Figure 2. e-HCFM-staining strategy is effective in revealing symbiotic interactions in marine protists.

These seven cells, fixed on board Tara and kept at 4°C for several years, were imaged manually using the e-HCFM workflow (Figure 1). Each cell is illustrated by two panels: the left side overlays all available fluorescent channels whereas the right side displays only the chlorophyll and the Hoechst fluorescence. Four fluorescent channels were recorded: (i) Green: cellular membranes (DiOC6(3)) indicate the core cell bodies; it also stains loricas of tintinnid ciliates (g); (ii) Blue: DNA (Hoechst) identifies nuclei; it also stains the cell-wall of thecate dinoflagellate (a, c); (iii) Red: chlorophyll autofluorescence resolves chloroplasts; (iv) Cyan: PLL-A546 is a generic counterstain for visualizing eukaryotic cells’ surface (not used in a, (c). 3D reconstructions were conducted with the software Imaris (Bitplane). Scale bar is 10 µm. (a) Association between the heterotrophic dinoflagellate Amphisolenia and unidentified cyanobacteria hosted inside the cell wall (arrow head). (b) The diatom Corethron sp. (Figure 2—video 1) harbors several epiphytic nanoflagellates living in small lorica and attached onto the diatom frustule (arrow head). These have been observed in association with different diatom species (see Figure 2—figure supplement 1). (c) The dinoflagellate Citharistes sp. has developed a chamber (phaeosome) for housing cyanobacteria (arrow head). (d) The diatom Thalassiosira sp. is surrounded by a belt chain of 14 coccolithophores (Reticulofenestra sessilis, arrow head). (e) A juvenile pelagic foraminifer hosts endosymbiotic microalgae (arrowhead), likely Pelagodinium dinoflagellates. (f) Colonies of Fragillariopsis sp. diatoms are regularly observed in close interaction with tintinnid Salpingella sp. ciliate (arrowhead). The tintinnid lorica is inserted inside the groove of the barrel formed by the diatom chain. (g) The lorica of the ciliate Tintinnopsis sp. aggregates several epiphyte pennate diatoms, which were still alive prior to fixation as chloroplast and nuclei are visible (arrow head).

Figure 2—figure supplement 1. e-HCFM reveals an unreported epibiose involving the diatom Chaetoceros simplex and an unidentified nano-flagellate from the 5–20 µm samples of the Tara Oceans expeditions.

The two first rows of the plate illustrate how the epiphytic nano-flagellate cells are attached on the diatom frustule. They live in small tubes (lorica) which are bound at the base of the setae (arrow head, scale bar is 10 µm). Their DNA signature indicates additional signal surrounding the nucleus, which suggest DNA from prey and a heterotrophic behavior. The rows 3 to 5 display few examples of this interaction that were automatically identified by supervised machine learning (Figure 3—source data 1) with a recall value of 88.5% (46 specimens were positively classified from 52 specimens in the learning set and six were false positives from the 18051 other specimens in the learning set). We also provide in the row six few examples of similar associations involving other diatom species (see also Figure 2b). Blue, channel Hoechst; green, Channel DiOC6; red, channel chlorophyll; cyan/purple, channel Alexa546; grey, bright field.

Figure 2—video 1. 3D-animation of a Diatom (Corethron sp.) which illustrates how the e-HCFM method supports investigation about microbial interactions.

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DOI: 10.7554/eLife.26066.018

The specimen (surface water, Tara Ocean station 137) was imaged manually from regular e-HCFM sample preparation with a Leica SP8 confocal laser scanning microscope (40X NA1.1 water). Four fluorescent channels are recorded: (i) Green: cellular membranes (DiOC6(3)) indicate the core cell bodies; (ii) Blue: DNA (Hoechst) is used to identify the nucleus; (iii) Red: chlorophyll (autofluorescence); (iv) Grey: PLL-Alexa546 is a useful counterstain for visualizing the specimen surface. 3D reconstruction, surface rendering and animation were conducted with the software Imaris (Bitplane).