For all experiments, axenic cultures were grown in seawater-based Hemi medium (see Table S1 in the supplemental material) supplemented with 1% horse serum and 0.025 g/liter LB broth powder (53 (link)). An artificial seawater medium lacking Sr2+, Ba2+, and sulfates was prepared from 288 mM NaCl, 8 mM KCl, 718 mM KBr, 100 mM MgCl2, 12 mM CaCl2, 40 mM HBO3, and 60 mM NaF, supplemented with 1% (vol/vol) heat-inactivated horse serum (Sigma-Aldrich) and 25 mg LB broth powder (Amresco). The medium was used as rinsing solution for preparation of ICP-MS samples and cell microcrystal depletion to be measured via quantitative phase imaging (QPI) (see below). For Ba2+ loading experiments, BaCl2 was added in equimolar amounts with respect to naturally occurring (88 μM) Sr2+ (12 (link)).
Axenic clonal cultures of 17 species of diplonemids were grown either at 27°C (Paradiplonema papillatum ATCC 50162), 22°C (Namystinia karyoxenos YPF1621), or 13°C (of D. aggregatum YPF1605, D. japonicum YPF1604, Flectonema sp. DT1601, Hemistasia phaeocysticola, Lacrimia lanifica JW1601, Lacrimia sp. YPF1808, Rhynchopus sp. YZ270 cl. 10.3, Rhynchopus sp. YZ270 cl. 9, Rhynchopus sp. DT0301, Rhynchopus humris YPF1505, R. euleeides ATCC 50226, R. serpens YPF1515, and Sulcionema specki YPF1618). P. papillatum and R. euleeides were isolated from coastal surface waters (United States) in 1985 and 1986, respectively. The remaining species originated either from coastal seawater around Japan or from Enoshima Aquarium (Kanagawa, Japan) and were continuously maintained in culture for 1 to 7 years prior to the analyses. The identity of not-yet-formally described species was established based on the 18S rRNA sequences as described previously (54 (link)). Dense cultures of trophic cells (55 (link)) were harvested by centrifugation at 3,000 × g for all subsequent analyses.
Light microscopy images and videos were taken with an Olympus BX53 microscope equipped with a DP72 microscope digital camera using CellSens software v. 1.11 (Olympus) and processed with GIMP v. 2.10.14, Irfan View v. 4.54, and Image J v. 1.51 software. Polarized microscopy was performed using crossed polarizers installed to a Raman microscope (as specified below).
Axenic clonal cultures of 17 species of diplonemids were grown either at 27°C (Paradiplonema papillatum ATCC 50162), 22°C (Namystinia karyoxenos YPF1621), or 13°C (of D. aggregatum YPF1605, D. japonicum YPF1604, Flectonema sp. DT1601, Hemistasia phaeocysticola, Lacrimia lanifica JW1601, Lacrimia sp. YPF1808, Rhynchopus sp. YZ270 cl. 10.3, Rhynchopus sp. YZ270 cl. 9, Rhynchopus sp. DT0301, Rhynchopus humris YPF1505, R. euleeides ATCC 50226, R. serpens YPF1515, and Sulcionema specki YPF1618). P. papillatum and R. euleeides were isolated from coastal surface waters (United States) in 1985 and 1986, respectively. The remaining species originated either from coastal seawater around Japan or from Enoshima Aquarium (Kanagawa, Japan) and were continuously maintained in culture for 1 to 7 years prior to the analyses. The identity of not-yet-formally described species was established based on the 18S rRNA sequences as described previously (54 (link)). Dense cultures of trophic cells (55 (link)) were harvested by centrifugation at 3,000 × g for all subsequent analyses.
Light microscopy images and videos were taken with an Olympus BX53 microscope equipped with a DP72 microscope digital camera using CellSens software v. 1.11 (Olympus) and processed with GIMP v. 2.10.14, Irfan View v. 4.54, and Image J v. 1.51 software. Polarized microscopy was performed using crossed polarizers installed to a Raman microscope (as specified below).
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