墨西哥辣鱼的行为跟踪与神经吻合
Summary
在这里, 我们提出了方法的高通量研究一系列的墨西哥紫鱼行为和重要染色的机械感官系统。这些方法使用自由软件和定制脚本, 为行为研究提供了一种实用且经济高效的方法。
Abstract
栖息在洞穴中的动物进化出一系列的形态和行为特征, 以适应其永恒的黑暗和食物稀疏的环境。在这些特征中, 觅食行为是行为特征进化功能优势的有益窗口之一。本文介绍了分析振动吸引行为 (VAB: 自适应觅食行为) 和适应血管的 tetra、 Astyanax mexicanus相关机械传感器成像的最新方法。此外, 还提出了对包括多动症和睡眠损失在内的一系列额外的空鱼行为进行高通量跟踪的方法。紫鱼也表现出性, 重复的行为和更高的焦虑。因此, 空鱼作为进化行为的动物模型。这些方法使用可应用于其他类型行为的自由软件和自定义脚本。这些方法为商业上可获得的跟踪软件提供了实用和具有成本效益的替代办法。
Introduction
墨西哥的特拉, (Tele特征 idae) 在鱼类中是独一无二的, 因为它有两种截然不同的替代形态–一种是有视力的、表面居住的变形和一种由几种不同的形态组成的盲目的、洞穴居住的变形人口1。虽然在形态和生理上不同, 但它们仍然是干扰素2,3。这些干涉形态似乎发展迅速 (约 20, 000年)4, 使其成为研究快速适应的理想模型系统。据悉, 紫鱼具有一系列不同的形态和行为特征, 包括味蕾密度的增加、机械传感器数量的增加、按振动刺激的特定频率调整的觅食行为、多动症和失眠。其中许多行为可能同时进化, 其中一些被认为在黑暗的洞穴中觅食5和在黑暗和食物稀疏的环境中节能 6,7。
在许多进化模型系统中, 由于大多数物种分布在复杂环境中的连续梯度上, 很难获得关于动物形态和行为如何改变环境的综合知识。然而, 洞穴和表面变形的对比, 在一个尖锐的生态带描绘的高度对比的环境中演变, 导致阿斯特安纳克斯成为一个很好的模型, 了解动物的进化。这使得它能够更容易地将基因和发育过程与环境中的适应性特征和选择联系起来。此外, 最近对Astyanax这些特征的生物医学调查表明, 这些特征可能与人类症状8、9、10 平行.例如, 失去社交性和睡眠, 以及多动症、重复行为和皮质醇水平的增加, 与自闭症谱系障碍8中观察到的情况相似。
为了解决许多行为和形态特征的复杂的共同进化, 它是有利的, 以检测其中的许多突出潜在的遗传和分子途径。本文介绍了一种表征阿斯蒂亚纳克斯表面、洞穴和形态的洞穴型行为表型程度的方法。分析表型特征的焦点行为有空化觅食行为 (振动吸引行为, 简称 vab) 和多动症/睡眠持续时间11,12。还提出了一种与 VAB13相关的感觉系统的成像方法。最近, 许多用于运行行为检测的开源跟踪软件已经出现了14,15.这些工作非常好的短视频, 不到10分钟长。但是, 如果视频较长, 因为密集的计算跟踪时间, 则会出现问题。有能力的商业软件可能是昂贵的。所介绍的方法主要使用免费软件, 因此被认为是具有成本效益和高吞吐量的方法。其中还包括基于这些方法的代表性结果。
Protocol
Representative Results
Discussion
这些方法易于访问, 但由于其免费软件起源的性质, 可能会很复杂。因此, 强烈建议在进行任何实际试验之前进行试验检测和分析。
一旦建立了实验和分析框架, 数据生成的速度就会很快。一旦建立, 它可以记录两条鱼在7分钟内的 VAB 检测, 30 鱼在24小时的活动/睡眠检测, 和一条鱼2.5 至3分钟的神经质体成像, 从 MS222 麻醉到最终的图像捕获。根据所使用的计算机的性能, 视频和图?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢包括 N ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ Cetraro, N. simon, C. valdez, C. Macapac, J. choi, l. lu, j. Nguyen, s. Podhorzer, H. Hernandes, j. fong, j. Kato 和 i. lorder, 用于本手稿中使用的实验鱼。我们还感谢基恩实验室成员, 包括 P. Masek 训练我组装红外 CCD 摄像机。最后, 我们要感谢夏威夷 a ‘ i Mānoa 大学通信学院媒体实验室, 特别是 B. smith、J. lam 和 S. white, 为制作视频提供了宝贵的帮助。这项工作得到了夏威夷社区基金会 (16CON-78919 和 18CON-90818) 和国家卫生研究所 (P20GM125508) 向我的赠款的支持。
Materials
| 4-Di-1-ASP (4-(4-(dimethylaminostyryl)-1-methylpyridinium iodide) | MilliporeSigma | D3418 | |
| 880 nm wave length black light | Advanced Illumination | BL41192-880 | |
| avfs | freeware | Version 1.0.0.6 | http://turtlewar.org/avfs/ |
| Avisynth | freeware | Version 2.6.0 | http://avisynth.nl/index.php/Main_Page |
| Cygwin | freeware | Version 2.11.0 | https://www.cygwin.com/ |
| Cylindrical assay chamber (Pyrex 325 ml glass dish) | Corning | 3140-100 | 10 cm diameter 5 cm high |
| Ethovision XT | Noldus Information Technology, Wageningen, The Netherlands | Version 14 | https://www.noldus.com/animal-behavior-research/products/ethovision-xt |
| Fish Aquarium Cylinder Soft Sponge Stone Water Filter, Black | Jardin (through Amazon.com) | NA | Sponge filter for Sleep/hyperactivity recording system |
| Grade A Brine shrimp eggs | Brine shrimp direct | BSEA16Z | |
| ImageJ | freeware | Version 1.52e | https://imagej.nih.gov/ij/ |
| macro 1.8/12.5-75mm C-mount zoom lens | Toyo | NA | Attach to USB webcam by using c-mount, which is printed in 3-D printer |
| Neutral Regulator | Seachem | NA | |
| Optical cast plastic IR long-pass filter | Edmund optics | 43-948 | Cut into a small piece to fit in the CCD of USB webcam |
| pfmap | freeware | Build 178 | http://pismotec.com/download/ (at “Download Archive” link at the bottom) |
| Reef Crystals Reef Salt | Instant Ocean | RC15-10 | |
| SwisTrack | freeware | Version 4 | https://en.wikibooks.org/wiki/SwisTrack |
| USB webcam (LifeCam Studio 1080p HD Webcam) | Microsoft | Q2F-00013 | Cut 2-2.5 cm of the front |
| WinAutomation | freeware | Version 8 | https://www.winautomation.com/ (free stand-alone app for this procedure) |
| Windows operating system | Microsoft | 7, 8 or 10 | https://www.microsoft.com/en-us/windows |
| x264vfw | freeware | NA | https://sourceforge.net/projects/x264vfw/ |
Referências
- Keene, A. C., Yoshizawa, M., McGaugh, S. E. . Biology and Evolution of the Mexican Cavefish. Biology and Evolution of the Mexican Cavefish. , (2015).
- Mitchell, R. W., Russell, W. H., Elliott, W. R. . Mexican eyeless characin fishes, genus Astyanax: Environment, distribution, and evolution.Special publications the museum Texas Tech University. (12), (1977).
- Wilkens, H. Evolution and genetics of epigean and cave Astyanax-fasciatus (Characidae, Pisces) – Support for the neutral mutation theory. Evolutionary Biology. 23, 271-367 (1988).
- Fumey, J., Hinaux, H., Noirot, C., Thermes, C., Rétaux, S., Casane, D. Evidence for late Pleistocene origin of Astyanax mexicanus cavefish. BMC Evolutionary Biology. 18 (1), 1-19 (2018).
- Yoshizawa, M., Gorički, S., Soares, D., Jeffery, W. R. Evolution of a behavioral shift mediated by superficial neuromasts helps cavefish find food in darkness. Current Biology. 20 (18), 1631-1636 (2010).
- Moran, D., Softley, R., Warrant, E. J. Eyeless Mexican cavefish save energy by eliminating the circadian rhythm in metabolism. PloS One. 9 (9), e107877 (2014).
- Moran, D., Softley, R., Warrant, E. J. The energetic cost of vision and the evolution of eyeless Mexican cavefish. Science Advances. 1 (8), e1500363 (2015).
- Yoshizawa, M., et al. The Evolution of a Series of Behavioral Traits is associated with Autism-Risk Genes in Cavefish. BMC Evolutionary Biology. 18 (1), 89 (2018).
- Riddle, M. R., et al. Insulin resistance in cavefish as an adaptation to a nutrient-limited environment. Nature. 555 (7698), 647-651 (2018).
- Protas, M. E., et al. Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism. Nature Genetics. 38 (1), 107-111 (2006).
- Yoshizawa, M., et al. Distinct genetic architecture underlies the emergence of sleep loss and prey-seeking behavior in the Mexican cavefish. BMC Biology. 13 (1), 15 (2015).
- Duboué, E. R., Keene, A. C., Borowsky, R. L. Evolutionary convergence on sleep loss in cavefish populations. Current Biology. 21 (8), 671-676 (2011).
- Fernandes, V. F. L., Macaspac, C., Lu, L., Yoshizawa, M. Evolution of the developmental plasticity and a coupling between left mechanosensory neuromasts and an adaptive foraging behavior. Biologia do Desenvolvimento. 441 (2), 262-271 (2018).
- Pérez-Escudero, A., Vicente-Page, J., Hinz, R. C., Arganda, S., de Polavieja, G. G. idTracker: tracking individuals in a group by automatic identification of unmarked animals. Nature Methods. 11, 743 (2014).
- Branson, K., Robie, A. A., Bender, J., Perona, P., Dickinson, M. H. High-throughput ethomics in large groups of Drosophila. Nature Methods. 6 (6), 451-457 (2009).
- Yoshizawa, M., Jeffery, W. R., Van Netten, S. M., McHenry, M. J. The sensitivity of lateral line receptors and their role in the behavior of Mexican blind cavefish (Astyanax mexicanus). Journal of Experimental Biology. 217 (6), (2014).
- Lee, A. . Virtualdub. , (2014).
- Schneider, C. A., Rasband, W. S., Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nature Methods. 9 (7), 671-675 (2012).
- Cavallari, N., et al. A blind circadian clock in cavefish reveals that opsins mediate peripheral clock photoreception. PLoS Biology. 9 (9), e1001142 (2011).
- Swimmer, B., Lang, H. H. . Surface Wave Discrimination between Prey and Nonprey by the Back Swimmer Notonecta glauca L. (Hemiptera , Heteroptera ). 6 (3), 233-246 (1980).
- Montgomery, J. C., Macdonald, J. A. . Sensory Tuning of Lateral Line Receptors in Antarctic Fish to the Movements of Planktonic Prey. 235 (4785), 195-196 (1987).
- Prober, D. A., Rihel, J., Onah, A. A., Sung, R. J., Schier, A. F. Hypocretin/orexin overexpression induces an insomnia-like phenotype in zebrafish. The Journal of Neuroscience. 26 (51), 13400-13410 (2006).
- Zhdanova, I. V., Wang, S. Y., Leclair, O. U., Danilova, N. P. Melatonin promotes sleep-like state in zebrafish. Brain Research. 903 (1-2), 263-268 (2001).
- Nussbaum-Krammer, C. I., Neto, M. F., Brielmann, R. M., Pedersen, J. S., Morimoto, R. I. Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans. Journal of Visualized Experiments. (95), e52321 (2015).
- Rasband, W. S. . Object Tracker. , (2000).
- Ferreira, T., Rasband, W. Create Shortcuts. ImageJ User Guide. , (2012).
- Lochmatter, T., Roduit, P., Cianci, C., Correll, N., Jacot, J., Martinoli, A. . SwisTrack. , (2008).
