We thank the Duke Physics Shop, especially Phil Lewis, for helping us build the behavioral apparatus and attachments and creating the drawing. This work is funded by the National Institutes of Health under award number R01GM100027.

1. Preparar Flies a ser testada <ol><li> Cultura voa em melaço padrão / media farinha de milho na incubadora a 25 ° C e 65% de humidade. Tome cuidado para não sobrecarregar os frascos. Por exemplo, colocar 8 mulheres e 6 homens para um frasco de alimentos estreita. Nota: O &quot;comida frasco estreito&quot; usado aqui tem um diâmetro interno de 2,3 cm. Nós normalmente dispensados ​​cerca de 10 ml de alimentos voar em cada frasco. A receita mosca alimentos que usamos é descrito aqui: http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/molassesfood.html. </li><li> 2 - 3 dias após a eclosão do sexo feminino, preparar frascos com pasta de levedura fresca, como mostrado na Figura 1E. Fazer a pasta de levedura fresco por mistura de 6 g de fermento activo com 10 ml de ácido proprionic 0,5%. Usar uma espátula para aplicar a pasta de levedura para a parede lateral dos frascos de alimentos. Colete 30 - 35 fêmeas eclosed juntamente com 20 - 25 machos em frascos. <ol><li> Em geral, recolher os machos de as mesmas frascos em que as fêmeas são recolhidos(De forma a poupar tempo e esforço). No entanto, se há uma preocupação com a fertilidade dos machos, use machos WT vez. Além disso, pasta de levedura é importante para estimular a produção de ovos, portanto, prepará-lo diariamente. </li></ol></li><li> Manter as fêmeas recolhidas / machos a 25 ° C, 65% de humidade, a menos que as moscas são sensíveis à temperatura. </li><li> Depois de ~ 4 - 5 dias, verifique os frascos para ver se as fêmeas estão prontas para experimentos de postura de ovos. Eles estão prontos quando a superfície do meio alimentar está molhado a partir de larvas escavando ativamente na alimentos (ver Figura 1E - F). Nota: As fêmeas geralmente reter colocar ovos uma vez a superfície do alimento torna-se molhado e cheio de larvas. Essa etapa assegura que as fêmeas estaria pronto para colocar muitos ovos quando se deparam com substratos com textura desejada (por exemplo., ~ 1 agarose%). </li></ol> 2. Câmara de Construção, Montagem, Configuração Assay <ol><li> Tem uma loja de máquina construir câmaras poedeiras acrílico (Figura 1A - D). O desenho de engenharia de diferentes peças foram mostrados em Suplementar Figura 1 -. 3 imagens de alta resolução também pode ser encontrada aqui (http://www.rebeccayang.org/pdf/chamber%20design.pdf). </li><li> Inserir folhas de plástico em carregamento (topo) pedaço da câmara, como mostrado na Figura 1C. Isso serve como a superfície inferior durante o carregamento voa em arenas individuais de postura de ovos. </li><li> Anestesiar fêmeas em uma almofada de CO 2 e carregá-los individualmente em cada arena de postura. Permitir ~ 30 min para as moscas para se recuperar do CO 2 e para se aclimatar ao novo ambiente. </li><li> Prepare os substratos de agarose. <ol><li> Por conveniência, manter uma garrafa de premade derretido 1% de agarose, num banho de água a 55 ° C. </li><li> Adicionar a quantidade desejada de solução de sacarose (2 M) em um tubo de 50 ml e misturando-a com a quantidade apropriada de agarose. Por exemplo, para preparar o substrato de sacarose 150 mM, PLAce 750 ul de uma solução 2 M de sacarose para dentro do tubo e, em seguida, encher o tubo com agarose para a marca de 10 mL. </li><li> Prepara-se o substrato liso, da mesma maneira, mas adicionar água destilada em vez da solução de sacarose. Nota: A concentração final de agarose, este protocolo é ligeiramente inferior a 1%. Na nossa experiência, a concentração exacta de agarose não interessa, desde que ele é controlado para estar dentro de ~ 0,9-1,1% e que os dois substratos são da mesma concentração de agarose. </li></ol></li><li> Aqui a peça do substrato (em baixo) da câmara de pipeta e 1000 ul de substrato de agarose em cada calha como pode ser visto na Figura 1D. </li><li> Permitir agarose para solidificar para ~ 30 min. </li><li> Uma vez que os substratos de agarose e moscas estão prontos, montar todas as três peças da câmara de postura e em seguida, retire as folhas de plástico. </li><li> Coloque as câmaras em incubadoras de voar. Nota: O comprimento de experimentos de postura de ovos pode variar dependendo expenecessidades rimental. Nós normalmente executar o experimento O / N (14-16 h). Além disso, não se observou qualquer impacto significativo de temporização circadiana nas preferências de postura de ovos. </li><li> Anestesiar fêmeas por injecção de CO 2 para dentro da câmara. Desmontar a câmara, descartar as moscas anestesiadas em necrotério mosca (ie., Um café vazia pode preenchido com um pouco de óleo de milho). Tire fotos dos resultados para manutenção de registros (ver Figura 2). </li><li> Contar o número de ovos manualmente e o cálculo dos índices de preferência para análise. Calcule preferência índice como (N a - N b) / (N a + N b) onde N a e N b representam número de ovos no local um site vs. b, respectivamente. </li></ol>

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	<li>Perisse, E., Burke, C., Huetteroth, W., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shocking+revelations+and+saccharin+sweetness+in+the+study+of+Drosophila+olfactory+memory.">Shocking revelations and saccharin sweetness in the study of Drosophila olfactory memory.</a> Curr. Biol. 23 (17), 752-763 (2013).</li><li>Yamamoto, N. C., Koganezawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+circuits+of+courtship+behaviour+in+Drosophila+males.">Genes and circuits of courtship behaviour in Drosophila males.</a> Nat. Rev. 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The authors have nothing to disclose.

As câmaras e protocolos aqui descritos têm várias melhorias em relação ensaios poedeiras anteriores. Em primeiro lugar, aumentar a taxa de transferência de doseamento preferências dos animais individuais de forma significativa. Cada câmara pode testar 30 fêmeas individuais e leva menos de uma hora de configurar. Em segundo lugar, eles aumentam a consistência das preferências de postura em relação aos métodos anteriores. A padronização das dimensões da arena, o tamanho dos substratos de postura e distân...

Drosophila melanogaster is a powerful genetic model organism to study the neural basis of behaviors. The rapid developments of genetic tools to manipulate neurons in a targeted manner and the emergence of sophisticated behavioral analysis tools have significantly improved our ability to dissect the circuit mechanisms that underlie the sensory-motor transformation processes of several innate and learned behaviors1-3.
Drosophila egg-laying is a suitable model to study the neural basis of simple decision making processes. In particular, Drosophila females have been shown to possess the ability to compare and rank their options before "committing" to depositing an egg onto a given option4-8. For example, when given only a plain (sucrose-free) substrate or only a sucrose-containing substrate, females readily accept either option for egg-laying. However, when presented with both options, females robust reject the sucrose substrate in some contexts7,9,10. Relatively little is known about the neural mechanisms that allow females to "choose the greater of two goods", however. A major obstacle has been the lack of an efficient method to assay egg-laying preferences such that one can use a systematic genetic screening approach to study this problem.
In this report, we describe the protocol we developed that allows egg-laying preferences of females to be assayed at single-animal resolution and with substantially improved throughput and consistency over previous methods. Specifically, we provide the blueprints for constructing the chambers we designed, the protocol for preparing the females so that each is primed to lay many eggs, and the protocol for using the chambers.

<table><tbody><tr><td>UltraPure Agarose&amp;nbsp;</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Water bath&amp;nbsp;</td><td>Fisher</td><td>15-462-6Q</td><td></td></tr><tr><td>LifeCam Cinema webcam</td><td>Microsoft</td><td>H5D-00013</td><td></td></tr><tr><td>Red LEDs</td><td>Cree</td><td>C503B-RAN-CA0B0AA1</td><td></td></tr><tr><td>Egg-laying chambers</td><td>Custom Built</td><td></td><td></td></tr><tr><td>Camera holders</td><td>Custom Built</td><td></td><td></td></tr><tr><td>LED holders</td><td>Custom Built</td><td></td><td></td></tr><tr><td>Fly vials (narrow)</td><td>Genesee</td><td>32-116BC</td><td></td></tr></tbody></table>

high throughput assay to examine egg-laying preferences of individual drosophila melanogaster

Recentemente, a preferência de postura de Drosophila tem emergido como um modelo geneticamente tratável para estudar a base neural dos processos de tomada de decisão simples. Ao selecionar locais para depositar seus ovos, as moscas fêmeas são capazes de classificar a atratividade relativa das suas opções e escolher o &quot;maior dos dois bens.&quot; No entanto, a maioria dos ensaios de preferência poedeiras não são práticos quando se quer ter uma abordagem sistemática rastreio genético para procurar a base circuito subjacente a este processo de tomada de decisões simples, como elas são e trabalhoso para configurar base populacional. Para aumentar a taxa de transferência de estudar de preferências de postura de ovos das fêmeas individuais, desenvolvemos câmaras personalizados que cada um pode simultaneamente ensaio preferências de postura de até trinta indivíduo voa, bem como um protocolo que garante a cada fêmea tem uma alta taxa de postura (de modo a que a sua preferência é facilmente discernível e mais convincente). Nossa abordagem é simples de executare produz resultados muito consistentes. Além disso, estas câmaras podem ser equipados com acessórios diferentes para permitir a gravação de vídeo os animais que põem ovos e fornecer luz para estudos optogenética. Este artigo fornece os planos para fabricar estas câmaras e ao processo de preparação das moscas a ser analisadas nestas câmaras.

As câmaras de postura de ovos são compostos de várias peças: um pedaço de substrato (inferior), um pedaço divisor (meio), uma carga (parte superior) peça, e 2 portas de correr (Figura 1A - D). Estas peças são usadas para independentemente moscas configuração e substratos antes experimentos poedeiras. Figura 1F mostra como frascos devem olhar quando as moscas fêmeas estão prontas para postura de ovos. Quando as moscas recebem uma escolha entr...

Watch this Scientific Journal Video about High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster at JoVE.com

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

High Throughput Ensaio para examinar Preferências de postura de Pessoa<em&gt; Drosophila melanogaster</em

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

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Neuroscience

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

7.9K visualizações.  Duke University. This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Vídeo: High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Métodos de Ensaio para Drosophila Comportamento

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Neurociência

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Luz Ensaio Preferências para estudar inata e circadiano Photobehavior Regulamentado<em&gt; Drosophila</em&gt; As larvas

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

A Single-fly Ensaio para comportamento de forrageamento em<em&gt; Drosophila</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Ensaio para a integridade da barreira hematoencefálica na Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Biologia do Desenvolvimento

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Medir e alterando Acasalamento Drive em Male<em&gt; Drosophila melanogaster</em

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Um gradiente de temperatura de ensaio para determinar preferências térmicas de larvas de Drosophila

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

Um ensaio rápido de preferência alimentar em Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

Fly Climbing Assay: A Method to Test General Locomotion in Drosophila melanogaster

Source: Gevedon, O., et al. <a href="https://www.jove.com/video/59720/in-vivo-forward-genetic-screen-to-identify-novel-neuroprotective">In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes a simple method to quantify fly locomotion and shows the climbing assay performed.&#160; The fly climbing assay is a general test of motor function which relies on a negative geotaxis response.

Ensaio de escalada com mosca: um método para testar a locomoção geral em Drosophila melanogaster

Source: Gevedon, O., et al. In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster. J. Vis. Exp. (2019).
This ...

JoVE Encyclopedia of Experiments

Biologia

Drosophila melanogaster (mosca da fruta)

Adulto

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

Source: Lee, S. Y. and Kang, K. <a href="https://www.jove.com/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2017).
C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.&#160; This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Ensaio de postura de ovos: um método para quantificar o comportamento de postura de C. elegans

Source: Lee, S. Y. and Kang, K. Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. 
...

Caenorhabditis elegans (nematódeo)

Metodologias Básicas

Island Assay: A High-Throughput Method to Evaluate Drosophila Locomotor Behavior

Source: Eidhof, I., et al. <a href="https://www.jove.com/video/55892/" target="_blank">High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay</a>. J. Vis. Exp. (2017).
This video describes the island assay, a behavioral method used to study Drosophila locomotion. The sample protocol features a setup for the assay compatible with semi-automated image analysis.

Island Assay: Um método de alto rendimento para avaliar o comportamento locomotor de Drosophila

Source: Eidhof, I., et al. High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay. J. Vis. Exp. (2017).
This video describes the ...

High Throughput Ensaio para examinar Preferências de postura de Pessoa

Resumo

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