+SWAT+watershed+applications.">The i_SWAT software package: a tool for supporting SWAT watershed applications.</a> , 66-69 .</li><li>Gitau, M. W., Veith, T. L., Gburek, W. J., Jarrett, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watershed+level+best+management+practice+selection+and+placement+in+the+Town+Brook+watershed,+New+York.">Watershed level best management practice selection and placement in the Town Brook watershed, New York.</a> J. Am. Water Resour. Assoc. 42, 1565-1581 (2006).</li><li>Jha, M., Rabotyagov, S., Gassman, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+placement+of+conservation+practices+using+genetic+algorithms+with+SWAT.">Optimal placement of conservation practices using genetic algorithms with SWAT.</a> Int. Agr. Eng. J. 18 (1-2), 41-50 (2009).</li><li>Jha, M. K., Wolter, C. F., Schilling, K. E., Gassman, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+total+maximum+daily+load+implementation+strategies+for+nitrate+impairment+of+the+Raccoon+River,+Iowa.">Assessment of total maximum daily load implementation strategies for nitrate impairment of the Raccoon River, Iowa.</a> J. Envir. Qual. 39, 1317-1327 (2010).</li><li>Kanna, M., Yang, W., Farnsworth, R., Onal, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost+effective+targeting+of+CREP+to+improve+water+quality+with+endogenous+sediment+deposition+coefficients.">Cost effective targeting of CREP to improve water quality with endogenous sediment deposition coefficients.</a> Am. J. Agr. Econ. 85, 538-553 (2003).</li><li>Kramer, R. A., McSweeny, W. T., Kerns, W. R., Stravros, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+alternative+policies+for+controlling+agricultural+nonpoint+source+pollution.">An evaluation of alternative policies for controlling agricultural nonpoint source pollution.</a> Water Resour. Bull. 20, 841-8446 (1984).</li><li>Lant, C. L., Kraft, S. E., Beaulieu, J., Bennett, D., Loftus, T., Nicklow, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+GIS-based+ecological-economic+modeling+to+evaluate+policies+affecting+agricultural+watersheds.">Using GIS-based ecological-economic modeling to evaluate policies affecting agricultural watersheds.</a> Ecol. Econ. 55, 467-484 (2005).</li><li>Muleta, M. K., Nicklow, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+algorithms+for+multiobjective+evaluation+of+watershed+management+decisions.">Evolutionary algorithms for multiobjective evaluation of watershed management decisions.</a> J. Hydroinform. 4 (2), 83-97 (2002).</li><li>Muleta, M. K., Nicklow, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decision+support+for+watershed+management+using+evolutionary+algorithms.">Decision support for watershed management using evolutionary algorithms.</a> J. Water Resour. Plan. Manag. 131 (1), 35-44 (2005).</li><li>Rabotyagov, S. S., Campbell, T., Jha, M., Gassman, P. W., Arnold, J., Kurkalova, L., Secchi, S., Feng, H., Kling, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Least+Cost+Control+of+Agricultural+Nutrient+Contributions+to+the+Gulf+of+Mexico+Hypoxic+Zone.">Least Cost Control of Agricultural Nutrient Contributions to the Gulf of Mexico Hypoxic Zone.</a> Ecol. Appl. 20, 1542-1555 (2010).</li><li>Rabotyagov, S., Jha, M., Campbell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+crop+rotations+on+optimal+selection+of+conservation+practices+for+water+quality+protection.">Impact of crop rotations on optimal selection of conservation practices for water quality protection.</a> J. Soil. Water Conserv. 65 (6), 369-380 (2010).</li><li>Rabotyagov, S. S., Jha, M., Campbell, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonpoint-source+pollution+for+an+Iowa+watershed:+An+application+of+evolutionary+algorithms.">Nonpoint-source pollution for an Iowa watershed: An application of evolutionary algorithms.</a> Canadian Journal of Agricultural Economics/Revue canadienne d'agroeconomie. , (2010).</li><li>Rabotyagov, S. S., Jha, M., Campbell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Searching+for+Efficiency:+Least+Cost+Nonpoint+Source+Pollution+Control+with+Multiple+Pollutants,+Practices,+and+Targets.">Searching for Efficiency: Least Cost Nonpoint Source Pollution Control with Multiple Pollutants, Practices, and Targets.</a> J. Nat. and Environ. Sci. 1, 75-90 (2010).</li><li>Ribaudo, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consideration+of+off-site+impacts+in+targeting+soil+conservation+programs.">Consideration of off-site impacts in targeting soil conservation programs.</a> Land Econ. 62, 402-411 (1986).</li><li>Ribaudo, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+conservation+reserve+program+to+maximize+water+quality+benefits.">Targeting the conservation reserve program to maximize water quality benefits.</a> Land Econ. 65, 320-332 (1989).</li><li>Srivastava, P., Hamlett, J. M., Robillard, P. D., Day, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watershed+optimization+of+best+management+practices+using+AnnAGNPS+and+a+genetic+algorithm.">Watershed optimization of best management practices using AnnAGNPS and a genetic algorithm.</a> Water Resour. Res. 38 (3), 1-14 (2002).</li><li>Veith, T. L., Wolfe, M. L., Heatwole, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+optimization+procedure+for+cost-effective+BMP+placement.">Development of optimization procedure for cost-effective BMP placement.</a> J. Am. Water Resour. Assoc. 39 (6), 1331-1343 (2003).</li><li>Whittaker, G., Confesor, R., Griffith, S. M., F&#228;re, R., Grosskopf, S., Steiner, J. J., Mueller-Warrant, G. W., Banowetz, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Hybrid+Genetic+Algorithm+for+Multiobjective+Problems+with+Activity+Analysis-based+Local+Search.">A Hybrid Genetic Algorithm for Multiobjective Problems with Activity Analysis-based Local Search.</a> Eur. J. Operat. Res. , (2007).</li><li>Zitzler, E., Laumanns, M., Thiele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPEA2:+Improving+the+Strength+Pareto+Evolutionary+Algorithm+for+Multiobjective+Optimization.">SPEA2: Improving the Strength Pareto Evolutionary Algorithm for Multiobjective Optimization.</a> CIMNE. , 95-100 (2002).</li></ol>

No hay conflictos de interés declarado.

Construimos un sistema integrado de simulación-optimización marco para buscar el sentido de Pareto eficientes conjuntos de configuraciones de las cuencas de menor costo que implican mezcla y ubicación de las prácticas agrícolas de conservación para lograr una serie de objetivos de nivel de cuencas de reducción de nutrientes. Un diagrama conceptual del sistema de simulación-optimización se presentan en la Figura 8. Simulación de cuencas, incluyendo la simulación de los impactos en la calidad d...

spatial multiobjective optimization of agricultural conservation practices using a swat model and an evolutionary algorithm

Encontrar los costos eficientes (es decir, el más bajo costo) formas de orientar las inversiones en conservación de práctica para el logro de determinados objetivos de calidad del agua en todo el paisaje es de una importancia primordial en la gestión de las cuencas hidrográficas. Métodos de la economía tradicional de encontrar la solución de más bajo costo en el contexto de la cuenca (por ejemplo, 5,12,20) asumen que el impacto fuera del sitio puede ser descrito con precisión como una proporción de la contaminación genera en el sitio. Estos planteamientos son poco probable que sea representativo del proceso de contaminación real en una cuenca, donde los impactos de las fuentes contaminantes suelen estar determinadas por complejos procesos biofísicos. El uso de los modernos basados ​​en la física, distribuidos espacialmente modelos de simulación hidrológicos permite un mayor grado de realismo en términos de representación del proceso, sino que requiere un desarrollo de un marco de simulación-optimización en el que el modelo se convierte en una parte integral de la optimización. Evoluciónalgoritmos ary parece ser una herramienta de optimización particularmente útil, capaz de tratar con la naturaleza combinatoria de una cuenca de simulación-optimización problema y permitiendo el uso del modelo de calidad de agua completo. Los algoritmos evolutivos tratar una distribución espacial particular de prácticas de conservación en una cuenca como una solución candidata y utilizar conjuntos (poblaciones) de soluciones candidatas iterativamente aplicando operadores estocásticos de la selección, la recombinación y mutación para encontrar mejoras con respecto a los objetivos de optimización. Los objetivos de optimización en este caso son dispersas para minimizar la contaminación procedente de la cuenca, al mismo tiempo reducir al mínimo el costo de las prácticas de conservación. Un conjunto reciente y creciente de investigación está tratando de utilizar los mismos métodos e integra los modelos de calidad del agua con la definición amplia de métodos de optimización evolutivas 3,4,9,10,13-15,17-19,22,23,25. En esta aplicación, se demuestra un programa que sigue Rabotyagov et al. &#39;S aproada e integra un moderno y de uso común SWAT modelo de calidad del agua 7 con un algoritmo evolutivo multiobjetivo SPEA2 26, y el usuario especifica un conjunto de prácticas de conservación y sus costos para buscar las fronteras compensación entre los costos completos de las prácticas de conservación y la calidad especificada por el usuario de agua objetivos. Las fronteras cuantificar las ventajas y desventajas que enfrentan los administradores de cuencas hidrográficas mediante la presentación de la totalidad de los costos relacionados con los objetivos de mejora de la calidad de agua diferentes. El programa permite una selección de configuraciones de cuencas han alcanzado determinadas metas de mejoramiento de calidad de agua y una producción de mapas de colocación optimizada de las prácticas de conservación.

Watch this Scientific Journal Video about Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm at JoVE.com

Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm

Este trabajo demuestra una integración de un modelo de calidad del agua con un componente de optimización utilizando algoritmos evolutivos para resolver óptimo (de menor costo) la colocación de las prácticas agrícolas para la conservación de un conjunto específico de objetivos de mejora de la calidad del agua. Las soluciones se generan utilizando un enfoque multi-objetivo, lo que permite la cuantificación explícita de compensaciones.

Optimización multiobjetivo espacial de las prácticas de agricultura de conservación que utilizan un modelo SWAT y un algoritmo evolutivo

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals ...

spatial-multiobjective-optimization-agricultural-conservation

Research

JoVE Journal

Biology

12.9K vistas.  University of Washington. Este trabajo demuestra una integración de un modelo de calidad del agua con un componente de optimización utilizando algoritmos evolutivos para resolver óptimo (de menor costo) la colocación de las prácticas agrícolas para la conservación de un conjunto específico de objetivos de mejora de la calidad del agua. Las soluciones se generan utilizando un enfoque multi-objetivo, lo que permite la cuantificación explícita de compensaciones.

Video: Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

La predicción de la eficacia de la estrategia de reemplazo de la población Utilizando modelos matemáticos

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

Investigación

Biología

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

No radiactivo In situ La hibridación aplicable para el abeto rojo protocolo y una serie de especies de plantas

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Un protocolo optimizado para cría Fopius arisanus, Un parasitoide de moscas de fruta tefrítidas

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

Ablación de una sola célula de ocho células de embriones de los crustáceos anfípodos<em&gt; Parhyale hawaiensis</em

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Medición Espacial y Temporal Ca<sup&gt; 2 +</sup&gt; Las señales en plantas de Arabidopsis

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

La observación y cuantificación de la conducta de apareamiento en el nematodo del pino,<em&gt; Bursaphelenchus xylophilus</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Expresión transitoria y localización celular de proteínas recombinantes en células cultivadas de insectos

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Técnicas Reproductivas para el Monitoreo y Control de La Ovacada en Anfibios

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of thermoregulation in birds across a variety of ecological circumstances. However, few studies characterize the contribution of the feathers alone to thermoregulation. Several previous studies have established methods for measuring the insulation value of animal pelts, but they require destructive sampling methods that are problematic for birds, whose feathers are not distributed evenly across the skin. More information is needed about 1) how the contribution of feathers to thermoregulation varies both across and within species and 2) how feather coats may change over space and time. Reported here is a method for rapidly and directly measuring the thermal performance of feather coats and the skin using dried whole skin specimens, without the need to destroy the skin specimen. This method isolates and measures the thermal gradient across a feather coat in a way that measurements of heat loss and metabolic cost in live birds, which use behavioral and physiological strategies to thermoregulate, cannot. The method employs a thermal camera, which allows the rapid collection of quantitative thermal data to measure heat loss from a stable source through the skin. This protocol can easily be applied to various research questions, is applicable to any avian taxa, and does not require destruction of the skin specimen. Finally, it will further the understanding of the importance of passive thermoregulation in birds by simplifying and accelerating the collection of quantitative data.

This protocol describes the quantification of heat transmission through a flat skinned avian specimen using a thermal camera and hot water bath. The method allows obtaining of quantitative, comparative data about the thermal performance of feather coats across species using dried flat skin specimens.

Uso de una cámara térmica para medir la pérdida de calor a través de abrigos de plumas de aves

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.