Abstract
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.