Plastic marine debris (PMD) affects spatial scales of life from microbes to whales. However, understanding interactions between plastic and microbes in the "Plastisphere"-the thin layer of life on the surface of PMD-has been technology-limited. Research into microbe-microbe and microbe-substrate interactions requires knowledge of community phylogenetic composition but also tools to visualize spatial distributions of intact microbial biofilm communities. We developed a CLASI-FISH (combinatorial labelling and spectral imaging - fluorescence in situ hybridization) method using confocal microscopy to study Plastisphere communities. We created a probe set consisting of three existing phylogenetic probes (targeting all Bacteria, Alpha-, and Gammaproteobacteria) and four newly designed probes (targeting Bacteroidetes, Vibrionaceae, Rhodobacteraceae and Alteromonadaceae) labelled with a total of seven fluorophores and validated this probe set using pure cultures. Our nested probe set strategy increases confidence in taxonomic identification because targets are confirmed with two or more probes, reducing false positives. We simultaneously identified and visualized these taxa and their spatial distribution within the microbial biofilms on polyethylene samples in colonization time series experiments in coastal environments from three different biogeographical regions. Comparing the relative abundance of 16S rRNA gene amplicon sequencing data with cell-count abundance data retrieved from the microscope images of the same samples showed a good agreement in bacterial composition. Microbial communities were heterogeneous, with direct spatial relationships between bacteria, cyanobacteria and eukaryotes such as diatoms but also micro-metazoa. Our research provides a valuable resource to investigate biofilm development, succession and associations between specific microscopic taxa at micrometre scales.

中文翻译：

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“塑料球”中的空间结构：用于对塑料海洋碎片上的微观群落进行成像的分子资源。


海洋塑料垃圾 (PMD) 影响从微生物到鲸鱼的生命空间尺度。然而，了解“塑料球”（PMD 表面的生命薄层）中塑料与微生物之间的相互作用一直受到技术的限制。对微生物-微生物和微生物-基质相互作用的研究需要了解群落系统发育组成的知识，还需要可视化完整微生物生物膜群落空间分布的工具。我们开发了一种 CLASI-FISH（组合标记和光谱成像 - 荧光原位杂交）方法，使用共焦显微镜来研究 Plastisphere 群落。我们创建了一个探针组，其中包含三个现有的系统发育探针（针对所有细菌、α-和γ-变形菌）和四个新设计的探针（针对拟杆菌、弧菌科、红细菌科和交替单胞菌科），总共标记有七个荧光团，并使用以下方法验证了该探针组：纯粹的文化。我们的嵌套探针组策略增加了分类识别的信心，因为目标是通过两个或多个探针确认的，从而减少了误报。我们在三个不同生物地理区域的沿海环境中进行定植时间序列实验，同时识别并可视化了这些类群及其在聚乙烯样品上微生物生物膜内的空间分布。将 16S rRNA 基因扩增子测序数据的相对丰度与从相同样品的显微镜图像中检索到的细胞计数丰度数据进行比较，显示细菌组成具有良好的一致性。微生物群落是异质的，细菌、蓝细菌和真核生物（例如硅藻）和微型后生动物之间具有直接的空间关系。 我们的研究为研究微米尺度上特定微观类群之间的生物膜发育、演替和关联提供了宝贵的资源。