Este trabalho foi financiado pelo National Institutes of Health Grant R01 GM54408 concedido a LC Griffith. Gostaríamos de agradecer a Fred Wolf para toda a sua ajuda na elaboração de nosso quarto quadrado e criação de nosso ensaio, Frank Mello na loja de máquina Brandeis University para a construção de nossa câmara, e Dan Valente e Tim Lebestky para conversas úteis em relação às questões de análise.

Parte 1: Alimentação e Gestão de moscas <ol><li> Moscas devem ser cultivadas em frascos contendo levedura livre de mídia padrão. Você vai precisar de um grande número de moscas, por isso atravessa precisa ser configurado de acordo. Moscas devem ser cultivadas em uma de 12 horas luz: ciclo escuro a 25 º C. </li><li> Moscas devem ser coletadas logo após a eclosão (1-3 dias). Moscas podem ser trabalhados com nesta fase usando um difusor de dióxido de carbono, e devem ser classificados em tubos de ensaio contendo meio autoclavado ou yeast-free. Usamos somente machos, armazenados de 10 a um tubo de ensaio. </li><li> Permitir que pelo menos um ou dois dias após o uso de dióxido de carbono antes de executar os experimentos comportamentais. Corremos voa em nosso ensaio comportamental 3-5 dias após a eclosão. </li><li> Ensaios deve ser sempre feito dentro da mesma janela de tempo de 2-3 horas por dia para evitar problemas de ritmo circadiano. </li></ol> Parte 2: Configurando o sistema de rastreamento em um ambiente controlado <ol><li> Todos os experimentos são feitos em uma sala de ambiente que mantém uma temperatura constante de 25 º C com umidade de 70%. </li><li> Suspender uma câmera de vídeo digital sobre a área a ser gravada (câmera virada para baixo). Desde o nosso software de rastreamento é baseado em contraste, nós suspendemos a câmera sobre uma caixa de luz. Registros os vídeos da câmera diretamente em um computador Dell através de uma conexão firewire. Nós usamos uma câmera de vídeo da Sharp digital, ligado a um computador Dell com o Windows Moviemaker (versão Vista) para aquisição de vídeo. </li><li> Pulsos de ar será administrado nesse protocolo, portanto, uma fonte de fluxo de ar deve estar presente na sala. Usando tubos de borracha, conecte a fonte de ar para um filtro de carbono para a filtração de ar. </li><li> Usando tubos de borracha mais, conecte o outro lado do filtro de carbono para um Erlenmeyer através de uma rolha de borracha oco, e encher o balão com cerca de meia polegada de água. Isto irá umidificar o ar. </li><li> O frasco também deve ter uma arma, que vai ligar a uma válvula em forma de Y através de tubos de borracha. </li><li> Um ramo do Y-válvula deve ser conectada a um fluxômetro. O outro ramo deve fornecer ar à câmara de locomoção quadrados. </li></ol> Parte 3: Disponibilizar voa para a câmara de locomoção <ol><li> A câmara de locomoção quadrado é baseado no projeto de Wolf et al. 2002 1, com a adição de bandejas pequena queda para impedir que as moscas de voar dentro da câmara. A câmara deve ser desmontado, e todos os componentes devem ser cuidadosamente limpo com álcool 70% e secadas antes de usar. </li><li> Anestesia imediatamente antes de realizar um ensaio de comportamento pode comprometer o desempenho. Para evitar isso, as moscas são gentilmente bateu para dentro da câmara usando um funil com uma pequena saída. Para fazer uma tomada do tamanho certo, coloque uma ponteira azul (para P1000) até o fim de um funil. Use uma tesoura para cortar o final da ponta da pipeta para fazer a abertura grande o suficiente para uma mosca passar. </li><li> No topo da nossa câmara é um pequeno pedaço de plexiglass garantidos por parafusos em torno das bordas. Quando os parafusos são removidos, os buracos podem ser usados ​​para entregar voa para a câmara. Posicione a parte superior de acrílico para que o orifício do parafuso fica diretamente sobre a câmara interna em vez de um parafuso, onde normalmente se senta. Pegue um tubo de ensaio contendo as moscas e colocá-lo de cabeça para baixo sobre o funil, que deve então ser colocado no orifício do parafuso. </li><li> Não mova o funil, pois isso poderia prejudicar ou danificar as moscas. Em vez disso, gentilmente bater toda a câmara, funil e todos, até que todas as moscas estão no interior da câmara. Este batendo deve ser feito em um mouse pad para absorver o choque deste batendo. Quando as moscas estão dentro, remova o funil, e re-posição superior a plexiglass sobre as camas parafuso. Em seguida, re-parafuso superior do plexiglass no lugar. </li></ol> Parte 4: Executando o Ensaio Locomotor <ol><li> Uma vez que as moscas são devidamente carregado, deixá-los se aclimatar na câmara por 30 minutos. Este período de aclimatação deve ocorrer em ambiente controlado (25 º C, umidade 70%), ea câmara deve ser colocada no topo da caixa de luz (que deve ser ligado). Acender as luzes outro quarto fora. </li><li> Após o período de aclimatação, mudar o Y-válvula no sistema de fluxo de ar de tal forma que o ar só de fluxo para o medidor de vazão. Ligue o ar, o ar e rampa até a velocidade desejada (4,0-6,0 L / min). Geralmente usamos 5,0-5,5 L / min, mas qualquer velocidade nessa faixa vai funcionar. </li><li> Uma vez que a velocidade desejada seja atingida, mudar o Y-válvula de modo que o ar flui para a câmara de locomoção. Esse tempo de pulso de ar por 15 segundos, e então abruptamente desligar o ar desligado. Como ligar o ar desligado, ligue a câmera ao modo de gravação. (Esta parte requer prática para fazer todas de uma vez). </li><li> Parar a gravação após a hora desejada e SAVE. Nós registramos 30 ensaios segundo em 10 quadros por segundo (pelo menos 8 ensaios por genótipo). </li></ol> Parte 5: Análise de Vídeos <ol><li> Usamos oSistema de Análise de dinâmica de imagem (DIAS) software 3.2 para o movimento de rastreamento 2. , A fim de usar este software, primeiro converter os nossos vídeos para um formato de arquivo AVI com o Quicktime 7.5.5. </li><li> Para acompanhar as moscas com base em contrário, usamos o &quot;AutoTrace pela Threshold&quot; função. Em seguida, usamos o &quot;Caminho Faça da Trace&quot; função para digitalizar esses traços. </li><li> Para a saída a velocidade instantânea de cada voar, usamos o &quot;Compute Parâmetros&quot; função de produzir um arquivo de banco de dados (DIAS-específicas). Usando esta função, também suavizar a dados usando um &quot;5,15,60,15,5&quot; Tukey Janela Smoothing. </li><li> Uma vez que esta informação é passada como um arquivo de banco de dados, ele pode ser aberto no Microsoft Excel. Nós usamos um script Matlab para compilar as velocidades instantânea dentro de excel e calcular parâmetros necessários para a compreensão dinâmica do movimento e da estrutura luta. </li></ol> Resultados representativos: A Figura 1 mostra os vestígios de organismos representativos tipo selvagem Canton S (Figura 1, painel esquerdo) e moscas nulo para o gene dCASK que foram gerados pelo cruzamento de duas grandes deleções sobrepostas (Df (3R) X307 e Df (3R) x313) (Figura 1 , à direita do painel). moscas nula dCASK já sido demonstrado que têm problemas de locomoção usando Paradigma de Buridan 3, e no nosso paradigma, em comparação com Canton S, eles mostram grande diminuição de locomoção. Corremos sempre organismos do tipo selvagem para ter certeza de que as condições e comportamento estão dentro da faixa normal em um determinado dia. Temos observado desvios padrão respostas em menos de 5% dos dias de testes. Essas diferenças podem normalmente ser atribuídas a problemas com os parâmetros de nosso ambiente controlado. <img src="/files/ftp_upload/1096/figure_1.png" alt="" width="600" height="298" /> Figura 1. DIAS gerado traços de ambos Canton S moscas do tipo selvagem (acima, à esquerda) e Locomotor deficiente Null dCASK moscas gerados a partir de exclusões sobrepostas (acima, à direita). Os traços representam o que é visto nos vídeos gravados. Ambos os traços imagens contrain 8-10 moscas executado por 30 segundos no teste de monitoramento de vídeo após um pulso de ar. <img src="/files/ftp_upload/1096/figure_2.png" alt="" width="600" height="438" /> Figura 2. Moscas tipo selvagem foram executados no ensaio locomotor (acima), após um pulso de ar (barras de roxo) e sem um pulso de ar (azul). Em todos os parâmetros calculados pelo programa de análise, não houve diferenças significativas entre as duas condições (determinado com bicaudal estudante t-teste). Isso demonstra que, após um pulso de ar, voa locomover normalmente em nossa configuração.

<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>

Em nossa configuração, um pulso de ar moderadamente forte transitoriamente pára movimento voar. Quando o pulso de ar termina, as moscas são liberados a partir deste estado estacionário e locomover normalmente, como mostrado na figura 2. Porque este pulso de ar efetivamente sincroniza a locomoção da população, nós usá-lo para iniciar o estudo para que possamos também estudar o início do movimento após um estímulo impulso quebrar. O ensaio pode, no entanto, ser feito sem um pulso de ar desde parâmetros de ...

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high-resolution video tracking of locomotion in adult drosophila melanogaster

Moscas fornecer um importante modelo para estudar o comportamento complexo devido à multiplicidade de ferramentas genéticas disponíveis para pesquisadores da área. Estudar o comportamento locomotor em<em&gt; Drosophila melanogaster</em&gt; Depende da capacidade de ser capaz de quantificar as mudanças no movimento durante ou em resposta a uma dada tarefa. Por esta razão, uma de alta resolução sistema de monitoramento de vídeo, como a que descrevemos neste artigo, é uma valiosa ferramenta para medir locomoção em tempo real. Nosso protocolo envolve o uso de um pulso de ar inicial para quebrar o ímpeto moscas, seguido por um período de filmagem trigésimo segundo em uma câmara quadrados. Um programa de rastreamento é então usado para calcular a velocidade instantânea de cada voar dentro da câmara em incrementos de 10 ms. Software de análise, em seguida, compila estes dados, e saídas de uma variedade de parâmetros como velocidade média, velocidade máxima, tempo gasto em movimento, aceleração, etc Este protocolo irá discutir alimentação adequada e manejo de moscas para tarefas comportamentais, manuseio voa sem anestesiados ou imobilização , a criação de um ambiente controlado, e correndo o ensaio do início ao fim.

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

O estudo do comportamento locomotor complexos em<em&gt; Drosophila melanogaster</em&gt; Depende da capacidade de quantificar as mudanças no movimento de uma mosca dado. Este artigo demonstra como fazer isso usando um sistema de rastreamento de alta resolução.

De alta resolução de vídeo de Rastreamento Locomotion em Adult 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 visualizações.  Brandeis. O estudo do comportamento locomotor complexos em Drosophila melanogaster Depende da capacidade de quantificar as mudanças no movimento de uma mosca dado. Este artigo demonstra como fazer isso usando um sistema de rastreamento de alta resolução.

Vídeo: 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.

Dissecção do CNS Larval em 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 ...

Biologia

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 resolução de Medição do Comportamento Odor-Driven em 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.

Visualmente Rastreamento Odor Mediated Durante o Voo em 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.

Dissecção da 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.

Isolamento de 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 ...

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.

Injeção de insulina e Extração de hemolinfa para medir a sensibilidade à insulina em 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.

Medição do Tempo de vida em<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.

Manutenção de um<em&gt; Drosophila melanogaster</em&gt; Gaiola População

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.

Determinação de área óptica transversal muscular dos músculos de voo indirecto adulto 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.

Infecção bacteriana e oral Shedding em 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 ...