Name: high throughput assay to examine egg-laying preferences of individual drosophila melanogaster
Uploaded: 2016-03-24T13:00:00.000+00:00
Duration: 3 min 58 s
Description: Watch this Scientific Journal Video about High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster at JoVE.com

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.准备苍蝇待测定<ol><li>培养蝇在标准糖蜜/在培养箱玉米面媒体设定为25℃和65％的湿度。小心不要过度拥挤的小瓶。例如，将8名女性和男性6成窄食瓶。 注意:这里使用的"窄食小瓶"有2.3厘米内径。我们通常分配大约10毫升飞食品到每个小瓶中。我们用的飞食品配方说明如下:http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/molassesfood.html。 </li><li> 2 - 3天后雌羽化， 如图1E制备用新鲜酵母膏小瓶中。通过混合6克活性酵母用10ml 0.5％丙酸使鲜酵母膏。用刮刀酵母膏施加到食品小瓶的侧壁。 20 35 eclosed女性在一起 - - 25雄性变成小瓶收集30。 <ol><li>一般情况下，收集来自那里的女性被收集在同一小瓶男性（以便节省时间和精力）。然而，如果有关于男性的生育能力的关注，使用WT男性代替。此外，酵母泥是刺激产蛋重要的，因此每日新鲜准备。 </li></ol></li><li>保持所收集的女性/男性在25℃，湿度65％，除非苍蝇温度敏感。 </li><li>经过4〜 - 5天，检查瓶，看看女性是准备好产蛋实验。它们是准备当食物介质的表面是从幼虫在食品积极挖洞湿（ 见图1E - F）。 注:通常女性扣留产卵一次食品表面变湿和幼虫爬行。此步骤可确保女性都准备就绪时，他们遇到所需的纹理基板打好的鸡蛋（ 例如 ，〜1％琼脂糖）。 </li></ol> 2.硐室施工，组装，实验设置<ol><li>有一个加工车间建设丙烯酸产蛋室（ 网络连接古尔1A - D）。不同部分的工程制图示于图补充1 - 3高分辨率照片也可以在这里找到（http://www.rebeccayang.org/pdf/chamber%20design.pdf）。 </li><li>插入塑料片成装载（顶部）片的腔室， 如图1C所示 。这可作为底面而载入飞入个人产蛋阿里纳斯。 </li><li>麻醉于二氧化碳垫女性，单独加载它们到每个产蛋舞台。允许〜30分钟，苍蝇从二氧化碳中恢复，并成为适应新的环境。 </li><li>准备琼脂糖基板。 <ol><li>为方便起见，继续融化的1％琼脂糖预制瓶在55℃水浴。 </li><li>添加的库存蔗糖溶液（2M）的所需量到50毫升锥形管，并与琼脂糖适量混合。例如，以制备150mM的蔗糖基板，PLACE 2M的蔗糖溶液进入管750微升，然后用琼脂糖填充管10 ml刻度线。 </li><li>制备中相同的方式在滑动基底但添加代替蔗糖溶液蒸馏水。 注意:在这个协议琼脂糖的最终浓度略小于1％。 1.1％，而在两个基板是相同的琼脂糖浓度 - 在我们的经验，琼脂糖的确切浓度不只要其被控制为〜0.9内无关紧要。 </li></ol></li><li>就拿基板（底）片室和吸管1000μl琼脂糖基板到每个槽， 如图1D所示。 </li><li>允许琼脂糖巩固约30分钟。 </li><li>一旦琼脂糖基板和苍蝇都准备好了，组装所有三块产蛋室，然后取出塑料片。 </li><li>放置在室内飞孵化器。 注:产蛋实验长度可根据EXPE变化rimental需求。我们通常运行实验O / N（14 - 16小时）。此外，观察昼夜定时上产卵的喜好没有显著影响。 </li><li>通过注入的 CO 2进入室麻醉雌性。拆卸腔，放弃麻醉苍蝇飞入太平间（ 即 ，一个空的咖啡能充满了一些玉米油）。就拿结果记录保存的照片（ 见图2）。 </li><li>手动计数卵的数目，并计算偏好指数分析。计算偏好指数（N A - N B）/（N A + N B），其中N 一和N B上分别代表蛋的数量在现场与网站B，。 </li></ol>

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The authors have nothing to disclose.

该商会在这里描述的协议具有比以前产蛋测定了一些改进。首先，它们提高显著测定单个动物的偏好的吞吐量。每个房间都可以分析30单身女性，它需要不到一个小时来设置。第二，它们增加超过以前的方法产蛋偏好的一致性。竞技场的尺寸的标准化，产蛋基板尺寸，和基片之间的距离可以更容易地不同实验天之间，并从不​​同的研究小组比较结果。第三，腔室可以配备附件以容纳更多的分析?...

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

近日， 果蝇产卵偏好已经成为一个听话的基因模型，研究简单的决策过程的神经基础。当选择的网站存入他们的蛋，雌蝇能居其选择的相对吸引力和选择的"较大的两种商品。"然而，如果一个人想采取系统的遗传筛选方法的搜索电路基础底层这个简单的决策过程，因为它们是基于人群的和费力的建立最产蛋偏好测定法是不实际的。为了增加单个雌性产卵偏好学习的吞吐量，我们开发了长达三十个人的每个可以同时测定产蛋偏好蝇以及确保每个女性具有高产蛋率的协议自定义腔室（使他们的偏好是容易辨别和更有说服力）。我们的方法是简单的执行并产生非常一致的结果。此外，这些室可以配备不同的附件，以允许视频记录产蛋动物，并为光遗传学研究提供光。本文提供了制造这些商会和程序准备在这些商会进行检测苍蝇的蓝图。

产蛋室由几个部分组成:一基底（底）片，一个分频器（中间）片，装载（顶部）片，和2滑动门（ 图1A - D）。这些作品被用于前产蛋实验独立设置苍蝇和基板。 图1F显示了当雌性果蝇准备好产蛋瓶的外观。当苍蝇给出一个普通基材和含有蔗糖的衬底之间的选择，女性鲁棒优选平原基板产蛋如图2。 <p class="jove_content" fo:keep-together.withi...

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

高通量检测，以检查个体产蛋首<em&gt;果蝇</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. ...

high-throughput-assay-to-examine-egg-laying-preferences-individual

Research

JoVE Journal

Neuroscience

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

7.9K 次观看.  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.

视频: 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.

对检测方法果蝇行为

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

科研

神经科学

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.

光偏好的检测，以研究的先天和昼夜调节感光特性<em&gt;果蝇</em&gt;幼虫

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.

单苍蝇检测方法的觅食行为的<em&gt;果蝇</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.

果蝇黑色素血脑屏障完整性分析

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

发育生物学

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

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.

测量并改变配套驱动马累<em&gt;果蝇</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.

温度梯度法测定果蝇幼虫的热偏好

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.

果蝇的快速食物偏好分析

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

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.

产卵测定:一种量化秀丽隐杆线虫产卵行为的方法

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. 
...

JoVE Encyclopedia of Experiments

生物学

秀丽隐杆线虫（线虫）

基本方法

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:一种评估果蝇运动行为的高通量方法

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

高通量检测，以检查个体产蛋首

摘要

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此视频中的章节

对检测方法果蝇行为

光偏好的检测，以研究的先天和昼夜调节感光特性

单苍蝇检测方法的觅食行为的

果蝇黑色素血脑屏障完整性分析

一个行为分析对机械感觉MARCM为基础的克隆

测量并改变配套驱动马累

温度梯度法测定果蝇幼虫的热偏好

果蝇的快速食物偏好分析

产卵测定:一种量化秀丽隐杆线虫产卵行为的方法

Island Assay:一种评估果蝇运动行为的高通量方法