We are grateful to anonymous reviewers for comments and suggestions that greatly improved this manuscript. We thank R. Bowes, R. Carreno, and T. Panhuis for assistance in collecting the fish. We also thank M. Lee for assistance with male preference trials. We are grateful to the Ohio Wesleyan University Department of Zoology for helpful advice and suggestions throughout this study and Arizona State University (McGraw Lab) for software advice.

所有实验均与俄亥俄卫斯理大学的机构动物护理和使用委员会的批准进行的。 用分光光度计鱼1.记录反射紫外光<ol><li>校准分光光度计和光源与已知白色标准以上的波长范围，以根据仪器或软件的建议来测定。 </li><li>通过在3-氨基苯甲酸乙酯甲磺酸盐的0.5％溶液放置麻醉鱼（MS-222）的缓冲用碳酸氢钠的等量直到鱼变得无法响应。一旦反应迟钝，立即将鱼放在一个黑色，无反射的背景。注:MS-222是有潜在毒性和手套应始终佩戴。 </li><li>拿在黑暗的房间所有的测量，以尽量减少多余光线超出了光谱仪的光源。 </li><li>使用针对生物体的体保持的光纤探针测量的反射率。使一定要形成光纤和所述主体之间的一致的45°角，如内置于一些探针的设计。注意:对于测量的一致性，可能希望在多个扫描（2 - 5）平均每身体区域。 </li><li>适当的反射分析软件记录反射率数据。注意:此处所使用的软件被设置为:积分时间= 2,000秒，平均扫描= 2，和像素平滑= 5的数据被复制并粘贴到电子表格。 </li><li>重复多个身体部位以确定有无反射紫外光和整个生物表型变异的范围。注意:初步扫描可能是必要的，以确定，如果有的话，身体区域一致显示紫外线标记。 </li><li>返回鱼良好的氧化控股水族馆，直到完全康复，由正常厣节奏和游泳的行为就是证明。注意:商业防护剂恢复粘膜涂覆到主体可以加入在皮肤恢复来帮助。 </li><li>众议员在多个个人吃的，包括如果需要男女双方。 </li><li>上，其中所有的数据都用散点图被复制，绘图数据，与在Y轴和波长上的X轴反射率的电子表格。在UV范围内幅度峰值（300 - 400纳米）表示反射紫外光对鱼的存在。 </li></ol> 2.二分交配试验注:执行控制的观察，其中焦鱼，UV过滤，和不透明滤波器到位。在这些试验中，在不透明过滤除去时，焦鱼将暴露于显示UV光的存在和不存在，但缺乏潜在交配伙伴罐部分。有关的分选择测试在本实验中的有效性详见Palmer和汉克森（2015）10，虽然注意到这些试验也有缺点11。 <ol><li>使用性接受个人作为焦点的有机体。使用女性P. latipinna &lt;48小时产后5次测试，以改善性动机的可能性之前，分离为至少24小时男性。 </li><li>将单焦点个体到相邻两个选择车厢中立地区。对于P. latipinna，使用75.7升试验水族馆（30.5厘米×30.5厘米×75厘米; 图1）由透明有机玻璃分区分为三段，形成了焦点个别由两个大小相等的选择隔间两侧的中心区域。确保没有水或嗅觉线索各个槽部之间共享。重复被观察每个个体。 </li></ol><img alt="图1" src="/files/ftp_upload/54453/54453fig1.jpg" /> 图1.实验水族馆设置一个长方形的水族馆分为三个部分:即持有焦点个人和持有的对象对两端车厢的中心区域（图中显示了与在两端对象男性中间有一个女）。可互换FILTER值，要么阻止或允许的UV反射率可以被放置在分割水族箱的男性和女性的部分中的玻璃隔板。这个数字是从Palmer和汉克森（2015年）10修改。 <a href="http://ecsource.jove.com/files/ftp_upload/54453/54453fig1large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <ol start="3"><li>在全谱照明条件（照明，其频谱延伸到UV-A范围），隔离在两端车厢（选择车厢）测试水族馆一个潜在的交配伙伴。确保潜在交配伙伴在大小，着色和显示特性相似彼此尽可能接近，使两个分离的配合伙伴之间的主要区别是它们是否出现一个滤波器，其阻止或传输紫外光后面。 注意:这可能是优选的以记录的反​​射率在可见光谱，以使可能的最佳色彩匹配，人虽然这里匹配用眼进行。 </li><li>适应视觉下的隔离（不透明过滤器）的个人15分钟。确保UV +和 - 过滤器在发生在这个时候。继驯化，去除不透明的过滤器。以确保侧偏差不存在（对于水箱的一侧，无论对象的固有偏好），随机地分配在UV +和紫外-槽的两侧的每个每次试验。 </li><li>通过使用秒表记录的时间的聚焦个人花费内的特定偏好区域（邻近的选择隔室区域，在这种情况下，大约两标准体长度）的量。对于女性的鱼，这是男性焦鱼它花费近每两个男性的时间量，而反之亦然。 注:事件记录器软件也可以用于记录。十五分钟试验在这个实验和鱼没有访问至少一次被排除在结果中的每一首区作为是使用反应迟钝。 </li><li>记录和分析鱼花近空UV +和UV的时间-隔室，以确保没有向可能影响交配选择鱼的一个特定的照明环境的固有偏差。使用配对t检验以确定是否个人偏好的特定紫外线条件。 </li></ol>

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The authors have nothing to disclose.

分光光度法成功地确定对P. UV标志latipinna。 P的两性latipinna具有沿其两侧UV标记。此外，一些男性对他们背鳍紫外标记，先前发现的性状是在雌性交配偏好7重要。 我们建议使用紫外分光光度法作为一种机制来检测紫外线标志的存在。进一步的测试可能确定其在社会互动，包括交配偏好（如这里详细介绍）的作用。可替代地，紫外线特性可能?...

了解线索，并在动物社会交往使用的信号可以让我们内部和物种之间理解的表型变异。此变化起着进化过程如人口发散，性选择，和形态的重要作用。通常，然而，研究人员正在限于探索最明显的人类感觉系统，特别是那些在视觉或听觉领域内的提示。光度法的使用，然而，使我们能够扩展超出人类可见光谱并进入波长可能在其他物种的社会互动重要我们的调查。 特别是，通过紫外线带来的短距离通信（UV; 300 - 400纳米）的灵敏度必须是择偶1中非常有利的可能性。鸟类和鱼类的许多视觉捕猎的食肉动物，比如，无法检测紫外线辐射。在男性精心显示系统到女性，这些男性会减少他们的捕食的风险，同时保持其通过利用UV光谱，而不是开发线索可检测在可见光谱2,3到吸引异性能力。如果一个发生故障考虑到生物体与每个通信的可能性其他使用这些"私人沟通渠道"，行为和进化显著驱动程序可能会被错过。 该协议概述了调查的茉莉花鳉， 孔雀鱼latipinna，已经没有先前已知的检测UV或利用UV标记能力的一夫多妻鱼使用UV线索的择偶。该鱼种有着密切的亲缘接近其它紫外线敏感的活轴承鱼4和有显微光谱证据表明P. latipinna，与其它茉莉物种如铜绿沿墨西哥和P.台塑 ，拥有一流的视锥细胞（负责统筹感光细胞LOR视觉）是最敏感的UV波长5。在这种性别二态性的物种，女性选择了在男性&#39;颜色鲜艳和扩大散热片6-9的进化中发挥了强有力的作用。这种方法使我们能够探索紫外线是由女性评估男性的质量的附加网上平台。 检测和P.紫外标记测量latipinna使用分光光度计用光纤探头这里详细描述。此外，接受女性mollies是否差异通过光学滤镜观察男性发射全光谱光源包括UV-A关联（[UV +]; 320 - 700纳米），并通过UV阻隔滤镜观察男性（[紫外线]; 400 - 700纳米）进行了讨论。该方法具有用于发现在鱼类和其他生物的UV敏感性和彩色图案，允许研究多种方法包括紫外线和其行为的作用的问题广泛的应用。

<table><tbody><tr><td>Spectrophotometer, P1000</td><td>Ocean Optics</td><td></td><td>newer models are availabe</td></tr><tr><td>DT 1000 xenon UV light source</td><td>Ocean Optics</td><td></td><td>newer models are availabe</td></tr><tr><td>Ocean Optics Overture Software</td><td>Ocean Optics</td><td></td><td>newer software is available</td></tr><tr><td>R200-Angle-UV bifurcated fiber-optic probe (Guided Wave)</td><td>Ocean Optics</td><td></td><td>newer models are availabe</td></tr><tr><td>Certified reflectance standard, white</td><td>labsphere</td><td></td><td></td></tr><tr><td>75.7 L Aquarium, divided</td><td>Experimental Builder</td><td></td><td></td></tr><tr><td>Full Spectrum Bulb</td><td>Nature&#039;s Sunlight</td><td></td><td></td></tr><tr><td>UV blocking sheet</td><td>GAM UV Sheet</td><td></td><td></td></tr></tbody></table>

assays to detect uv-reflecting structures and determine their importance in mate preference using the sailfin molly poecilia latipinna

许多生物过程中的社会交往使用暗示和信号超出人类的敏感性。试图了解他们的行为和生态环境时，要考虑到生物体是如何看待他们的世界是很重要的。灵敏度的紫外光谱（UV; 300 - 400 nm）的跨鸟类，鱼类，爬行类，两栖类，甚至哺乳动物的多个属找到。这个协议描述用于检查生物体紫外线反射结构的存在和用于测试这些线索是否被用作在择偶的上下文社会信号的方法的技术。分光光度计被用来检测反射紫外光和变异在个体和两性之间的反射强度的存在。提出这种技术的一个例子，其中一个二分法的择偶测试暴露性接受个人异性个体，其外观可以通过过滤器，无论是传输全谱或阻挡紫外线的波长进行操作。这个系统允许的女性，而不是男性，sailfin mollies（孔雀鱼latipinna）采用UV的标记，作为其交配的决定的一部分的决心。这些类型的研究，有助于扩大我们的，利用UV生物体的各种知识和洞察UV如何发挥他们生活中的作用。

<html> 图1和2示出了交配偏好水族馆建立和UV测量部位为我们的实验。 测量允许的决心，紫外线反射P. latipinna做具有紫外线的特性，特别是沿其身体（ 图3）的侧面，除了在这些性状的个体差异。一旦UV性状发现，测试显示，女性，而不是男性，P. latipinna在它们的交配决定使用?...

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Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

这个协议概括了使用分光光度法来检测生物体紫外线反射结构（在本例中，茉莉花鳉孔雀latipinna），并描述了对分选择测试鱼类允许要在紫外线线索的伴侣选择期间的作用作出推论。

试验以检测紫外线反射结构，并使用茉莉花鳉确定其重要性伴侣偏好<em&gt;孔雀鱼latipinna</em

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Neuroscience

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

6.5K 次观看.  Ohio Wesleyan University. 这个协议概括了使用分光光度法来检测生物体紫外线反射结构（在本例中，茉莉花鳉孔雀latipinna），并描述了对分选择测试鱼类允许要在紫外线线索的伴侣选择期间的作用作出推论。 

视频: Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

多模态光学显微镜方法在加勒比海礁珊瑚显示息肉组织形态和结构

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

科研

环境科学

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

Tigriopus桡足的个体培养及其交配行为的定量分析

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a ...

Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate hosts into raising their young and how hosts detect parasitism provide important insights in the field of coevolutionary biology. Brood parasites, such as cuckoos and cowbirds, gain an evolutionary advantage because they do not have to pay the costs of rearing their own young. However, these costs select for host defenses against all developmental stages of parasites, including eggs, their young, and adults. Egg rejection experiments are the most common method used to study host defenses. During these experiments, a researcher places an experimental egg in a host nest and monitors how hosts respond. Color is often manipulated, and the expectation is that the likelihood of egg discrimination and the degree of dissimilarity between the host and experimental egg are positively related. This paper serves as a guide for conducting egg rejection experiments from describing methods for creating consistent egg colors to analyzing the findings of such experiments. Special attention is given to a new method involving uniquely colored eggs along color gradients that has the potential to explore color biases in host recognition. Without standardization, it is not possible to compare findings between studies in a meaningful way; a standard protocol within this field will allow for increasingly accurate and comparable results for further experiments.

This protocol provides guidelines for running egg rejection experiments: outlining techniques for painting experimental egg models to emulate the colors of natural bird eggs, conducting fieldwork, and analyzing the collected data. This protocol provides a uniform method for conducting comparable egg rejection experiments.

基于表型梯度的鸡蛋排斥试验对鸡蛋识别极限的探讨

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate ...

行为学

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the appearance and behavior of visual stimuli, compared to manipulating live animals. Here, we present the FishSim Animation Toolchain, a software framework developed to provide researchers with an easy-to-use method for implementing 3D computer animations in behavioral experiments with fish. The toolchain offers templates to create virtual 3D stimuli of five different fish species. Stimuli are customizable in both appearance and size, based on photographs taken of live fish. Multiple stimuli can be animated by recording swimming paths in a virtual environment using a video game controller. To increase standardization of the simulated behavior, the prerecorded swimming path may be replayed with different stimuli. Multiple animations can later be organized into playlists and presented on monitors during experiments with live fish.
In a case study with sailfin mollies (Poecilia latipinna), we provide a protocol on how to conduct a mate-choice copying experiment with FishSim. We utilized this method to create and animate virtual males and virtual model females, and then presented these to live focal females in a binary choice experiment. Our results demonstrate that computer animation may be used to simulate virtual fish in a mate-choice copying experiment to investigate the role of female gravid spots as an indication of quality for a model female in mate-choice copying.
Applying this method is not limited to mate-choice copying experiments but can be used in various experimental designs. Still, its usability depends on the visual capabilities of the study species and first needs validation. Overall, computer animations offer a high degree of control and standardization in experiments and bear the potential to &#39;reduce&#39; and &#39;replace&#39; live stimulus animals as well as to &#39;refine&#39; experimental procedures.

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.

利用fishsim动画工具链调查鱼类行为--以鱼翅钼中的选物复制为例

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the ...

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

墨西哥辣鱼的行为跟踪与神经吻合

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

一个简单的行为分析在检测视觉功能<em&gt;非洲爪蟾</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

神经科学

Swimming Performance Assessment in Fishes

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.

The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.

鱼类游泳绩效评估

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, ...

生物学

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

试验以检测紫外线反射结构，并使用茉莉花鳉确定其重要性伴侣偏好

摘要

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