果蝇 guttifera的分期蛹期和机翼色素沉着的测量方法
Summary
介绍了果蝇 guttifera的分期蛹周期和机翼色素沉着的测量方法。色素沉着的分期和量化为研究成人性状的发育机制提供了坚实的基础, 并使性状发育的种间比较得以实现。
Abstract
不同种类的果蝇(水果蝇) 提供了研究进化机制和遗传变化的机会。特别是, 成人阶段是一个丰富的形态特征来源的种间比较, 包括翼色素沉着比较。为了研究物种间的发育差异, 需要进行细致的观察和适当的分期, 以便进行精确的比较。在这里, 我们描述的蛹周期的分期和量化的翅膀色素在一个波尔卡斑点果蝇,果蝇 guttifera的协议。首先, 我们描述了详细的形态学观察方法和定义的蛹阶段基于形态。该方法包括一种去除蛹外壳的蛹的技术, 以便对蛹形态进行细致观察。其次, 我们描述了测量定义的蛹阶段的持续时间的方法。最后, 我们用数字图像和 ImageJ 软件描述了基于图像分析的机翼色素沉着定量方法。通过这些方法, 我们可以为比较蛹阶段成人性状的发育过程奠定坚实的基础。
Introduction
果蝇的某些形态特征在种类中是多种多样的1,2,3,4,5。我们可以通过比较这些形态的生成机制来探讨形态多样性的产生问题。这种形态的例子有幼体毛、成人性梳子、外生殖器、腹部色素沉着和翅膀色素沉着6、7、8、9、10,11,12,13,14,15. 为了研究成人之间的形态学差异, 观察和分析蛹阶段是很重要的, 因为成虫性状的命运决定于晚期幼体阶段, 随后的形态发生在蛹时期。
在果蝇的发育生物学研究中, “小时 APF” (蛹形成后的小时) 是表示蛹阶段16的常用方法。该系统采用蛹形成后的绝对时间, 便于常规实验。但是, 蛹的发育速度可能不同, 可能会受到轻微的遗传、后生或微差异的影响, 因此在蛹形成后同样的绝对时间不保证蛹在同一发展阶段。在许多情况下, 由形态学特征定义的阶段比比较多个个体更可取。特别是, 物种之间的比较需要精确的分期和相应的 (同源) 阶段之间的比较。
班布里奇和 Bownes17根据果蝇蛹的形态学特征, 认可了20蛹阶段 (P1 至 P15 (ii))。这种分期是最广泛使用的系统形态发育分期18。在先前的研究中, 我们进行了蛹分期的果蝇 guttifera , 以建立一个基础的机翼色素沉着研究19。D. guttifera的翅膀上有一个黑色的圆点图案, 它是机翼色素沉着形成的模型物种之一20。虽然我们提到了班布里奇和 Bownes 的研究17中描述的形态学标准, 但我们直接用串行观测19来测量阶段的持续时间, 而不是使用班布里奇和 Bownes 的阶段工期估计从观察的频率。在这里, 我们描述了在卜et al19中使用的果蝇的蛹阶段的蛹分期和测量的方法。
研究翼色素沉着的发育机制, 需要了解蛹或成年期色素沉着的发生。卜et al。19通过对机翼图像进行图像分析, 在蛹和成人阶段对色素沉着进行量化的光学密度 (ODs)。果蝇翅膀的色素沉着被认为是由黑黑色素堆积引起的21。为量化 ODs, 使用了灰度图像和 ImageJ 软件 (https://imagej.nih.gov/ij/)22 。为了识别和量化斑点特定的色素沉着 (ΔOD), 我们从一个斑点内的 od 中减去外径。为了使这种方法重现性和客观, OD 测量的地方应该确定使用翼静脉作为地标。在这篇文章中, 我们详细描述了这种定量的方法, 机翼色素在果蝇 guttifera。
Protocol
1. 飞行股票 对下列所有协议使用果蝇 guttifera 。 使用塑料瓶 (直径25毫米 x 高度96毫米) 和纤维素插头 (直径 23 mm x 高度 26 mm) 进行库存维护。使用标准的玉米面/糖/酵母/琼脂食品, 并遵循一个出版物描述了这个物种的三其他替代食谱2。注: D. guttifera (股票编号 15130-1971.10) 由加州大学圣地亚哥分校的果蝇物种储备中心提供。尽管D. guttifera属于…
Representative Results
将D. guttifera的蛹期划分为17阶段 (P1-P15 (ii); 三个具有代表性的阶段 (P1、P5-6、P10) 的图像显示图 3, 所有17阶段都在图 4中说明。虽然班布里奇和 Bownes17在d. 黑腹中识别了20阶段, 但这些阶段中的某些步骤无法应用于d. guttifera。两个发育事件的顺序, 黄色身体的出现 (在中肠26内?…
Discussion
我们在这里描述蛹阶段的定义的协议, 去除蛹为详细的观察, 测量蛹阶段的期间和测量黑斑点的强度在翼在D. guttifera。这些协议可以适用于许多果蝇和相关的蝇类, 特别是具有翼色素沉着的物种。
深入观察和描述更详细的发展事件将使阶段进一步细分。在许多情况下, 需要解剖或切片的蛹的发展事件不适合阶段定义, 因为一个人必须杀死一个蛹的分期, 并进一步使?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢肖恩·卡罗尔和托马斯. 沃纳为您提供飞行股票、设备的直保险丝、在拍摄中的帮助、清阿加塔的指导和伊丽莎白中岛的英语编辑。这项工作得到了 KAKENHI 17K19427 和武田科学基金会的支持。
Materials
| Drosophila guttifera | The Drosophila Species Stock Center at the U.C. San Diego | 15130-1971.10 | Drosophila guttifera, a fruit fly species used in this article |
| Plastic vial | Hightech | MKC-30 | Plastic vial, for fly stock maintenance |
| Buzz plugs vial and bottle closures for glass vials | Fisher Scientific | AS-271 | Cellulose plug, for fly stock maintenance |
| White soft sugar | Mitsui Sugar | J-500g | White soft sugar, for standard cornmeal/sugar/yeast/agar food |
| Corn flour | Nippon Flour Mills | F | Corn flour, for standard cornmeal/sugar/yeast/agar food |
| Corn grits – C | Nippon Flour Mills | GC | Corn grits – C, for standard cornmeal/sugar/yeast/agar food |
| Agar powder | Matsuki Kanten Sangyo | No.602 | Agar powder, for standard cornmeal/sugar/yeast/agar food |
| Dry beer yeast | Asahi Food & Healthcare | Y2A | Dry beer yeast, for standard cornmeal/sugar/yeast/agar food |
| Butyl p-hydroxybenzoate | Nacalai Tesque | 06327-02 | Butyl p-hydroxybenzoate, for standard cornmeal/sugar/yeast/agar food |
| Ethanol | Wako | 057-00456 | Ethanol, for standard cornmeal/sugar/yeast/agar food |
| Flat bottom microtube | Ina Optica | CF-0150 | 1.5 mL microtube, for collecting pupae |
| CAPSULEFUGE | Tomy | PMC-060 | Mini microcentrifuge, for collecting pupae |
| Sterilized Schale NB | Sansei Medical | 01-013 | Plastic Petri dish (diameter 90 mm x height 15 mm) |
| Serum tube rack | Iwaki | 9796-050 | Used as a moist chamber, for observation of pupa |
| Corning Falcon Easy-Grip tissue culture dish | Corning | 353001 | Plastic Petri dish (diameter 35 mm x height 10 mm) |
| Falcon standard tissue culture dish | Corning | 353002 | Plastic Petri dish (diameter 60 mm x height 15 mm) |
| Push-pin | Kokuyo | 51233709 | Push-pin, for making pinholes on the microtube lid |
| Stereomicroscope | Olympus | SZX16 | Stereomicroscope, for morphological observation |
| Digital camera | Olympus | DSE-330-A | Digital camera, for imaging |
| NICETACK double sided tape | Nichiban | NW-15SF | Double sided tape, for removing puparium |
| Dumont #5 forceps | Fine Science Tools | 11252-20 | Forceps, for removing puparium |
| Van Gogh VISUAL Paint brush | Talens Japan | GWVR-#5/0 | Paint brush, for removing puparium |
| Greiner CELLSTAR 12 well cell culture plate | Merck | 665-180 | 12-well cell culture plate, for measuring durations of pupal periods |
| NaCl | Wako | 191-01665 | NaCl, for PBS |
| KCl | Nacalai Tesque | 285-14 | KCl, for PBS |
| Na2HPO4·12H2O | Wako | 196-02835 | Na2HPO4·12H2O, for PBS |
| KH2PO4 | Nacalai Tesque | 28721-55 | KH2PO4, for PBS |
| Stepped Neutral Density (ND) Filter 0.04 – 3.0 | Edmund Optics | 64-384 | Stepped density filter, for calibration of pigmentation measurement |
| ImageJ software | NIH | 1.8.0-101 | ImageJ software, for measurement of intensity of black spots on a wing (https://imagej.nih.gov) |
| FINE FROST glass slide | Matsunami Glass Ind | FF-001 | Glass slide, for measurement of intensity of black spots on a wing |
| Square microscope cover glass 18 x 18 | Matsunami Glass Ind | C018181 | Cover slip, for measurement of intensity of black spots on a wing |
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Citazione di questo articolo
Fukutomi, Y., Matsumoto, K., Funayama, N., Koshikawa, S. Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera. J. Vis. Exp. (131), e56935, doi:10.3791/56935 (2018).