量化腹部色素沉着<em>黑腹果蝇</em
Summary
这项工作提出了一种使用数字图像分析快速准确量化黑腹果蝇腹部色素沉着的方法。该方法简化了表型获取和数据分析之间的过程，包括样本安装，图像采集，像素值提取和特征测量。
Abstract
色素是一种形态上简单但高度可变的特征，通常具有适应性意义。它广泛地作为了解形态表型发育和演化的模型。果蝇黑腹果蝇的腹部色素沉着症特别有用，使研究人员能够鉴定出形态学上和种内变异之间的基因座。然而，迄今为止， 黑腹果蝇腹部色素沉着已经在很大程度上通过评分而不是定量地测定，这限制了可用于色素数据的统计分析的形式。这项工作描述了一种新的方法，可以量化成年人黑腹果蝇腹部色素沉着模式的各个方面。该协议包括样品安装，图像捕获，数据提取和分析。用于图像捕获和分析的所有软件都是宏用于开源图像分析。这种方法的优点是能够使用在不同成像系统中高度可重复的方法精确测量色素沉着特征。虽然该技术已经被用于测量成年黑腹果蝇的terga色素沉积模式的变化 ，但是该方法是灵活的并且广泛适用于无数不同生物体中的色素沉着模式。
Introduction
染色显示物种，种群和个体之间以及甚至在个体发育1,2,3,4,5,6期间的个体内的巨大表型变异。尽管在各种各样的动物中有无数的色素沉着研究，但色素沉淀在黑腹果蝇中可能已被最好地研究，其中分子遗传学的全部功能被用于阐明调节色素沉着的发育和生理机制以及这些机制如何演变1 ， 6 。已知关于调节黑腹果糖7,8中颜料的生物化学合成的基因以及控制时间和空间di的基因这种生物合成的贡献9,10,11,12,13。此外，遗传作图已经确定了黑腹果蝇14,15,16,17中色素沉着的基因和种间差异的遗传基因座。色素沉着和多效性之间的关系，如行为18,19和免疫19,20也已经被探索，色素形态15,21,22的适应性意义。因此，黑腹果蝇的色素沉积已经成为一个强大但简单的model用于复杂表型的发展和演变。
成年人黑腹果蝇的色素沉着特征在于身体黑色化的特征，特别是在胸部和背部胸部和腹部。每个角质层（tergite）在背腹的色素沉着是受到最多研究关注的。由于遗传17,23和环境24,25因素，这种色素沉着（ 图1A -F ）有相当大的变化。腹部扁桃体的角质层由前后发育室（ 图1G ）组成，每个隔室可以根据色素沉着和装饰26进一步细分。前房包括六个角质层类型（a1-a6），后隔室包括三（p1-p3）（ 图1G ）。其中，p1，p2和a1角质层通常在未拉伸腹部的tergite下折叠，使得它们被隐藏。可见的可见角质层的特征在于一层重的色素沉着，这里称为“色素带”，由角质层类型a4（毛状的中度刷毛）和a5（毛状的大刷毛）组成，带的后缘比前边缘更强烈地着色（ 图1G ）。该带的前面是轻度着色的毛状角质层的区域，后面具有刷毛（a3）但不是前面的（a2）。在色素的强度和颜料带的宽度方面均观察到苍蝇之间色素沉着的变化。一般来说，大部分后段（腹部段5,6和7）的变化最大，前段较多（腹部gments 3和4） 24 。此外，黑腹果蝇色素沉着中存在性二态性，男性通常具有完全着色的第五和第六腹部白细胞（ 图4C ）。
在黑腹果蝇腹部色素沉着的大多数研究中，色素沉着已被视为一种分类或顺序性状，其特征是定性地测定了27,28,29或半定量地在14,15,16,17,24,30 ， 31,32,33,34,35， 36,37 。这些方法不可避免地缺乏精确度，并且由于它们依赖于色素沉着的主观评估，因此难以比较研究中的数据。一些作者量化了色素沉积的空间维度38,39 ，特异性角质层23,25,39,40的色素沉着强度，或整个腹部T细胞的平均色素沉积强度41,42,43 。然而，这些量化方法不能同时测量腹部色素沉着的强度和空间分布，因此不能捕捉染色体如何在整个abd中变化的细微差别主体tergite。此外，这些量化方法38,41,42,43中的几个需要腹部角质层的解剖和安装。这样做既耗时又破坏样本，使其不能用于额外的形态分析。随着对腹部色素沉着的发展和进化的理解加深，需要更复杂的工具来快速，准确地测量色素沉着的空间分布和强度。
该方法的总体目标是利用数字图像分析获得黑腹果蝇腹部色素沉着的可复制和更准确的测量。该方法包括三个阶段。首先，成年飞行是非破坏性的，并且背部腹部的数字图像被拍摄。其次，使用ImageJ宏，用户定义了从第二个角质层的前部延伸到第三个和第四个腹部节段上的a5角质层（绿色框， 图1G ）的后部的前后后条带。然后沿其长轴提取穿过该条带的宽度的平均像素值，产生一个轮廓，其捕捉到从tergite的前部到后部变化时的色素沉积的空间分布和强度。第三，R脚本用于使用三次样条数学地描述色素分布。然后，R脚本使用花键及其第一和第二导数来提取a2-a5角质层的宽度，颜料带的宽度以及色素沉着的最大和最小水平。因此，该方法量化了腹部色素沉着的空间特征和深度。
这种方法量化了第三和第四腹部白细胞的色素沉着，这些都是许多以前的研究1,15,23,24,25,28,33,39,42的重点，无论是专门还是与更多的后白蛋白组合。尽管比第五和第六腹部白细胞少变量，但第三和第四锥体在男性中并不完全着色，因此该方案可适用于男性和女性。然而，如图所示，方案可用于测量女性第五和第六腹部白细胞的色素沉着。此外，用于提取色素分布特征的脚本的微小修改应允许该方法用于量化各种其他色素沉着的变化生物。
Protocol
Representative Results
Discussion
该方法允许以适合多次下游分析的定量形式精确，快速和可重复地获取色素数据。该方法已被用于获取温度对苍蝇等基因系中腹部色素沉着的影响的数据。然而，该方法可以用于前向遗传学研究，以鉴定基因，这些基因是个体，群体或物种之间色素沉着差异的基础，或反向遗传研究，以探索特定基因对色素沉着模式的影响。尽管如上所述，已经有无数研究探讨了黑腹果蝇色素沉着的发展和进化，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由国家科学基金会授予IOS-1256565和IOS-1557638资助给AWS。感谢Patricia Wittkopp和三位匿名评论者对本文早期版本的有益评论。
Materials
| Dumont #5 Biology Forceps | FST | 11252-30 | |
| Agar | Sigma-Aldrich | 5040 | |
| Dissecting Scope | Leica | MZ16FA | |
| Base | Leica | MDG41 | |
| Camera | Leica | DFC280 | |
| Gooseneck Cold Light Source | Schott | ACE 1 | |
| Image Acquisition Control Software | Micro-Manager v1.3.20 | https://micro-manager.org/ | |
| Image Analysis Software | ImageJ | https://imagej.nih.gov/ij/ | |
| Data Analysis Software | R 3.3.2 | https://www.r-project.org/ | |
| LED | Thor Labs | LEDWE-15 | |
| Multimeter | Fluke | Fluke 75 Series II | |
| 60 x 15 mm Petri dish | Celltreat Scientific Products | 229663 | |
| Stage micrometer | Klarman Rulings, Inc. | KR-867 |
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