Este trabajo fue financiado por los Institutos Nacionales de Salud Grant R01 GM54408 otorgado a LC Griffith. Nos gustaría agradecer a Fred Wolf por toda su ayuda en el diseño de nuestra cámara cuadrada y la creación de nuestro ensayo, Frank Mello en el taller de máquinas para la construcción de la Universidad de Brandeis nuestra cámara, y Dan Valente y Lebestky Tim para las conversaciones útiles con respecto a temas de análisis.

Parte 1: alimentación y manejo de las moscas <ol><li> Las moscas deben ser cultivadas en frascos de levadura libre de los medios de comunicación estándar. Usted necesitará un gran número de moscas, por lo que cruza es necesario establecer en consecuencia. Las moscas deben ser cultivadas en un 12 horas de luz: oscuridad ciclo a 25 º C. </li><li> Las moscas deben ser recogidos poco después de la eclosión (1-3 días). Las moscas pueden ser trabajado en esta etapa con un difusor de dióxido de carbono, y deben ser ordenados en tubos de ensayo que contiene los medios de comunicación en autoclave o sin levadura. Utilizamos sólo los machos, almacena 10 a un tubo de ensayo. </li><li> Deje por lo menos un día o dos después de su uso de dióxido de carbono antes de ejecutar los experimentos de comportamiento. Se corre vuela en nuestro ensayo de comportamiento de 3-5 días después de la eclosión. </li><li> Los ensayos se debe hacer siempre en la misma ventana de tiempo de 2-3 horas cada día para evitar problemas de ritmo circadiano. </li></ol> Parte 2: Configuración del sistema de seguimiento en un ambiente controlado <ol><li> Todos los experimentos se hacen en una sala de medio ambiente que mantiene una temperatura constante de 25 º C con una humedad del 70%. </li><li> Suspender una cámara de vídeo digital sobre la zona a grabar (la cámara hacia abajo). Desde nuestro software de seguimiento basado en un contraste, se suspenderá la cámara a través de una caja de luz. La cámara graba vídeos directamente en un ordenador Dell a través de una conexión FireWire. Usamos una cámara de vídeo digital de Sharp, conectado a una computadora Dell con Windows MovieMaker (versión de Vista) para la adquisición de video. </li><li> Pulsos de aire será administrado en el presente Protocolo, por lo que una fuente de flujo de aire debe estar presente en la sala. Con mangueras de caucho, conecte la fuente de aire a un filtro de carbono para la filtración de aire. </li><li> El uso de más tubos de goma, conecte el otro lado del filtro de carbón a un erlenmeyer a través de un tapón de goma hueco, y llenar el recipiente con aproximadamente media pulgada de agua. Esto humedecer el aire. </li><li> El frasco debe también tener un arma, que se conectará a una válvula en forma de Y a través de un tubo de goma. </li><li> Una rama de la Y de la válvula debe estar conectado a un medidor de caudal. La otra rama que suministra aire a la cámara de locomoción cuadrados. </li></ol> Parte 3: Entrega de las moscas a la cámara de locomoción <ol><li> La cámara de locomoción cuadrados se basa en el diseño de Wolf et al. 2002 1, con la incorporación de bandejas de pequeña gota para evitar que las moscas que vuelan dentro de la cámara. La cámara debe ser desarmado, y todos los componentes deben ser cuidadosamente limpiados con etanol al 70% y se seca antes de su uso. </li><li> Anestesia inmediatamente antes de la realización de un ensayo de comportamiento que podría comprometer el rendimiento. Para evitar esto, las moscas se golpeó suavemente en la cámara usando un embudo con una pequeña salida. Para hacer una salida del tamaño adecuado, colocar un azul punta de la pipeta (para P1000) hasta el final de un embudo. Utilice unas tijeras para cortar el extremo de la punta de la pipeta para hacer la abertura lo suficientemente grande como para volar a pasar. </li><li> La parte superior de nuestra cámara es una pequeña pieza de plexiglás asegurada con tornillos en los bordes. Al retirar los tornillos, los agujeros pueden ser utilizados para presentar las moscas en la cámara. Coloque la parte superior de plexiglás, para que el agujero del tornillo se encuentra directamente sobre la cámara interior, en lugar de que un tornillo se encuentra normalmente. Tome un tubo de ensayo que contienen las moscas y colocarlo boca abajo sobre el embudo, el cual debe ser colocado en el agujero del tornillo. </li><li> NO SE DEBE mover el embudo, ya que esto podría perjudicar o dañar a las moscas. En cambio, suavemente golpe toda la cámara, un embudo y todos, hasta que todas las moscas dentro de la cámara. Este golpear se debe hacer en una alfombrilla de ratón para absorber el impacto de esta golpeando. Cuando las moscas están en el interior, retire el embudo, y volver a colocar la tapa de plexiglás sobre las camas de tornillo. A continuación, volver a atornillar la tapa de plexiglás en su lugar. </li></ol> Parte 4: Al realizar el análisis Locomotor <ol><li> Una vez que las moscas se han cargado correctamente, deje que se aclimate en la cámara durante 30 minutos. Este período de aclimatación debe tener lugar en el ambiente (25 º C, 70% de humedad), y la cámara debe ser colocado en la parte superior de la caja de luz (que debe estar encendido). Encienda las luces de sala de otros. </li><li> Tras el período de aclimatación, el parámetro-y de la válvula en el sistema de flujo de aire de tal manera que el aire sólo el flujo del medidor de caudal. A su vez en el aire, y la rampa de aire hasta la velocidad deseada (4.0-6.0 L / min). Por lo general, el uso 5.0-5.5 L / min, pero cualquier velocidad en ese rango de trabajo. </li><li> Una vez que la velocidad deseada se alcanza, el parámetro-y la válvula para que el aire fluye a la cámara de locomoción. Esta vez el aire de latidos durante 15 segundos, y luego, abruptamente, a su vez el flujo de aire. A medida que gira el aire apagado, conecte la cámara al modo de grabación. (Esta parte requiere de práctica para hacer todo a la vez). </li><li> Detener la grabación después de la hora deseada y guardarla. Registramos 30 en segunda a 10 cuadros por segundo (al menos 8 ensayos por genotipo). </li></ol> Parte 5: Análisis de Videos <ol><li> Usamos elImagen Sistema de Análisis Dinámico (DIAS) software 3.2 para el seguimiento del movimiento 2. Para poder utilizar este software, lo primero que convertir nuestros videos a un formato de archivo AVI con QuickTime 7.5.5. </li><li> Para realizar el seguimiento de las moscas basado en el contraste, se utiliza el &quot;Autotrace por Umbral&quot; función. A continuación, utilizar el &quot;Hacer trazado de Trace&quot; función para digitalizar estas huellas. </li><li> Para dar salida a la velocidad instantánea de cada vuelo, se utiliza el &quot;Cálculo de los parámetros de&quot; la función de producir un archivo de base de datos (DIAS específicos). Con esta función, también sin problemas los datos mediante un &quot;5,15,60,15,5&quot; Tukey ventana suavizado. </li><li> Una vez que esta información es emitida como un archivo de base de datos, se puede abrir en Microsoft Excel. Nosotros usamos un script de Matlab para compilar la velocidad instantánea en Excel y calcular los parámetros adicionales necesarios para la comprensión de la dinámica del movimiento y la estructura de combate. </li></ol> Los resultados representativos: La figura 1 muestra las huellas representante de los organismos de tipo salvaje Cantón de S (Figura 1, panel izquierdo) y las moscas nulos para el gen dCASK que fueron generados por el cruce de dos grandes deleciones superpuestas (Df (3R) X307 y Df (3R) X313) (Figura 1 , panel derecho). moscas dCASK nulos han sido demostrado que tienen problemas del aparato locomotor utilizando paradigma de Buridán 3, y en nuestro paradigma, en comparación con el Cantón de S, que muestran gran disminución de la locomoción. La humanidad siempre corre organismos de tipo salvaje primero para asegurarse de que las condiciones y el comportamiento están dentro del rango normal en un día determinado. Hemos observado desviaciones estándar de las respuestas en menos de 5% de los días de prueba. Estas diferencias en general se puede atribuir a problemas con los parámetros de nuestro entorno controlado. <img src="/files/ftp_upload/1096/figure_1.png" alt="" width="600" height="298" /> Figura 1. DIAS generado por las huellas de ambos Cantón S moscas de tipo salvaje (arriba, izquierda) y la locomotora con deficiencia de Null dCASK moscas generadas por las supresiones superposición (arriba, derecha). Las huellas representan lo que se ve en los vídeos grabados. Ambos rastros imágenes contraindicaciones 8-10 moscas funcionar durante 30 segundos en el ensayo de seguimiento de vídeo después de un pulso de aire. <img src="/files/ftp_upload/1096/figure_2.png" alt="" width="600" height="438" /> Figura 2. Moscas de tipo silvestre se realizaron en el ensayo del aparato locomotor (arriba) después de un pulso de aire (barras moradas) y sin un impulso de aire (azul). En todos los parámetros calculados por el programa de análisis, no hubo diferencias significativas entre las dos condiciones (determinado con dos colas prueba t de Student). Esto demuestra que después de un impulso de aire, las moscas de locomoción normal en nuestra configuración.

<ol>
	<li>Wolf, F. W., Rodan, A. R., Tsai, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Analysis+of+Ethanol-Induced+Locomotor+Stimulation+in+Drosophila.">High-Resolution Analysis of Ethanol-Induced Locomotor Stimulation in Drosophila.</a> J Neurosci. 22 (24), 11035-11044 (2002).</li><li>Soll, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+computers+in+understanding+how+animal+cells+crawl.">The use of computers in understanding how animal cells crawl.</a> International review of cytology. 163, 43-104 (1995).</li><li>Martin, J. R., Ollo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+Drosophila+Ca2+/calmodulin-dependent+protein+kinase+(Caki)+is+localized+in+the+central+nervous+system+and+implicated+in+walking+speed.">A new Drosophila Ca2+/calmodulin-dependent protein kinase (Caki) is localized in the central nervous system and implicated in walking speed.</a> The EMBO journal. 15 (8), 1865-1876 (1996).</li><li>Levine, J. D., Funes, P., Dowse, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+the+Circadian+Clock+by+Social+Experience+in+Drosophila+melanogaster.">Resetting the Circadian Clock by Social Experience in Drosophila melanogaster.</a> Science. 298, 2010-2012 (2002).</li></ol>

En nuestra configuración, un impulso de aire moderadamente fuerte transitoriamente detiene el movimiento de volar. Cuando el pulso termina de aire, las moscas son liberados de este estado estacionario y moverse con normalidad, como se muestra en la figura 2. Debido a que este impulso de aire efectivamente se sincroniza la locomoción de la población, lo usamos para comenzar el juicio por lo que también podemos estudiar el inicio del movimiento después de un estímulo impulso sin precedentes. El ensayo puede, sin emb...

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<td>Square Chamber</td>
<td>Tool</td>
<td>Machine Shop</td>
<td>N/A</td>
<td>Design from Wolf et al. 20021</td>
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<td>Digital Camera</td>
<td>Camera</td>
<td>Sharp</td>
<td>ViewcamZ VL-23</td>
<td>&nbsp;</td>
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<td>Flowmeter</td>
<td>Tool</td>
<td>Cole Parmer</td>
<td>SY-32003-12</td>
<td>&nbsp;</td>
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<td>Light Box</td>
<td>Tool</td>
<td>DNASTAR, Inc.</td>
<td>Seq-Easy</td>
<td>&nbsp;</td>
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<td>Charcoal Filter</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>09-744-37</td>
<td>&nbsp;</td>
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high-resolution video tracking of locomotion in adult drosophila melanogaster

Las moscas proporcionar un importante modelo para estudiar el comportamiento complejo debido a la gran cantidad de herramientas genéticas disponibles para los investigadores en este campo. Estudiar el comportamiento del aparato locomotor en<em&gt; Drosophila melanogaster</em&gt; Depende de la capacidad para poder cuantificar los cambios en marcha durante o en respuesta a una tarea dada. Por esta razón, un sistema de video de alta resolución de seguimiento, como el que se describe en este documento, es una valiosa herramienta para la medición de la locomoción en tiempo real. El protocolo incluye el uso de un pulso de aire inicial para romper el impulso moscas, seguido por un período de rodaje treinta segundos en una cámara cuadrada. Un programa de seguimiento se utiliza para calcular la velocidad instantánea de cada vuelo dentro de la cámara en incrementos de 10 ms. Software de análisis a continuación, compila estos datos, y genera una serie de parámetros como la velocidad media, velocidad máxima, tiempo en movimiento, aceleración, etc Este protocolo discutirá la alimentación adecuada y el manejo de las moscas para las tareas de conducta, manejo de moscas sin anestesia o inmovilización , la creación de un ambiente controlado y la ejecución de la prueba de principio a fin.

Watch this Scientific Journal Video about High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster at JoVE.com

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

El estudio del comportamiento complejo en el aparato locomotor<em&gt; Drosophila melanogaster</em&gt; Depende de la capacidad de cuantificar los cambios en el movimiento de una mosca dado. Este artículo muestra cómo hacer esto con un sistema de rastreo de alta resolución.

Vídeo de alta resolución de Seguimiento de la locomoción en adultos de Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

high-resolution-video-tracking-locomotion-adult-drosophila

Research

JoVE Journal

Biology

13.4K vistas.  Brandeis. El estudio del comportamiento complejo en el aparato locomotor Drosophila melanogaster Depende de la capacidad de cuantificar los cambios en el movimiento de una mosca dado. Este artículo muestra cómo hacer esto con un sistema de rastreo de alta resolución.

Video: High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

La disección del sistema nervioso central de larvas de Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Investigación

Biología

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

De alta resolución de medición del comportamiento de olor-Driven en larvas de Drosophila

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Seguimiento visual mediada olor durante el vuelo en Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

La disección de Oenocytes de Adultos Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

El aislamiento de Drosophila melanogaster Testículos

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Inyección de insulina y extracción hemolinfa para medir la sensibilidad a la insulina en adultos Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Medición de la esperanza de vida en<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

El mantenimiento de una<em&gt; Drosophila melanogaster</em&gt; Jaula Población

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

Determinación de zona óptica transversal muscular de los músculos de vuelo indirectos adultos de Drosophila Melanogaster

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Oral infección bacteriana y vertimiento en Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...