Three-dimensional micro-mapping of microorganisms in the plastisphere using laser scanning confocal fluorescence microscopy

Abstract

We investigated the attachment patterns of microorganisms within plastispheres on plastic surfaces through three-dimensional (3D) micro-mapping of the plastisphere using multichannel laser scanning confocal fluorescence microscopy (DNA staining, chlorophyll autofluorescence, exopolysaccharides staining, and reflection). This approach allowed us to focus on information derived from the 3D structures of the plastisphere. Four-channel observations of four types of plastics—polyethylene terephthalate (PET), polyvinyl chloride (PVC), polymethyl methacrylate (PMMA), and polycarbonate (PC)—and glass plates immersed in coastal Sagami Bay for 10 days revealed the layered 3D structure of plastispheres composed mainly of diatoms and bacteria. Some dinoflagellates were also observed in the plastispheres. Morphological characteristics and chlorophyll content indicated that the observed dinoflagellates were in a vegetative form and included potentially toxic and/or red-tide-causing harmful dinoflagellates. This reaffirms previous concerns about environmental changes caused by toxic dinoflagellates captured on plastics and drifting in the ocean as hitchhikers, thereby spreading over a wider area than their original dispersion. Quantitative analysis of the 3D micro-mapped plastispheres suggested that a biofilm layer a few micrometers thick was required for dinoflagellates to attach to the plastic surface. Since extracellular polymeric substances (EPS) may be associated with the stickiness of the plastisphere, we performed five-channel observations, including exopolysaccharide staining to label EPS. These results suggested that the viscosity of EPS was responsible for these dinoflagellates’ attaching to plastic surfaces. These results provided a new perspective on the potential for the formation of plastispheres containing dinoflagellates.