No experimento de elección: <ol><li> Cuidadosamente adjuntar la planta Arabidopsis de interés en un vaso de plástico. En este caso: Col-0 (WT) y un mutante con discapacidad en la producción de JA. Asegúrese de limitar el daño a las hojas. Para que el uso final de este pincel. </li><li> Use un pincel fino para dar un larvas recién nacidas de la colonia en la col. </li><li> Coloque cuidadosamente la larva en las plantas de Arabidopsis. </li><li> Peso larva en el día 4, 7, 10. Sugerencia: crecer sus plantas en condiciones de día corto (8 horas de luz) para aumentar el crecimiento vegetativo (1 larva puede cumplir su ciclo de vida completo de una planta). </li></ol> Elección de experimentación: <ol><li> Dos plantas se seleccionan, uno de cada genotipo. Lo mejor es hacer crecer plantas en la misma olla, para limitar otros factores, tales como los efectos bandeja, etc </li><li> Colocar las plantas en una jaula de mariposas. </li><li> Seleccione una hembra fértil. Por lo general la captura de una mujer que está activamente poniendo los huevos en la colonia de cría. </li><li> Liberación de la mujer en la jaula con la elección de dos plantas de 24 hrs. </li></ol> CONSEJO 1: Para eliminar un efecto de aprendizaje, no vuelva a usar las mujeres para las pruebas de selección posterior. TIP 2: Sólo se puede comparar las plantas que tienen un fenotipo de crecimiento similar. Como alternativa, puede montar una hoja de cada genotipo en la parte superior de un erlenmeyer y comparar su atractivo para una mujer. Por favor, visite <a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.arplant.59.032607.092825">las revisiones anuales de Biología Vegetal</a> para obtener más información acerca de este protocolo y la inmunidad de plantas a la herbivoría de insectos.

choice and no-choice assays for testing the resistance of a. thaliana to chewing insects

Las larvas de la mariposa de la col blanca pequeña son una plaga en actividades agrícolas. Esta especie de oruga se alimenta de las plantas de la familia del repollo, que incluyen muchos cultivos como el repollo, brócoli, coles de Bruselas de cría, etc de los insectos se lleva a cabo en las plantas de repollo en el invernadero. Al menos dos jaulas son necesarias para la cría de Violación de Pieris. Una de las larvas y el otro para contener a los adultos, las mariposas. Con el fin de investigar el papel de las hormonas vegetales y sustancias químicas tóxicas de plantas en la resistencia a esta plaga de insectos, se demuestra dos experimentos. En primer lugar, la determinación del papel del ácido jasmónico (JA - una hormona vegetal a menudo se indica en la resistencia a insectos) en la resistencia a la masticación de insectos Violación de Pieris. El crecimiento de Caterpillar se puede comparar en las plantas de tipo salvaje y mutante con discapacidad en la producción de JA. Este experimento se considera &quot;otra opción&quot;, porque las larvas se ven obligados a subsistir con una sola planta que sintetiza o es deficiente en JA. En segundo lugar, se demuestra un experimento que investiga el papel de los glucosinolatos, que se utilizan como oviposición (puesta de huevos) señales. Aquí, nosotros usamos Arabidopsis WT y mutantes alteración en la producción de glucosinolatos en una &quot;elección&quot; experimento en el que las mariposas hembras se les permite elegir para poner sus huevos en las plantas de ambos genotipos. Este video muestra el montaje experimental para ambos ensayos, así como obtener resultados representativos.

Watch this Scientific Journal Video about Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects at JoVE.com

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Resistencia de las plantas a insectos herbívoros de mascar puede ser probado de varias maneras. En este sentido, se muestra cómo configurar una opción y no un experimento de elección con la planta modelo Arabidopsis thaliana para identificar la resistencia contra la plaga de especies Violación de Pieris.

Elección y no elección-Los ensayos para probar la resistencia de A. thaliana a insectos masticadores

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

choice-no-choice-assays-for-testing-resistance-thaliana-to-chewing

Research

JoVE Journal

Biology

17.1K vistas.  Cornell University. Resistencia de las plantas a insectos herbívoros de mascar puede ser probado de varias maneras. En este sentido, se muestra cómo configurar una opción y no un experimento de elección con la planta modelo Arabidopsis thaliana para identificar la resistencia contra la plaga de especies Violación de Pieris.

Video: Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Investigación

Biología

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit ensayos para las convulsiones en la epilepsia de los mutantes Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Los ensayos farmacológicos y genéticos funcionales para manipular la regeneración de la planaria Japonica Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

Los ensayos biofísicos investigará las propiedades mecánicas del núcleo celular Interfase: Aplicación del sustrato deformación y manipulación de Microneedle

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making malaria control an omnipresent problem in tropical countries. The emergence of insecticide resistant populations warrants the exploration of novel drug target pathways and compounds for vector mosquito control. Epigenetic drugs are well established in cancer research, however not much is known about their effects on insects. This study provides a simple protocol for examining the toxicological effects of 3-Deazaneplanocin A (DZNep), an experimental epigenetic drug for cancer therapy, on the malaria vector, Anopheles gambiae. A concentration-dependent increase in mortality and decrease in size was observed in immature mosquitoes exposed to DZNep, whereas the compound reduced the fecundity of adult mosquitoes relative to control treatments. In addition, there was a drug-dependent decrease in S-adenosylhomocysteine (SAH) hydrolase activity in mosquitoes following exposure to DZNep relative to control treatments. These protocols provide the researcher with a simple, step-by-step procedure to assess multiple toxicological endpoints for an experimental drug and, in turn, demonstrate a unique multi-prong approach for exploring the toxicological effects of water-soluble epigenetic drugs or compounds of interest against vector mosquitoes and other insects.

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

Los ensayos toxicológicos para efectos de prueba de un epigenética de Drogas sobre el Desarrollo, Fecundidad y Sobrevivencia de los mosquitos de malaria

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

Diseño y Desarrollo de aptámeros y oro nanopartículas basados ​​en ensayos colorimétricos para aplicaciones en-el-campo

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Métodos para la extracción de endosimbiontes de la mosca blanca<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protocolos para las pruebas de la toxicidad de la novela química insecticida para mosquitos

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

Un Protocolo de Pantalla Mutante de Señalización de Señalización de Fuerza Táctil Y Repetible para el Estudio de la Thigmomorfogénesis de una Planta Modelo Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

A Finite Element Approach for Locating the Center of Resistance of Maxillary Teeth

The center of resistance (CRES) is regarded as the fundamental reference point for predictable tooth movement. The methods used to estimate the CRES of teeth range from traditional radiographic and physical measurements to in vitro analysis on models or cadaver specimens. Techniques involving finite element analysis of high-dose micro-CT scans of models and single teeth have shown a lot of promise, but little has been done with newer, low-dose, and low resolution cone beam computed tomography (CBCT) images. Also, the CRES for only a few select teeth (i.e., maxillary central incisor, canine, and first molar) have been described; the rest have been largely ignored. There is also a need to describe the methodology of determining the CRES in detail, so that it becomes easy to replicate and build upon.
This study used routine CBCT patient images for developing tools and a workflow to obtain finite element models for locating the CRES of maxillary teeth. The CBCT volume images were manipulated to extract three-dimensional (3D) biological structures relevant in determining the CRES of the maxillary teeth by segmentation. The segmented objects were cleaned and converted into a virtual mesh made up tetrahedral (tet4) triangles having a maximum edge length of 1 mm with 3matic software. The models were further converted into a solid volumetric mesh of tetrahedrons with a maximum edge length of 1 mm for use in finite element analysis. The engineering software, Abaqus, was used to preprocess the models to create an assembly and set material properties, interaction conditions, boundary conditions, and load applications. The loads, when analyzed, simulated the stresses and strains on the system, aiding in locating the CRES. This study is the first step in accurate prediction of tooth movement.

This study outlines the necessary tools for utilizing low-dose three-dimensional cone beam-based patient images of the maxilla and maxillary teeth to obtain finite element models. These patient models are then used to accurately locate the CRES of all the maxillary teeth.

Un enfoque de elementos finitos para localizar el centro de resistencia de los dientes maxilares

The center of resistance (CRES) is regarded as the fundamental reference point for predictable tooth movement. The methods used to estimate the CRES of ...