自动图像处理确定河流大型无脊椎动物群落大小结构
Summary
本文基于创建适应协议，以使用半自动成像程序扫描，检测，分类和识别与底栖河流大型无脊椎动物相对应的数字化物体。该程序允许在大约1小时内获得大型无脊椎动物群落的个体尺寸分布和尺寸指标。
Abstract
体型是一个重要的功能性状，可用作评估自然群落扰动影响的生物指标。群落规模结构响应生物和非生物梯度，包括跨分类群和生态系统的人为扰动。然而，手动测量底栖大型无脊椎动物等小型生物（例如，>500 μm 至几厘米长）非常耗时。为了加快群落规模结构的估计，我们开发了一种协议来半自动测量受保护的河流大型无脊椎动物的个体体型，这是评估淡水生态系统生态状况最常用的生物指标之一。该协议改编自现有的方法，该方法旨在使用专为水样设计的扫描系统扫描海洋中游动物。该协议包括三个主要步骤：（1）扫描河流大型无脊椎动物的子样本（细样本和粗样本量分数）并处理数字化图像以个性化每个图像中的每个检测到的对象;（2）通过人工智能创建、评估和验证学习集，以半自动地将大型无脊椎动物的单个图像与扫描样本中的碎屑和人工制品分开;（3）描绘大型无脊椎动物群落的大小结构。除协议外，这项工作还包括校准结果，并列举了几个挑战和建议，以使该程序适应大型无脊椎动物样品并考虑进一步改进。总体而言，研究结果支持使用所提出的扫描系统对河流大型无脊椎动物进行自动体型测量，并表明其尺寸谱的描述是淡水生态系统快速生物评估的宝贵工具。
Introduction
底栖大型无脊椎动物被广泛用作确定水体生态状况的生物指标1。大多数描述大型无脊椎动物群落的指数都侧重于分类指标。然而，鼓励使用整合体型的新生物评估工具，为分类学方法提供替代或补充视角2，3。
体型被认为是与其他重要特征（如新陈代谢、生长、呼吸和运动）相关的元特征4.此外，体型可以决定营养位置和相互作用5。群落中个体体型与按大小等级标准化生物量（或丰度）之间的关系定义为尺寸谱6，并遵循随着个体大小在对数尺度上增加而归一化生物量线性减少的一般模式7。这种线性关系的斜率在理论上得到了广泛的研究，跨生态系统的实证研究将其作为群落规模结构的生态指标4。在生物多样性生态系统功能研究中成功使用的群落规模结构的另一个综合指标是群落规模多样性，它表示为大小谱或其类似物的大小类别的香农指数，该指数是根据个体大小分布8计算的。
在淡水生态系统中，不同动物群落的大小结构被用作共济失调指标，以评估生物群落对环境梯度9，10，11和人为扰动12，13，14，15，16的响应。大型无脊椎动物也不例外，它们的大小结构也对环境变化17，18和人为扰动做出反应，例如采矿19，土地利用20，或氮（N）和磷（P）富集20，21，22。然而，测量数百人以描述社区规模结构是一项繁琐且耗时的任务，由于时间不足，通常避免将其作为实验室的常规测量。因此，已经开发了几种半自动或自动成像方法来分类和测量标本23，24，25，26。然而，这些方法中的大多数都侧重于分类学分类，而不是生物体的个体大小，并且尚未准备好用于所有种类的大型无脊椎动物。在海洋浮游生物生态学中，扫描图像分析系统已被广泛用于确定浮游动物群落的大小和分类组成27，28，29，30，31。该仪器可以在世界各地的几个海洋研究所找到，它用于扫描保存的浮游动物样品以获得整个样品的高分辨率数字图像。本议定书调整了该仪器的使用，以快速自动的方式估计河流中大型无脊椎动物群落的大小范围，而无需投资创建新设备。
该协议包括扫描样品和处理整个图像，以自动获得样品中对象的单个图像（即晕影）。形状、大小和灰度特征的几种测量表征了每个对象，并允许将对象自动分类为类别，然后由专家进行验证。每个生物体的个体大小是使用椭球体生物体积（mm3）计算的，该生物体积来自以像素为单位测量的生物体面积。这允许快速获得样品的尺寸谱。据我们所知，这种扫描成像系统仅用于处理中浮游动物样品，但该设备可能允许处理淡水底栖大型无脊椎动物。
因此，本研究的总体目标是引入一种方法，通过调整以前用于海洋中游生物27，32，33的现有协议，快速获得保存的河流大型无脊椎动物的个体大小。该过程包括使用半自动方法，该方法与扫描设备一起扫描水样和三个开放式软件来处理扫描的图像。本文提出了一种经过调整的协议，用于扫描、检测和识别数字化河流大型无脊椎动物，以自动获取群落规模结构和相关规模指标。还根据从伊比利亚半岛东北部（东北）三个盆地（Ter、Segre-Ebre和Besòs）收集的42张河流大型无脊椎动物样本的扫描图像，对提高效率的程序和准则进行了评估。
这些样品是在100米长的河段收集的，遵循西班牙政府对可涉足河流中底栖河流大型无脊椎动物进行实地取样和实验室分析的协议34。在多栖息地调查后，用苏伯采样器（框架：0.3 m x 0.3 m，目数：250 μm）收集样品。在实验室中，将样品通过5毫米和500μm网清洁和筛分，以获得两个子样品：粗子样品（5毫米目）和精细子样品（500微米目），它们储存在单独的小瓶中并保存在70%乙醇中。将样品分成两个大小的分数可以更好地估计群落大小结构，因为大型生物比小型生物更稀有且更少。否则，扫描样品具有大尺寸分数的偏差表示。
Protocol
Representative Results
Discussion
Gorsky等人2010年描述的河流大型无脊椎动物方法的调整允许在估计淡水大型无脊椎动物的群落规模结构时具有很高的分类准确性。结果表明，该方案可以将估计样品中个体大小结构的时间减少到约1小时。因此，拟议的协议旨在促进常规使用大型无脊椎动物大小光谱作为快速和综合生物指标，以评估淡水生态系统中扰动的影响。大型无脊椎动物的体型谱已经被用作评估沿海泻湖生态状况的成功指标<s…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了西班牙科学、创新和大学部的支持（批准号RTI2018-095363-B-I00）。我们感谢CERM-UVic-UCC成员Èlia Bretxa，Anna Costarrosa，Laia Jiménez，María Isabel González，Marta Jutglar，Francesc Llach和Núria Sellarès在大型无脊椎动物现场采样和实验室分类方面的工作，以及David Albesa在样品扫描方面的合作。最后，我们感谢Josep Maria Gili和海洋科学研究所（ICM-CSIC）使用实验室设施和扫描仪设备。
Materials
| Beaker | Labbox | Other containers could be used | |
| Dionized water | Icopresa | 8420239600123 | To dilute the ethanol |
| Funnel | Vitlab | 41094 | |
| Glass vials 8 ml | Labbox | SVSN-C10-195 | 1 vial/subsample |
| ImageJ Software | Free access | Version 4.41o/ Image processing software | |
| Large frame | Hydroptic | Provided by ZooScan | 24.5 cm x 15.8 cm |
| Monalcol 96 (Ethanol 96) | Montplet | 1050JE001 | |
| Plankton Identifier Software | Free access | Version 1.2.6/ Automatic identification software | |
| Sieve | Cisa | 26852.2 | Nominal aperture 500µ and nominal aperture 0,5 cm |
| Tweezers | Bondline | B5SA | Stainless, anti-magnetic, anti-acid |
| VueScan 9 x 64 (9.5.09) Software | Hydroptic | Version 9.0.51/ Sacn software | |
| Wooden needle | Any plastic or wood needle can be used | ||
| Zooprocess Software | Free access | Version 7.14/Image processing software | |
| ZooScan | Hydroptic | 54 | Version III/ Scanner |
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